Comp.Biochem. Physid

0300-9629/92 %S.OO+O.OO

Vol. lOlA,No. 2,pp. 259-268,1992

0 1992Pergamon Press plc

Printed in Great Britain

STATOLITH HAIR MOVEMENTS AND THE REGULATION OF TONIC GRAVITY REFLEXES IN THE LOBSTER, HOMARUS AMERICANUS MARION L. PATTON* and ROBERT F. C&o@’ *Department of Biology, Occidental College, Eagle Rock, CA 90041, U.S.A. and ERCB, 5510 Morehouse Dr., San Diego, CA 92121, U.S.A.; tSouthem California Edison Co., Rosemead, CA 91770, U.S.A. (Received 31 May 1991) Abstract-l. The irregularity of the statolith of the lobster, Homarus americanus, probably causes a large and haphazard variation in the response of the individual statocyst receptors to body rotation. 2. Lobster gravity reflexes are regulated by the summed responses of the statocyst receptors; this probably compensates for the haphazard variation in the sensory input.

INTRODUCTION Lobster balance organs, like most animal balance organs, are hollow, fluid-filled sacs that have sensory hairs projecting into their lumens. Dense bodies, called statoliths, are fastened to the tips of these hairs. The statoliths sink toward the bottom of the sacs so that, as the animal changes its orientation relative to gravity, the statoliths move within the sacs and bend the sensory hairs. The hairs have a flexible joint, called the ‘casque’, at their bases (Schiine and Steinbrecht, 1968), so the lith movement changes the angle of the hairs relative to the cyst wall (to simplify discussion, this will be called the ‘hair angle’). Hypothetically, this change in hair angle provides hair receptors with their adequate stimulus (Lowenstein, 1936, 1950; Dijkgraaf, 1956; Schiine, 1959; Gemandt, 1959; Young, 1960; Stein, 1975; Ozeki et al., 1978). The responses of these receptors regulate the gravity reflexes which compensate for or correct body tilt (Schiine, 1959; Davis, 1968; Takahata and Hisada, 1982a,b; Newland, 1989). Different receptors respond differently to body tilt; this may be caused by the irregularity of the statolith (Cohen, 1954, 1955, 1960). The statolith is irregular because it consists of sand grains, which are also irregular, of different sizes (Cohen, 1955). Since the tips of the statolith hairs are inserted between these sand grains, lith irregularity should result in a haphazard variation in the position of the hair tips. A haphazard variation in the position of the tips of the hairs would, of course, produce a haphazard variation in hair angle. Receptor response is a consistent function of hair angle (Cohen, 1960; Patton and Grove, in press), and the tonic hair receptors are non-adapting (Cohen, 1955; Patton and Grove, 1992). Therefore, a haphazard variation in the hair angle would be reflected in a haphazard variation in the responses of the receptors of different hairs to body tilt. This haphazard variation in receptor response might be large compared to the change in receptor activity during body tilt because lith excursion is probably very small (Cohen, 1955).

Despite this haphazard variation, and despite the probability that this sensory input changes when the statocyst is shed during ecdysis, the lobster’s gravity reflexes are very consistent (Schiine, 1959; Davis, 1968). In this communication, we verify the haphazard variation in the hair angle and attempt to learn how the consistent motor output is generated from an inconsistent sensory input. Using anatomical measurements, we tried to show that the variation between hairs in hair angle was large compared to the average change in hair angle during normal tilt, and that this variation was uncorrelated with hair location in the statocyst. A variation in hair angle that was correlated with hair location in the statocyst would not really be haphazard; it would probably be genetically determined and related to statocyst function. We also tried to show that the effect of deflecting several hairs in concert equalled the sum of the effects of their separate deflexions. This would indicate that the lobster generates its consistent motor output from inconsistent sensory input by summing the responses of many sensory receptors. It is obvious that a summation of the discharge of many receptors would be more consistent than the discharge of a single receptor, because averages based on large samples are less variable than averages based on small samples (Sokal and Rohlf, 1973). This hypothesis would also explain why the lobster, an animal with few neurons (Wiersma, 1952), has a very large number (> 2000) of statocyst receptors (Cohen, 1954; Bush and Laverack, 1982). We also examined alternative hypotheses that could explain the large number of statocyst receptors. Receptor response curves do not completely overlap and thus the lobster could measure body tilt by determining which receptors were active (Cohen, 1954, 1955). The lobster could use this method of measuring tilt if differences in hair angle, and therefore in the response curve of the receptors, were determined genetically and not by lith configuration. If this were true, deflexion of different hairs or groups of hairs in the same lobster should elicit different

