J. Exp. Biol. (1964), 41, 1-14 IWith 2 text-figures

Printed in Great Britain

CONTROL OF LUMINESCENCE IN HEMICHORDATES ANDSOME PROPERTIES OF A NERVE NET SYSTEM

BY CHARLES H. BAXTER* AND PETER E. PICKENSf

Department of Zoology, University of California, Los Angeles

{Received 17 May 1963)

INTRODUCTION

Since 1875, when Panceri first described luminescence in a hemichordate, Glosso-balanus minutus, four other species of enteropneusts have been observed to emit lightDelage & Herouard (1898) reported that luminescence of Balanoglossus clavigerus fromthe coast of Brittany was bright enough to be seen even in the presence of candlelightKuwano (1902) described luminescence in Balanoglossus misakiensis from Japan;Crozier (1920) and Harvey (1926) found the response in Ptychodera bahamensis fromBermuda, and Rao (1954) in Ptychodera flava from India. Judging from the speciesexamined which do not show luminescence, Harvey suggested (1952) that this responseis confined to the family Ptychoderidae, to which these genera belong.

Crozier (1920) reported that Ptychodera shows a diurnal rhythm in the productionof luminescence and could be made to emit light during the day only if subjected toelectrical or to very strong mechanical stimuli. Harvey (1926) was unable to observethis rhythmicity, and found that species from both Bermuda and the Mediterraneanwould luminesce during the day without unduly strong stimulation. Crozier, Harveyand Rao have agreed that luminescence is inhibited by light and that it was necessaryto keep animals in darkness a few hours in order to evoke a response by mechanicalstimulation. The production of light occurs all over the body except in the gill region(Crozier, 1917; Rao, 1954); it has been thought to be caused by the emission of aluminous slime.

Virtually nothing has been reported on the control of luminescence in the group.Panceri comments that he could observe no luminescent waves in these animals, aswere sometimes present on the surface of pennatulid coelenterates. Dahlgren (1917)described goblet cells in the epidermis of Ptychodera bahamensis which he concludedmust be the photogenic cells, and Bullock (1940) has described the intra-epidermalnervous system, but anatomical connexions have not yet been found between the two.

The present study was undertaken to investigate the nature of the luminescentresponse in greater detail, particularly with regard to the role played by the nervoussystem in eliciting the response. This is of special interest since little is known of thephysiological organization of the enteropneust nervous system. Knowledge of thecharacteristics of the nerve net of enteropneusts would allow a comparison with thewell-studied coelenterate nets to determine common denominators of neural activityexisting at similar levels of organization in phylogenetically diverse groups.

• Present address: Department of Biological Science, Stanford University, Stanford, California,t Present address: Department of Zoology, University of Arizona, Tucson, Arizona.

1 Exp. BioL 41, 1

2 CHARLES H. BAXTER AND PETER E. PICKENS

The nervous system of enteropneusts consists of a fine-textured nerve net lying inthe base of the epidermis. The thickness of this net is quite variable with body region.Along the entire length of the trunk this epidermal net is thickened into mid-dorsaland mid-ventral cords. The ventral cord differs from the dorsal in being of greatercross-sectional area, having a greater lateral extent, and a more gradual transition intothe general net. While in both cords the predominant orientation of fibres is longi-tudinal, the ventral cord contains more numerous diversely oriented fibres. Theventral cord terminates anteriorly at the collar and at this point is connected with thedorsal cord by a circumenteric nerve ring. The dorsal cord continues anteriorly toconnect with the richly developed plexus of the proboscis. Descriptions of, andreferences to, the gross anatomical structure of the nervous system of enteropneustsmay be found in Hyman (1959) and an excellent comparative treatment of the finestructure is presented by Bullock (1945). Silen (1950) and Knight-Jones (1952)provide recent accounts of the histology of the nervous system.