259

260

MARION L. PATTONamd ROBERTF. GROVE

responses, but deflexion of the same hairs in different lobsters should elicit similar responses. That is, if the function of a hair is determined genetically, the response elicited by a given hair should be related to its location in the statocyst. We tested this hypothesis by deflecting hairs in different locations in the statocyst and measuring the magnitude of the reflex response. Even if the differences in the response ranges of the receptors were determined by lith configuration and not genetically, the lobster could still measure its body tilt by determining which receptors were active by re-learning, after the lith was shed in each ecdysis, which body tilts were signaled by the new response curves of the receptors. Since every statolith is different, this hypothesis predicts that different hairs should have different effects in the same lobster and that the relationship between hair location and the effect of its receptors on the gravity reflexes should be different in different lobsters. We tested this hypothesis by, once again, deflecting hairs in different locations and measuring the reflex response. A third exnlanation for the large number of statocyst neuronsis that the statocyst f;nctions as a ‘noise

filter’ that compensates for the arrythmic discharge of the statocyst receptors (Cohen, 1960). If the statocyst functioned in this way, a reflex response would appear only if two or more receptors were activated simultaneously. A temporary increase in the discharge of a single receptor could be produced by the arrythmic nature of the statocyst receptor discharge, but a simultaneous increase in the discharge of several receptors would probably represent a stimulus. We tested this hypothesis by deflecting single hairs and measuring the response. To test these hypotheses it was, of course, unnecessary to measure the effects of all 800 statolith hairs on all the many gravity reflexes (Davis, 1968) during all possible postural changes. The response of the eyecups to roll tilt was used as a model of the lobster’s gravity reflexes because it is relatively simple. The eyecups have only one joint, and over a range of tilts, about 45” each way from the animal’s normal position, both eyecup movement and the movement of the appendages involved in righting reflexes (Schiine, 1959; Davis, 1968; Neil, 1982) are almost directly__oronortional to imposed _ tilt. ’

Fig. 1. Dorsal aspect of the right statocyst. The anterior end is on the upper right. The base of the antennule and the dorsal wall of the cyst has been removed. The statolith hairs are arranged in a crescent around the statolith which is cemented to the cyst floor. Hairs of the outermost row, “Row l”, do not touch the lith. The fine lines projecting from the medial cyst wall represent the thread hairs which also do not contact the statolith (after Cohen, 1960).

Statocyst regulation of lobster gravity reflexes The eyecups and the statocysts respond to both pitch and roll (Cohen, 1955, 1960; Schiine, 1959; Davis, 1968; Neil, 1982). To avoid confounding summation within a reflex with the elicitation of two reflexes, we first identified those statolith hairs involved in the roll reflex. In the crayfish, the response to roll is regulated by the hairs lying laterally to the lith (Takahata and Hisata, 1979, 1982a,b). To learn whether the roll reflex was elicted by the same hairs in the lobster, we removed parts of the lith and examined the response of the eyecup to movements of the remaining lith. Other preliminary experiments were necessary. In order to use extracellular muscle recordings to measure reflex response, it was necessary to demonstrate that the muscle we used was innervated by a single tonic axon. Finally, many statocyst hairs drive three receptor neurons with different response characteristics (Patton and Grove, in press), and thus it was necessary to identify which of the three receptors regulated the tonic response to roll. Following these preliminary observations, we performed experiments that supported the hypothesis that the magnitude of the tonic gravity reflexes was regulated by the linear sum of the input from a given set of statocyst receptors, but did not support the other hypotheses.

lmm

MATERULS

261 AND METHODS

Anatomy Gross statocyst anatomy was described in the classical work of Cohen (1954, 1955, 1960) (Figs 1 and 2). The statolith hairs are attached to a raised and roughly crescentshaped area of the cyst floor called the sensory cushion. To simplify discussion, we will distinguish ‘row’ hairs, arranged in a hook around the posterior and lateral sides of the lith, from the small ‘matt’ hairs that form the anterior limb of the crescent that lies under the lith (Fig. 2). There are four complete rows of ‘row’ hairs and one partial row. Starting from the outside, the rows will be referred to as rows 1, 2, 3-A (the partial row), 3 and 4. This anatomical study was largely focused on the row 3 and 4 hairs because they were the easiest hairs to stimulate with micromanipulators and because such stimulation elicted gravity reflexes. Mature male and female lobsters were shipped by air from Maine and kept in aerated sea water at 10°C until use. All experiments were done at 9 f 05°C. First, lith movement was measured in unfixed statocysts. Each statocyst was excised and the membranous top of the cyst removed. The statocyst was then pinned in a petri dish in such a way that it could be immersed in chilled, aerated lobster Ringer solution while it was tilted and observed. Lith movement was measured with an ocular micrometer. To measure the movements of sensory hairs and statoliths in fixed preparations, the statocysts were first excised and pinned to a cork. The membranous top of the cyst was removed and the exoskeleton carefully chipped away from

-E

Fig. 2. Dorsal view of the lobster statocyst with the lith removed. Anterior side is on the left. Hairs appear foreshortened in this dorsal view. The ‘row’ hairs surround the lith and project into it from the side, while the ‘matt’ hairs lie beneath it.

262

MARIONL. PATTONand ROBERTF. GROVE

the antennule base beneath the cyst. The statocysts were then tilted to the desired position, fixed in glutaraldehyde, and embedded in EPGN (Sjostrand, 1967). Dissection and tilting were done in chilled lobster Ringer solution (Cole, 1941). Hair angles and lith movement were measured from cross sections obtained by grinding the embedded statocysts on a geologist’s lap wheel. Hair angles were measured with the aid of a camera lucida. Hair angle was operationally defined as the angle between a line parallel to the lateral wall of the base of the hair above the bulbous casque and a line parallel to the cyst floor below the casque. Hair location was determined by examining the statocysts from above. The row 2 hairs were numbered starting at the anterior end of the row. This study included the hairs most clearly involved in the eyecup response to roll tilt: the hairs lying between hair #20 and X35, i.e. between the matt hairs and the point where the hair rows turn to round the posterolateral lith margin (Fig. 2). Physiology To determine which statolith hairs were involved in the roll response of the eyecups, the lobster was fastened down in a dish of chilled, aerated sea water, the statocyst was opened by removing its membranous top, and the lith was split into sections which were moved separately by a stainless steel wire loop fastened into a micromanipulator. We found it unnecessary to monitor the discharge of all the motoneurons innervating all eyecup muscles. Changes in the tonic response of one muscle that lifts the eyecup during the response to roll tilt, 23B (Cochran, 1935; Burrows and Horridge, 1968), were adequate to test our hypotheses. For intracellular recording from the isolated eyecup, both eyecups, the brain, and the attached nerves were pinned out in Cole’s (1941) solution. The muscle was exposed by chipping away the exoskeleton from the base of the eyecup. The muscle was impaled from its dorsal side; the motoneurons were stimulated with a suction electrode. Muscle responses were displayed and photographed using standard methods. For intracellular recording from the intact lobster, the animal was fastened down in a dish of chilled, aerated sea water, the eyecups were waxed to the carapace, and the muscle cells exposed by cutting the membranous joint at the base of the eyecup. Muscle cells were impaled, and junction potentials (jp’s) were displayed and photographed using standard methods. To stimulate the receptor hairs during intracellular recording, the roof of the statocyst was removed, the hth washed out, and the receptors stimulated by jets of water (Davis, 1968; Patton and Grove, in press). For extracellular recording from the intact lobster, the animals were blinded with black rubber cement and immobilized in aerated sea water. The extracellular muscle potentials of muscle 23B were recorded with #40 copper wire insulated to the tip. The wire was waxed into a hole drilled over the insertion of the muscle. The ground wire was waxed into a hole drilled above the eye socket. After an experiment, the wires were cut at their point of entry and the eyecup was removed and fixed in sea water Bouin’s solution. Subsequent dissection confirmed the position of the electrode tip. Muscle potentials were displayed and photographed using standard methods. To stimulate the statocyst hairs, the rostrum was removed and one eyecup was waxed to the carapace to expose the statocyst contralateral to the eye where the recording was made. The contralateral statocyst was usually used because

the eyecup lies immediately dorsal to the statocyst and it is difficult to simultaneously stimulate the statocyst and record from the ipsilateral eyecup. The statocyst was opened dorsally and the lith washed out. The individual hairs were visualized with transmitted light and manipulated with a stainless steel pin fastened to a micromanipulator. The position of the pin was monitored with a photocell transducer.

201

0

-100

PREPARATION

100

TILT

(“1

I

Fig. 3. Change in the ‘hair angle’ of the row 3 and 4 hairs during statocyst rotation about the long axis. ‘Hair angle’ is the angle between a line parallel to the lateral wall of the base of the hair and a line that is parallel to the cyst floor beneath the hair and perpendicular to the long axis of the animal. Before examination, preparations were tilted, fixed, embedded in Epon, and ground down in the plane of a cross section. The examined hairs were attached to the lateral side of the statolith between row 2 hairs #20 #35 (See Fig. 2). Side-up tilt represented as negative. N = 12 (preparations) at o”, N = 4 (preparations) at -90” and at f90” f SE. RESULTS Hair movement

during tilt

Hair angles of the row 3 and 4 hairs did not significantly differ, so the data were pooled. During an 180” roll of the body from a 90” side-up tilt to a 90” side-down tilt, the bases of the row 3 and 4 hairs moved through an angle of about 60” (Figs 3 and 4). The row 2 hairs were unexamined because they did not appear to be involved in the eyecup reflexes (see below). Statolith hairs were tapering, apparently flexible, and curved toward the lith (Fig. 5). This curvature decreased in a side-up rotation, when the lith moved medially away from the hair bases, and increased in side-down rotation. At rest and in sidedown rotation the flexible distal half of the hairs appeared to be appressed to the lith. Thus, though the lith moved more in side-up rotation, the hair angle changed more in side-down rotation. Lith movements were the same in fixed and unfixed preparations (Fig. 6). Fixed statolith hairs resembled unfixed hairs; there was no obvious twisting or

.I 1

I

0

0 .

. 0

20420

’ r 26 LOCATIO: (HAIR NUMBER)

1 35

Fig. 4. Variation in the ‘hair angle’ of row 3 and 4 hairs attached to the lateral side of the statolith in 8 untilted, f&d statocysts. Hair location was determined by counting the number of row 2 hairs between the examined hair and the anterior end of the statocyst (Fig. 2).

Statocyst regulation of lobster gravity reflexes

We observed no anatomical distinction between hairs of the same row, and hairs of different rows appeared to differ only in size. For the row 3 and 4 hairs, the variation in hair angle between hairs was large compared with the change in this angle during tilt (Fig. 4). The position and size of the sand grains seemed important in determining the angle the hairs made with cyst floor. Hairs attached to a large grain embedded in a lith of small grains often had hair angles that differed greatly from the average hair angle. There was no clear pattern in the arrangement of sand grains (Fig. 4) or in hair position. There was no significant relationship between hair location and the hair angle of the row 4 hairs (one-way ANOVA, F = 0.17, P > 0.99) or of the row 3 hairs (one-way ANOVA, F = 0.81, P > 0.38).