MATERIALS AND METHODS

The majority of the observations reported in this study utilized specimens ofPtychodera flava collected at Kaneohe Bay, Oahu, Hawaii. Collections made inJanuary, March, and April were maintained in Los Angeles in standing sea water at2O°-23° C , some for as long as 6 weeks, while being used in experiments on theluminous response. Studies were also conducted on Ptychodera during 2 weeks ofOctober and November at the University of Hawaii Marine Laboratory in KaneoheBay. Some additional observations were carried out in Los Angeles on a local luminousspecies of Balanoglossus.

Prior to use in experiments individuals were placed separately in dishes of sea waterin the dark for 30-60 min. A Grass S4 stimulator and silver electrodes were used forelectrical stimulation with a loudspeaker arranged to monitor the stimuli by ear.Pulses of 1 msec, duration were used unless otherwise specified. The individual beingstimulated was either in air and kept moist by irrigation, or under water. Observationsof luminescent response were made after the investigator's eyes had been dark-adapted.

Considerable individual variability exists in luminous responsiveness within a groupof specimens of one collection maintained under similar conditions. It has not beenpossible to correlate these individual differences with any characteristics of the animalor its previous treatment. The differences between specific individuals usually per-sisted for at least the 2-5 hr. of an experimental session.

RESULTS

Diurnal rhythm

Ptychodera will usually luminesce to tactile or vibrational stimulation. A light tapto the body characteristically produces a brief local glow of a greenish-blue colour.In agreement with Crozier (1920) our work suggests a diurnal rhythmicity of ability toluminesce. This rhythm is most apparent in recently collected specimens and dis-appears after 3—7 days when they are kept in trays of running water under laboratory

Control of luminescence in hemtchor dates 3

conditions. Dark-adapted animals stimulated during daylight hours show increasedthreshold and reduced area of spread for luminescence when contrasted with animalsstimulated at night.

The excitability of the rapid withdrawal response of Ptychodera was investigated asa function of the time of day to obtain evidence on whether the diurnal rhythm of theluminescence was specific for the luminescent response or was only one facet of adiurnal fluctuation of nervous excitability. The withdrawal response consists of acontraction of the longitudinal muscles of the anterior part of the body when a relaxedindividual is stimulated by a tap on the proboscis. The characteristics of the responseand the pathways involved have been described for other species by Bullock (1940,1944). The response of light-adapted individuals indicated no detectable diurnalrhythm either in threshold or vigour of the response. Since the same neural pathwaysin the main appear to mediate the muscular and luminous phases of this reflex (seebelow and p. 6), we suggest that the diurnal rhythm reflects a fluctuation of respon-siveness in the photocytes—either at the neural junction or in the photogenous process.

Responses to mechanical stimuli

The characteristics of the response to mechanical stimulation depend on the para-meters of the stimulus, the region of the body stimulated and the previous history ofthe individual. In an animal which has been undisturbed for several minutes theusual response to a light tap on the proboscis is a glow in this entire structure and abrighter and more maintained luminescence in the branchio-genital region, exceptingthe gill area, from the collar back to the hepatic region. This will be called the startleluminescence. Such a stimulus also produces the quick withdrawal response (seeabove); examination under a dim red light reveals that this response is correlated withluminescence in the proboscis following the stimulus and in the branchio-genitalregion when contraction occurs. The startle luminescence fatigues rapidly and thentactile stimulation of the proboscis produces luminescence of the entire proboscis onlyor, in the case of weak stimuli, only of a local area surrounding the stimulated site.

The intensity, area, and duration of luminescence are correlated with the strengthof the mechanical stimulus. Repetition of a mechanical stimulus produces an increasein intensity and a spread of the luminous response which may be local, or extensive,or produce response in distant areas without luminous continuity between the stimu-lated and responding sites. Spread is polarized, favouring the posterior direction(Fig- 1).

The body regions possess the following order of sensitivity for luminescent responsesto single and repetitive mechanical stimuli (from high to low): proboscis, caudal tip,collar and genital wings, remainder of the trunk. This agrees with Crozier's findings(1915) on threshold to mechanical stimulation (presumably for local muscularresponse).