263

distortion.

Statocyst

region involved in the roll response

The ‘lith dissection’ experiments indicated that, as we expected, roll responses of the eyecups were mainly driven by the statocyst hairs lying lateral to the lith, between hairs #20 and #45. We removed sections of the lith and moved the remaining sections with micromanipulators. Eight preparations were examined. The posterior part of the lith appeared to be involved in the pitch reflex. When the part of the lith attached to the posterior limb of the sensory crescent, posterior and lateral to hair #45, was

SIDE

UP

x h.,,

( Se) an$l,* =270

I

i

‘001

I

-2~05izzgq

Fig. 6. Displacement of the statolith during roll tilt about the long axis in fixed and unfixed preparations. Lith movement measured as displacement from the postion occupied in the untilted preparation. Medial lith movements and side-up rotation represented as negative. N = 8 unfixed preparations, N = 5 fixed preparations &SE.

moved as it moves in pitch, i.e. in an anterior or posterior direction, eyecup movements clearly resembling those occurring in pitch were elicited. When this part of the statolith was moved laterally, i.e. in the same way it moves in roll tilt, the eyecups either did not move or made slight movements slightly resembling those observed during pitch. Lateral movements of the part of the lith between hair #20 and hair #45 always elicted typical and obvious roll responses of the eyecups. No movements of the part of the lith anterior to hair # 20, the part covering the matt hairs, elicted any response in the eyecup at all. Furthermore, no movement of the matt hairs produced any change in the discharge of muscle 23B in any context. To be certain that the reflex response to pitch was not elicted, we only used the hairs lying between row 2 hairs #20 and #35 to test our hypotheses. Innervation of eyecup muscle 238

hair4

SIDE

DOWN

3 3& 2

( SO’)

.f half m91e=S2°

Fig. 5. Movement of hairs lying lateral to the statolith during roll tilt. Drawing of cross-sections taken near row 2 hair #25 in fixed statocysts.

An effort was made to sample cells in all parts of the muscle. The average resting potential was about 55 mV, inside negative. Pulses were small initially, facilitated slowly, and showed no apparent adaptation. The pulses, initially about 0.2-0.5 mV in height, had a slow and smooth facilitation, requiring about 13 pulses to attain maximum size (Fig. 7). After a 60 set interval, the effects of facilitation had disappeared. All pulses recorded were similar in height (2-6 mV at a 20 Hz stimulation rate), duration (about 50msec), and facilitation rate. We were unable to produce any fatigue at stimulation rates of less than 50 Hz, although some preparations could eventually fail to respond to every stimulus at 50 Hz. We obtained no evidence from the isolated preparation that suggested that 23B was innervated by more than one axon. In muscle cells with multiple innervation, the different axons usually have different stimulus thresholds and produce different kinds of pulses (Davis, 1968); in such muscles, a gradual increase in stimulus voltage or duration will produce, at some point, compound pulses or an abrupt change in the size or shape of the junction potential. Neither compound pulses nor abrupt changes in the size of

MARION L. PATC~N and ROBERTF. GROW

264 1

L_

mV

5mS

Fig. 7. Superimposed intracellular responses of muscle 23B of the isolated lobster eyecup to stimulation of the eyecup motoneuron at 20Hz. The first responses are small; successive responses show a gradual facilitation.

Fig. 8. Intracellular response of muscle 23B of the eyecup of an intact lobster to a brief jet of water directed into the ipsilateral statocyst.

the junction potential were evoked by any change in stimulus amplitude, stimulus duration, or polarity. No difference in stimulus frequency, duration of stimulus trains, or any inter-train interval produced any qualitative change in the muscle junction potential. One aging preparation showed two spontaneous trains of pulses that sometimes joined together to form compound pulses. In this preparation, nerve stimulation neither produced new pulses nor affected the ongoing pulses. Further, in no other cell was more than one kind of pulse recorded. Recordings made on the intact preparation also indicated that, under the conditions of this study, only a single motor axon is active in muscle 23B after the first 0.5 set of statocyst stimulation. Some impalements revealed spontaneous activity that was, once again, unaffected by statocyst stimulation, but no jet of water directed into the statocyst ever produced more than one kind of pulse. Stimulation of the ipsilateral statocyst produced a pulse that, in all but one case, was very similar to the pulse elicited with nerve stimulation (Fig. 8). In one impalement, stimulation elicited a pulse which facilitated and adapted rapidly: a prolonged jet of water produced only 2-3 pulses. This unit appeared to recover from adaptation in a few seconds. Stimulation of the contralateral statocyst, which slows the discharge of muscle 23B in the intact lobster (see below), produced no observable pulses.

Statocyst receptors regulating the response to roll

The next step was to determine which rows of statolith hairs had an effect on the response of the eyecup to roll. When the contralateral lith was removed, the discharge of 23B rose from about 20 Hz to about 36 Hz and began a slow decline. Stimulation of some statocyst hairs caused a decrease in this discharge; when the hairs were released, the inhibited discharge exhibited a tonic post-inhibitory rebound (Fig. 9). The ‘rebound’ discharge also had a longterm adaptation that was too slow to affect experimental results. The most sensitive and consistent measurement of the effects of the statocyst stimulation was the difference between this tonic inhibited discharge and the tonic ‘rebound’ discharge. Except where noted, this difference was used in all the following experiments. The stimulus protocol used in these experiments was followed, except where noted, in subsequent experiments. The hairs were deflected laterally for 7 set, returned to rest for 7 set, again deflected for 7 set, and again returned to rest. ‘Rest’ is the angle the hairs naturally assume after the lith has been washed out: about 50”. The average discharge rate during the last 5 set of both deflexions was compared with the discharge rate during seconds 2-7 following both returns to rest. The preparations were allowed to remain at rest for 60 set before the next set of stimuli.

Fig. 9. Extracellular recording of the response of muscle 23B (arrow) to deflexion of row 3 and 4 hairs of the contralateral statocyst. Upper trace is the muscle response. Lower trace is hair movement. Upward movement of the lower trace indicates a lateral deflexion; downward movement of this trace indicates a return to rest. Time calibration 0.5 sec.

Statoeyst regulation of Iobster gravity retlexes

The effects of deflexions of the hairs attached to the lateral side of the statolitb were examined row by row. The row 1 hairs are twisted and do not touch the lith; they were not examined. The row 2 hairs are the largest and the hairs of rows 3-A, 3 and 4 are successively smaller. The influence of these rows of hairs on muscle discharge was tested by removing all rows but one and deflecting the remained row 125 p, about 40”, from rest. Significant changes in muscle discharge were produced by deflexions of row 3-A (t =4.8, P cO.Ol), row 3 (t = 3.82, P < 0.01) and row 4 (t = 4.5, P < O.OOl), but not row 2 (t = 0.3, P > 0.35). The next experiments showed that only one of the three receptors driven by the smaller statocyst hairs the ‘type A’ receptor, produced a tonic change in the muscle discharge under our experimental conditions. The muscle discharge changed when the discharge of type A receptors was probably ch~~ng, but not when the discharge of ‘type B’ receptors was changing. In this experiment, all hairs but row 4 hairs were removed and these row 4 hairs were deflected laterally to an angle of 140” and returned to rest in a series of 7 set steps. The muscle discharge during the last 6 set of each step was plotted against hair angle (Fig. 10). Under these conditions, the type A receptors’ tonic discharge is almost directly proportional to hair angle between 140” and 100” and is nearly constant and very slow between 80” and 50”. Conversely, the type B receptor’s discharge is silent between 140” and lOO”, and almost inversely proportional to hair angle between 80” and 50” (Patton and Grove, in press). Between 140” and loo”, the inhibition of the muscle discharge was related to hair angle; between 80” and SO”, the muscle discharge was almost constant (Fig. 10). Further, a deflexion that pushed the hairs medially and down toward the center of the statocyst, which stimulates some type B receptors but no

I

_-_

26

$0

I

1

80 HAIR

120 ANGLE

160 I

I

Fig. 10. Mean tonic response of muscle 23B as 3 to 10 hairs of row 4 in the contralateral statocyst were returned to rest in steps from a lateral deflexion, and the mean response of type A and type B statolith hair receptors to the same deflexion schedule. ‘Rest’ is the position the hairs assume after the lith is washed out. The hairs were held still for 7 set at each step; ‘tonic response’ was the mean discharge rate during the last 6 sec. N = 6 preparations. Response of type A and B receptors taken from Patton and Grove (1992) f SE,

36

41

Pig. 11. Tonic response of muscle 23B to lateral degexion of groups of 3 to I2 hairs of rows 3 and 4 in different locations in the contr~ater~ statocyst. Row 3 hairs were deflected from a hair angle (See Fig. 3) of 50” to lOO”,and row 4 hairs were deflected from a hair angle of 90” to 141)“. The hair angle at rest was 50” for both rows. The hairs were deflected for 7 set; ‘tonic response’ was the mean discharge rate during the last 5 sec. Mean of six preparations; values averaged to obtain the mean were averages of the response of a given preparation to two to four deflexions of the same group of hairs *SE. type A receptors, produced no eyecup movements or changes in muscle discharge. It is of course, unlikely that the type C receptors, which are phasic, were involved in the tonic response of muscle 238.

All the hairs lying lateral to the Iith and posterior to the matt hairs had similar effects on muscle discharge (Fig. 11). Groups of 3 to 12 row 3 and row 4 hairs lying in different locations between hair #23 and hair #43 were deflected laterally 125 fi from the rest postion for the row 3 hairs. This deflexion moves the row 4 hairs from about 90” to about 140”. It moves the row 3 hairs from 50” (rest) to about 100”. The effects of the deflexion of each group of hairs was measured 2-4 times in each of six preparations. A two-way AIUOVA that used preparation and hair number as blocking variables indicated that, though there was some difference between the response of different preparations (l; = 3.06, P = 0.025), hair location had no effect on the muscle response (F = 1.2, P = 0.320). The interaction term was not s~g~~~nt (F = 1.18, P = 0.329}, i.e. the relationship between hair location and the motor effects of its deflexion was not different in different preparations. Summation

40

31

HAIR LOCATION

of sensory inputs

The next two experiments indicated that the effect of the deflexion of several hairs on the discharge of muscle 23B was the sum of their individual effects. in the first experiment, two micromanipulators were used to deflect two groups of row 3 and 4 hairs 125 fl lateralfy from the rest position of the row 3 hairs. The number of hairs in the two groups was systematically varied. The sum of the changes produced by separate deflexions of each pair of hair groups was plotted against the change produced by simultaneous deflexion of both groups (Fig. 12); the relationship appeared to be close to that predicted for linear summation, a straight line of unit slope. A regression of the sum of changes in frequency