Responses to single electrical stimuli

Threshold voltages for luminescence to single shocks were measured by starting atsubthreshold values and gradually increasing the voltage while delivering test shocksat intervals of 10-30 sec. When threshold was reached an interval of 2-4 min. wasallowed to elapse, and then threshold was redetermined by giving another series of

4 CHARLES H. BAXTER AND PETER E. PICKENS

stimuli, starting with a voltage somewhat below that used to obtain the initial response.Electrodes were spaced several millimetres apart and time was allowed for tactileadaptation before stimulation. Stimuli were confined as closely as possible to thesame site and results were discarded when excessive movement of the specimenoccurred.

Stimulatingelectrodes

Local spread

% % ^ % % % % # ^ ^

Extensive spread

Startle luminescence

Disjunctive luminescence

Initial spread of luminescence

Increment of spread with more intense stimulation

Fig. i . Types of spread of luminescence to electrical stimulation observed in Ptychodera.

A threshold voltage for single shocks exists, but after a response has been elicitedfrom a given spot several times the threshold increases and the preparation may evenbecome refractory. It is then impossible to produce luminescence with single shocksdelivered to this site, even though ten to twenty times the strength initially effective.In these instances mechanical stimulation of moderate intensity applied directly abovethe site of the electrodes, or repetitive electrical stimulation, will usually produce animmediate, bright luminescence indicating that the refractoriness is not attributableto inability of the photocytes to respond. This non-responsiveness to single electricalshocks was first explained by a decline in excitability at the neuro-effector junctionduring the series of trials so that it becomes a facilitating junction requiring repetitiveexcitation (provided by the train of impulses evoked by the mechanical stimulus).However, further experiments show that this is not the primary cause and thatrepeated stimulation produces a gradual decline in responsiveness of the nerve net,or more probably of sensory elements that excite the net, with the consequent reduc-tion and disappearance of repetitive discharge to single electrical shocks (seediscussion).

Control of luminescence in hemichordates 5

The luminous response at threshold voltage is of long latency, low intensity, con-fined closely (1 mm. or less) to the region of the stimulating electrodes, and of about1 sec. duration. When the voltage of the single-shock stimulus is increased abovethreshold, the luminescence decreases in latency, increases in intensity in the originalarea, spreads a few millimetres, and increases in duration. The maximum spread isreached at a voltage only slightly above threshold, and there is an increase of brightnessin this area to successively stronger shocks until saturation occurs at several timesthreshold voltage.

The limited response to single shocks described above holds true for the entireanimal except the proboscis. Here, in a fresh individual, the first single shock whichproduces a response usually results in the startle response and its characteristicluminescence, as described above under mechanical stimulation. When the preparationhas been stimulated previously, only a local glow is obtained to single shocks. Thisdiffers from that produced in other body regions in that its duration is somewhatprolonged.

The order of sensitivity of body regions (for threshold of luminescence) determinedfor single electrical shocks is the same as that for mechanical stimulation.

All the above determinations were made with pulses of 1 msec, duration. As theduration of the pulse is increased, the threshold voltage declines. This suggests thata strength/duration curve could be plotted for the response though such measurementswould be complicated by the lability of threshold to repeated stimulation.

Responses to repetitive electrical stimulation(a) Facilitation

The fact that initially subthreshold shocks produce luminescence upon repetitionindicates facilitation. Luminescence usually appears on the second to fourth stimulusthough it may come much later. Maximum intervals between subthreshold pulsesyielding a response on repetition are of the order of 5 sec.

During repetitive stimulation (o-3-io/sec.) the intensity of the luminescent responseincreases for the first few responses at a given frequency. At frequencies of 1-10/sec.the plateau intensity of luminescence exhibits an increase with increasing frequency.The maximum response occurs at a frequency of 6—8/sec. giving the interval formaximum interaction between pulses of about 0-15 sec. The plateau intensity isgraded with stimulus frequency and the ultimate level of luminosity differs greatlyover the frequency range. A consequence of facilitation and the processes leading toa plateau of intensity for each frequency is that the number of responses required toreach the plateau is roughly the same, independent of frequency.

(b) Local spreadRepetitive stimulation usually induces a spread of luminescence. This spread will be

considered under four categories which may correspond with different pathways ofconduction or may represent different physiological states of the same neural units(Fig. 1). The first type of spread is confined to the local area of stimulation, usuallyprogressing 0-5-2 cm. The velocity of propagation varies between 0-5 and 2 cm./sec.(at 220 C). Luminous spread is partially polarized, in that a lower frequency,intensity, or duration of stimulation is required for posterior spread than for anterior.

6 CHARLES H. BAXTER AND PETER E. PICKENS

Spread is obtained using stimulation frequencies of o-5-io/sec. with the higherfrequencies tending to produce spread sooner and to a greater distance. The usualpattern is for the luminescence to appear under the electrodes and remain there fromfour to ten stimuli, and then to spread out to the ma-yimnm area at a uniform ratewithout exhibiting increments correlated with arriving impulses. The luminous areafades, either before the end of stimulation or a second or so after, and does not exhibitthe after-discharge noted elsewhere. This local spread was characteristically obtainedwhen using animals collected some weeks before their use or with animals previouslysubjected to a period of stimulation.

(c) Extensive spread

In experiments with fresh specimens spread is often characterized by waves ofluminescence which arise at the site of stimulation and sweep extensively throughoutthe length of the animal at a velocity of 3-4 cm./sec. (at 220 C) . The polarization islike that described in the preceding paragraph. Stimulation frequencies of i-10/sec.are effective; the higher frequencies produce spread sooner and more reliably. Thewaves progress at a uniform velocity until extinguished at an anatomical boundary(caudal tip or collar) or simply fade out after an excursion of some centimetres. Thesewaves usually leave in their wake local areas which exhibit a faint persistent glow, oftenmade up of small bright spots enduring for variable periods of time, from seconds tominutes. If stimulation is continued, the primary wave may be followed by secondaryand tertiary waves at 1 or 2 sec. intervals.

Section of the ventral cord anywhere along its length abolishes propagation of therapid, extensively conducted, luminous wave past the point of section. A slow waverequiring much facilitation does spread a few centimetres posterior to the section ora lesser distance in the anterior direction. Subsequent section of the dorsal cordproduces some decrement in the remaining response.

Section of the dorsal cord of an individual which exhibits extensive conduction ofa luminous wave does not abolish this ability. Subsequent section of the ventral cordthen produces the results described above. Controls with lesions in the lateral bodywall still show extensive spread of the luminous wave. These results show the essenti-ality of the intact ventral cord for propagation of the rapid wave of luminescence anda lack of dependence on the continuity of the dorsal cord.

(d) Startle luminescence

The conducted luminous wave associated with the startle response to proboscisstimulation has been discussed previously under the section on mechanical stimulationand single electrical shocks. The startle luminescence is the only conducted luminousresponse elicited by a single shock; it spreads rapidly back to the hepatic region.Several stimuli become necessary to elicit the startle response and its associatedluminescence in older preparations. The rapid spread of luminescence, its all or nonenature, and concomitance with the startle response contraction lead to the conclusionthat they are both mediated by the same neural mechanism. Bullock (1944) describedthe giant fibre system of enteropneusts and its participation in the startle withdrawalof some species.

Control of luminescence in hemichordates 7

(e) Disjunct luminescence

Using repetitive stimulation, in several cases a rapid spread occurred over longdistances without luminous continuity between the electrodes and a distant lumin-escing area. Luminescent pulses, usually involving the caudal tip, were correlatedwith successive electrical stimuli applied to regions 5-12 cm. anterior. In these casesthe stimulation frequency was o-5-o-2/sec. The conduction velocity for this responsewas quite high and could only be estimated at about 1 m./sec. The high conductionvelocity suggests mediation by extensive spread in long-fibred tracts in the dorsal orventral cord.