266

MARION

L. PATTON and

ROBERT F. GROW

muscle discharge. This prediction was not fulfilled. Deflexion of single row 4 hairs 40” from rest produced a significant change in muscle discharge (t = 4.5, P < 0.001). DISCUSSION . -18

0 -9 -16 SIMULTANEOUS DEFLEXIONS (Hz)

Fig. 12. Sum of the tonic responses of muscle 23B to separate deflexions of two groups of row 3 and row 4 hairs of the contralateral statocyst compared with the response to simultaneous deflexion of the same two groups. Each point represents the mean response to deflexion of a different pair of hair groups. One or two pairs of hair groups were deflected in each of eight animals. Deflexion schedule as in Fig. 11.

produced by separate deflexions against the changes produced by simultaneous deflexions yielded a regression coefficient of 1.06 with a standard error of estimate of 0.16. The results of ANOVA rejected the hypothesis that the coefficient was zero (F = 41.85, P < 0.0001). The hypothesis that the regression coefficient was 1 was not rejected (F = 0.14, P = 0.714). The summation hypothesis also predicts that the change in muscle discharge should be linearly related to the number of hairs deflected. This was tested by deflecting different numbers of row 3 and 4 hairs 125~ laterally from the rest postion for the row 3 hairs (Fig. 13). The deflexions were presented in random order and were separated by an interval of 60 sec. Linear regression of the change in muscle discharge rate on number of hairs deflected yielded a coefficient of 0.28 with a standard error of 0.06. The results of ANOVA indicated that the coefficient was not zero, i.e. that the change in discharge rate was linearly related to the number of hairs deflected (F = 7.67, P < 0.001). ‘Noise Jilter’ hypothesis

The next experiment tested the ‘noise filter’ hypothesis which predicted that deflexions of single hairs would not produce a significant change in

0

i! 5 j ;; SE

-5

8

-10

8 -E

-15

0

10 20 30 NO. HAIRS DEFLECTED

40

Fig. 13. Mean tonic response of muscle 23B to deflexion of different numbers of row 3 and 4 hairs of the contralateral statocyst. Mean of 7 preparations; values averaged to obtain the mean were averages of the response of a given preparation to two to six deflexions of the same group of hairs. Deflexion schedule as in Fig. 11 St SE.

The observed lith movements and the apparent change in hair angle during tilt confirm the hypothesis that these movements constitute adequate stimulus of the statocyst position receptors. If we assume that the lith and the statocyst hairs were not distorted by fixation and ‘sectioning’, our results also indicate that there is a haphazard variation in hair angle that is related to statolith configuration and large compared to the change in hair angle during tilt. Our assumption is probably valid, because the appearance of the lith and the hairs was similar before and after fixation and because lith movements in fresh preparations were very similar to lith movements measured in fixed preparations. The eyecup reflexes appeared to be driven by type A receptors of rows 3-A, 3 and 4 hairs. We obtained no evidence indicating that either the row 2 hairs, the ‘matt’ hairs, or the type B or C receptors of the row 3-A, 3 and 4 hairs had any effect on the tonic gravity reflexes of the eyecup. It is possible that the row 2 hairs, the matt hairs, and the type B and type C receptors are involved in other tonic gravity reflexes or in the phasic response to tilt (Davis, 1968). During a 180” body rotation from side-up to side-down, the row 3 and 4 hairs move through a hair angle of about 60”. The discharge of single type A receptors is continuously variable only through a hair angle of 40” (Patton and Grove, 1992). Therefore it might be objected that the type A receptors could not regulate the eyecup reflexes because these reflexes are continuous throughout a 180” body rotation (Schiine, 1959). The discharge of type A receptors is a saturating linear function of hair angle (Patton and Grove, 1992) however, and there is much variation in hair angle caused by lith irregularity. During body rotation, therefore, the number of active type A receptors would change as well as the response of individual receptors, and the total discharge from all the type A receptors would, like the eyecup reflexes, change continuously throughout a 180” rotation. The junction potentials recorded from 23B resemble the ‘slow’ junction potentials (jp’s) recorded from the crustacean limbs (Hoyle and Wiersma, 1958a,b). They decayed more slowly than typical ‘fast’ jp’s, showed considerable facilitation in amplitude, and never showed any spike-like activity. The jp’s of 23B were slightly unusual in that they were of appreciable size, about 3 mV, at a stimulation rate of 10 Hz, whereas crustacean limb muscle jp’s are usually vanishingly small at such a rate of stimulation (Hoyle and Weirsma, 1958a,b). Motoneuron stimulation in the isolated eyecup suggested that stimulation of the statocyst hairs produced a tonic response in only one slow axon of 23B. In those crustacean muscles innervated by more than one axon, most individual muscle cells are also innervated by more than one axon; complex pulses and pulses of more than one kind can be recorded from such cells (Hoyle and Wiersma, 1958a,b). We

Statocyst regulation of lobster gravity reflexes

were only able to elicit single, similar pulses from 23B. However, in a few crustacean limb muscles the cells are innervated by either a fast or a slow axon but not both. In such muscles, double pulses or pulses of different sizes would not be observed in intracellular recordings. However, in this sort of a muscle, fast and slow jp’s are very different (Hoyle and Weirsma, 1958a,b), and all pulses evoked from 23B were similar. Stimulation of the statocyst in the intact preparation also produced a tonic response in only one slow axon in 23B. Most ipsilateral statocyst stimulation elicited only one kind of pulse, which was very similar to the pulses elicited by motoneuron stimulation. Stimulation of the contralateral statocyst, which slows the discharge of the motoneuron in the intact preparation, produced no observable pulses, i.e. produced no evidence that the muscle was innervated by an inhibitory neuron. We found only one muscle cell in the intact preparation that had a response resembling a fast jp. This single instance is not good evidence for double innervation of 23B because no fast jp’s were recorded in response to motoneuron stimulation of the isolated eyecup. This fast jp would not effect the results of later experiments, even if it was not an artifact, because it only appeared during the first 0.5 set of stimulation. Spontaneous pulses appeared in a few impalements in both the intact and isolated preparations. These impulses were probably artifactual since they usually appeared in muscle cells that did not respond to stimulation. Even if they were not artifactual, they would not effect the results of later experiments because they were unaffected by stimulation. In the crustacean MRO, different receptors apparently signal different limb positions (Mill, 1976; Bush and Laverack, 1982). We obtained no evidence, however, indicating that different statocyst receptors signal different angles of tilt. Activity in different statocyst receptors could signal the magnitude of body tilt if the relationship between receptor discharge and body tilt were determined genetically. If this were true, then the same hair receptor would have the same response to tilt and the same effect on the gravity reflexes in different lobsters. However, if the results we obtained from the row 3 and 4 hairs are typical, hair angle is largely determined by the irregular configuration of the statolith. Since receptor response is a consistent function of hair angle (Patton and Grove, in press), it can be concluded that differences in receptor responses to tilt are also a function of statolith configuration and not determined genetically. Further, all hairs seem to elicit similar responses in the eyecup muscle. If different receptor hairs signalled different body positions, this result would not have been obtained. The lobster could still measure body tilt by determining which receptors were active if it re-learned, after ecdysis, what angle of tilt elicited the maximal response from each receptor. It this were true, deflexion of different hairs would elicit different responses in the same lobster and the relationship between hair location and the reflex response to hair deflexion would be different in different lobsters. We found, conversely, that this reflex response to hair move-

267

ment was similar in different hairs and different preparations. Moreover, it is difficult to see how tilt could be measured by determining which receptors are active, because the relationship between body tilt and recep tor response is apparently continuously changing. Cohen (1955) published records of a receptor response that changed with repeated tilt. The response of the statocyst receptors to hair deflexion does not change with repeated stimulation (Cohen, 1960; Patton and Grove, 1992) so the change in the above receptor was probably due to a spontaneous change in the way its sensory hair was inserted into the statolith. If these responses change with repeated tilt, they probably also change with body movements such as the violent escape response. Such variability in the response curves of the receptors would not be unexpected because the lith is loosely fastened. together and the hairs are loosely fastened to the lith (Cohen, 1960; Patton, personal observation). We also obtained no evidence suggesting the existence of the kind of ‘noise filter’ that only permits a response when two receptors are excited simultaneously. Instead, stimulation of single statocyst hairs produced significant changes in tonic muscle discharge. If it is assumed that the response of muscle 23B to tonic statocyst hair deflexion is an accurate reflection of the lobster’s gravity reflexes, our results are consistent with the hypothesis that the lobster uses a linear summation of statocyst inputs to measure body tilt. A simple summation of sensory inputs has also been demonstrated for the statocyst of the crayfish, which also has a sand-grain statolith (Takahata and Hisada, 1982a,b, 1985). This ‘summation’ hypothesis explains why there are so many statocyst receptors, why there is probably a haphazard variation in the response of different receptors to body tilt, why the response of individual receptors changes with repeated stimulation, and why this sensory discharge is arrythmic. The results of the summation of a large number of variable inputs would, of course, be more consistent than the results of the summation of a small number, and neither an arrythmic discharge, nor a difference between receptors in the response to tilt, nor temporal changes in the response curves of individual receptors would have much effect on the summed response of a large number of receptors. This summation of a large number of inputs can be seen as an evolutionary solution to the haphazard variation in sensory response necessarily produced by an irregular statolith of sand grains. That is, through evolution, a poor statolith design has been compensated for by summing the inputs of a large number of statocyst receptors. This appears to be a clumsy and metabolically expensive solution to a problem that seems unnecessary. After all, many successful organisms, e.g. vertebrates (Lowenstein, 1950) and cephalopods (Budelmann, 1975), have calcareous statoliths. Such ‘inelegant’ adaptations, however, are common in nature (Gould, 1989). Acknowledgements-This work was submitted in partial satisfaction of the requirements for the degree of Ph.D. in Comparative Physiology, University of Oregon at Eugene, 1972.

268

MARIONL. PAWN and ROBERTF. GROW

We are grateful to Dr M. J. Cohen, Dr S. B. Kater, Dr H. B. Hartman, and Dr M. Burrows for advice and support, to Dr. R. Eokert for hospitality, and to Mr L. Vernon for technical assistance. Supportedby USPHS grant ROl NBO 1624 to M. J. Cohen, NIH training grant 671 2Tl GM336 and NIH grant 1 F02 NS47, 792-01 to M.L.P.