Luminescence in isolated pieces

Some observations were made on the responses of pieces of the lateral body wall ofthe trunk removed so as to exclude the dorsal and ventral cords. These pieces con-sisted of the genital wing and sometimes a portion of the hepatic area. When isolatedand placed in sea water the fragments exhibited much spontaneous muscular activitywhich involved curling and uncurling as well as local movements. Pieces werefrequently observed in continuous writhing movements for periods of a few minutesto over hah0 an hour. The fragments would only remain quiescent for a few minutesat a time.

After a period of dark-adaptation isolated pieces were checked for luminescentresponses. Spontaneous luminescence was never observed, but application of mildtactile stimuli or suprathreshold single shocks produced a local response confinedwithin a few millimetres of the stimulated area. The intensity of the response in agiven area and its spread of a few millimetres to the adjacent area are graded withstimulus intensity as described previously for unoperated individuals. The luminousresponse endures for 1-10 sec. depending on the intensity of the stimuli.

Repetitive mechanical or electrical stimulation causes a marked increase in thebrightness, spread, and duration of luminescence. Spread of a bright, steady lumin-escence has been observed to extend posteriorly a distance of 2 cm., and the furtherinvolvement of an additional 1-3 cm. as small scattered patches of light was common.The existence of polarity in the general plexus is indicated by the higher thresholdand reduced extent of spread for anterior luminescence.

Autoexcitation

Certain individuals, after a series of mechanical or electrical stimuli, exhibit post-stimulatory luminescent displays. They range in duration from less than a minute toan exceptional individual which performed for almost \ hr., but displays of 1-5 min.were most frequent. The display consists of luminescent waves which begin at a fixedor shifting locus and sweep over all, or a portion of, the body. Often waves arise froma site at quite constant intervals. The most spectacular case occurred when the hepaticregion of an animal was stimulated at a frequency of o-25/sec. After several pulses aseries of luminescent waves began at the region of the electrodes and passed back tothe tail. These waves continued after cessation of stimulation; a rather steadily glowingarea in the hepatic region remained the source of the successive waves. Their velocitywas about 1 cm./sec. (220 C) . The waves varied locally in intensity but passed to the

Control of luminescence in hemichordates 9

colony of polyps, or the luminescence of extensive areas of tissue. In each of thesecases the observed response represents the integrated activity of myriad unit effectorsand one does not know the fine pattern of effector response. The knowledge of thecharacter of the response in terms of its components permits an assignment of para-meters of neural function to the net which necessitates fewer assumptions than ifworking only with the integrated response.

The normal response of each photocyte to a single shock somewhat above thresholdis, after a variable latent period, the rapid development of luminescence (peak timeis a few tenths of a second) followed by a gradual decline (disappearing in i to severalseconds). The active photocytes may vary somewhat in their peak intensities, butthere is no correlation between intensity and their position in the responding field.On the average, photocytes appear as bright at the edge of the luminous area as they

Stimulatingelectrode

Threshold 1-5 x thresholdDiameter of field of view equals 3 mm.

4 x threshold

Fig. 2. Idealized patterns of luminous photocytes responding to single shocks ofdifferent strengths at a fixed site.

are at the locale of the electrodes. The density of active photocytes is graded withdistance from the site of stimulation. It is highest next to the electrodes and, dependingon the preparation, declines gradually, or rather abruptly, or patchily.

With increasing stimulus strength the latency of the first observed luminousresponse declines from values of around a second at threshold to a few tenths of asecond at several times threshold voltage. We could not detect by eye any appreciableincrement in the intensity of individual luminous points. The duration of luminescencein the activated photocytes shows an increase at higher intensities of stimulation.

Observation of spread of the local response as a function of stimulus intensity ofsingle shocks has been extremely informative on the nature of activity in the generalplexus (Fig. 2). At threshold, the area of response is confined to within £ mm. or lessof the electrodes, and the photocytes are fairly simultaneous in appearance after aprolonged latent period. Within a certain range of values above threshold, increase ofthe voltage produces graded spread of response out to a distance of 2-5 mm. There isalso a marked increase in density of photocytes in the area previously responding tothe weaker stimuli. The latency of most photocytes increases with their distancefrom the electrodes; however, it was characteristic for some of the more distant onesto respond before some of those located more proximally to the stimulated site.

In favourable preparations saltatory spread was observed; waves moved outwardfrom the activated locale and successively involved additional subequal increments

io CHARLES H. BAXTER AND PETER E. PICKENS

while the density of active photocytes increased in the previously luminescing area.The frequency and number of these waves is graded with the strength of a single shockup to values which are several times threshold. The frequencies observed varied fromo-5/sec. to 3/sec. and the number of waves from i to 4. The increments covered by thewaves were estimated to be from 0-5 to 1-5 mm. However, in most cases spread wasnot by these discrete waves. The usual development of luminescence could more aptlybe described as the gradual involvement of a greater area with an increase in thedensity of active photocytes.

When preparations were stimulated excessively, the spread declined in later trials;finally only a very local response or none at all appeared even to high shock strengths.It was possible to obtain results similar to those described above when employingeither mild tactile or repetitive electrical stimulation. This is true primarily for theinitial portion of the response and when using moderate strengths and frequencies ofstimulation. Repetitive electrical stimulation of fresh, responsive individuals usuallyinitiates a more rapid spread which appears to involve the maximum density of photo-cytes as it passes across the limited field of view. Repetitive stimulation of fatiguedor aged preparations produced responses as obtained with single shocks.

Observation of the area responding to a single shock demonstrates that regions ofactive photocytes tend to fade out in the inverse of the order of their appearance. Thisis not an inviolable rule, and frequently large intermediate areas would fade appreci-ably before patches of luminous photocytes which were located at the outer limit ofthe responsive field. That region situated most proximal to the electrodes was in-variably among the last to disappear. The duration of luminescence of a photocyteafter a single shock was a function of stimulus strength and ranged between values of1 sec. and 10—15 8ec<

Source of luminescence

When stimuli just above threshold for response to a single shock are used, theduration of the luminous pulse is about 1 sec. This is much shorter than valuesreported for animals described as producing luminescence by discharge of a luminoussecretion {Chaetopterus, Nicol, 1952; Pholas, Harvey, 1952); these cases all fall in therange of minutes. The duration in Ptychodera is well within the range described fromanimals with intracellular light production (Ackoloe, Nicol, 1954; Renilla, Nicol, 1955).Also characteristic of extracellular luminescence is the rapid fatigue due to exhaustionof the luminous secretion, whereas intracellular photogeny may persist throughhundreds of successive flashes. In this respect the long continued autoexcitatorydisplay of Ptychodera corresponds in numbers of replications to intracellular photogeny.

The comparison of response in Ptychodera and Balanoglossus is very instructive asto the site of luminescence. In some cases during experiments on Ptychodera, materialexhibiting luminescence adhered to the instruments or electrodes, and the glow enduredfor several seconds. Rao (1954) observed luminous slime left behind by Ptychodera.To mild stimulation, the paremeters of the luminous response of Balanoglossus arevery similar to Ptychodera. However, after strong stimulation of an individual lyingon moist tissue paper we have observed a luminous secretion to flow out from thetrunk, and this material continues to glow for approximately fifteen minutes. Thesecretion does not exhibit the points of light visible under x 25 magnification of the

Control of luminescence in hemichordates 11

body. This strengthens the conviction that these points represent internal activity ofindividual photocytes, for when the luminescence is extracellularized, it becomesdiffuse and no longer shows lability of duration. The evidence indicates that photogenyin enteropneusts is in general a brief intracellular phenomenon, but that under certainconditions long-lasting luminescence occurs in secreted mucus in these two genera.