REFERENCES Budelmann G.-U. (1975) Gravity receptor function in cephalopods with particular reference to Sepia oficinalis. Fortschr. 2001. 23, 85-89.

Burrows M. and Horridge G. A. (1968) Motoneuron discharges to the eyecup muscles of the crab Carcinus. J. exp. Biol. 49, 251-267.

Bush M. M. H. and Laverack M. S. (1982) Mechanoreception. In The Biology of Crustacea: Neural Integration and Behavior (Edited by Sandeman D. C. and Atwood H. L.), Vol. III, pp. 399-460. Am. Mus. Natl. Hist., New York. Cochran D. M. (1935) The skeletal musculature of the blue crab, Callinectes sapidus, Rathbun Smithsonian misc. CON. 92, l-76.

Cohen M. J. (1954) Equilibrium receptors of invertebrates: an electrophysiological analysis of the statocyst of the lobster, Homarus americanus. Dissertation UCLA, Los Angeles. Cohen M. J. (1955) The function of receptors in the statocyst of lobster; Homarus americanus. J.Physiol. 130, 9-34. Cohen M. J. (1960) The response patterns of single receptors in the crustacean statocyst. Proc. Roy. Sot. B. 152,30-49. Cole W. H. (1941) A perfusing solution for the lobster (Homarus) heart and the effects of its constituent ions on the heart.‘J. gen. Physiol. 25, 1-16. Davis W. J. (1968) Lobster righting responses and their neural control. Proc. Roy Sot. B 70, 435-456. Dijkgraaf S. (1956) Structure and functions of the statocyst in crabs. Experientia (Basel) 12, 394-399. Gemandt B. E. (1959) Vestibular mechanisms. In Handbook of Physiology (Edited by Magoun H. W. and Hall V. E.), DD. 549-563. Waverlv Press. Baltimore. MD. Gould S. J. (1989) Ever Since Darwin. .W. W. Norton, New York. Hoyle G. and Wiersma C. A. G. (1958a) Excitation at neuromuscular junctions in crustacea. J. Physiol. 143, 403-425.

Hoyle G. and Wiersma C. A. G. (1958b) Inhibition at neuromuscular iunctions in crustacea. J. Physiol. 143, 426-449. Lowenstein 0. (1936) The equilibrium function of the vertebrate labyrinth. Biol Rev. 11, 113-145.

Lowenstein 0. (1950) Labyrinth and equilibrium. Symp. Sot; exp. Biol. 4, 60-82. Mill P. J. (1976), Chordotonal oraans of crustacean appendages. In Structure and Function of Proprioreceptors in the Invertebrates (Edited by Mill P. J.), pp. 243-297. Chapman and Hall, London. Neil D. M. (1982) Compensatory eye movements. In The Biology of Crustacea: Neural Integration and Behavior (Edited by Sandeman D. C. and Atwood H. L.), Vol. IV, pp. 133-161. Am. Mus. Natl. Hist., New York. Newland P. L. (1989) The uropod righting reaction of the crayfish Procambarus clarkii (Girard): an equilibrium response driven by largely independent reflex pathways. J. camp. Physiol. 54, 685-696.

Ozeki M, Takahata M., and Hisada M. (1978) Afferent patterns of the crayfish statocyst with ferrite grain statolith to magnetic field stimulation. J. camp. Physiol. 12, l-10. Patton M. L. and Grove R. F. The response of statocyst receptors of the lobster, Homarus americanus, to movements of the statolith hairs. Camp. Biochem. Physiol. lOlA, 249-257.

Schiine H. (1959) Die Lageorientierung mit statolithenorganen and augen. Ergb. der Biol. 21, 161-209. Schiine H. and Steinbrecht R. A. (1968) Fine structure of statocyst receptors of Astacus fulviatilis. Nature (Lond). 220, 184-186.

Sjiistrand, F. S. (1967) Electron Microscopy of Cells and Tissues. pp. 462. Academic Press, New York. Sokal R. R. and Rohlf F. J. (1973) Introduction to Biostatistics. pp. 368. W. H. Freeman and Co., San Francisco. Stein A. (1975) Attainment of positional information in the crayfish statocyst. Fortschr. Zool. 23, 109-119. Takahata M. and Hisada M. (1979) Functional polarization of statocyst receptors in the crayfish Procambarus clarkii Girard. J. camp. Physiol. 130, 201-207. Takahata M. and Hisada M. (1982a) Statocyst intemeurons in the crayfish Procambarus clarkii Girard. I. Identification and the response characteristics. J. camp. Physiol. 149, 287-300.

Takahata M. and Hisada M. (1982b) Statocyst inter-neurons in the crayfish Procambarus clarkii Girad. II. Directional sensitivity and its mechanism. J. camp. Physiol. 149, 301-306.

Takahata M. and Hisada M. (1985) Interactions between the motor systems controlling uropod steering and abdominal posture in crayfish. J. camp. Physiol. 157, 547-554. Cold Spring Harb. Symp. quant. Biol. 17, 155-163~ Young J. Z. (1960) The statocysts of Octopus vulgaris. Proc. R. Sot. 152, 78-87.

Wiersma C. A. G. (1952) Neurons of arthropods.