DISCUSSION

Nerve net basis. The properties of spread of luminescence in the lateral body wallare consistent with the interpretation that spread occurs in a nerve net. Furthermorethese properties suggest a nerve net with synapses, as has been inferred for somecoelenterates (Pantin, 1935a; Horridge, 1957). Bullock (1945) described a fine-textured nerve plexus within the epidermis throughout the body of enteropneusts andgave physiological evidence of diffuse conduction (1940). Such a net could be thebasis for the observed autonomy of luminescence in fragments, the diffuse conductionand decrement of spread—if we accept the probability of repetitive impulses tophysiological stimuli.

Threshold facilitation. The results described point to an essentially all-or-nothingcharacter of the individual photocyte response. Though different points of light varyin intensity, each has a characteristic intensity which is not heightened by increasingeither the frequency or number of stimuli. These serve only to prolong the glow.Nevertheless, photocyte discharge requires facilitation which is apparently in theneuro-effector transmission. Individual photocytes can have different facilitationthresholds, i.e. require different intervals between stimuli.

Patchiness of response. The results indicate a significant tendency to patchiness ofluminescence. This suggests a distribution of excitabilities at the photocyte junctionsthroughout the general plexus. Since this variability is probably a function of factorsin the recent past, it would not logically be expected to be random, especially whenconsidered over a reasonably large area in which two fairly distinct recent historiesmay have prevailed. It is not entirely possible to separate the characteristics ofluminous response which reside in the neuro-effector junction from those which areonly a property of the nerve net. We feel that the patchy appearance of luminescentareas during the early portion of a response is more likely to be predominantly afunction of the lability of the photocytes than of the net.

Repetitive discharge. The fine pattern of development of luminosity to single shocksprovides the explanation for gradation of intensity and spread with stimulus growth.At threshold the response is very local, delayed in appearance, and of short duration.Increase in stimulus strength produces spread of the response, decreases the latentperiod for the first observed photocytes, prolongs the luminous responses, and causesnew photocytes to appear over an extended interval. These features suggest repetitivedischarge of the nerve net to single electrical shocks. The observation of spreading,luminous waves from the stimulated locus, with their number and frequency gradedby shock strength, constitutes corroboration of repetitive discharge. The ability tomimic the effects of intense single shocks by mild repetitive shocks or weak tactilestimulation indicates that the patterns of neural activity evoked by the three forms ofstimulation are essentially similar. Thus after-discharge appears to be a property of

12 CHARLES H. BAXTER AND PETER E. PICKENS

some nerve nets of both coelenterates (Pantin, 19356; Josephson, 1961) and entero-pneusts.

Limitation of spread. The general plexus in both Ptychodera and Balanoglossusexhibits marked interneural facilitation, and spread has been observed to occur inBalanoglossus by successive, subequal increments. However, local spread throughthe general plexus of enteropneusts only proceeds to a limited distance within whichluminescence may be maintained by continuation of stimulation. What properties ofa net would produce appreciable spread which soon ceases though response is main-tained in the previously active area? This characteristic of limited spread to main-tained stimulation resembles the response for polyp retraction in Porites, describedby Horridge (1957) and also one form of response of a simulated model of a nerve netprogrammed for a digital computer (Josephson, Reiss & Worthy, 1961). Horridgeproposes a mathematical model to describe activity in a net mediating the responseseen in Porites but does not clarify the reaction to repetitive stimulation sufficientlyto characterize the behaviour. Josephson's model demonstrates limited spread whenprogrammed for a low effective value of interneural facilitation. It seems likely inconsidering the present results in detail that some modification of this model wouldduplicate the luminescent behaviour of enteropneusts.

Comparison zoith pennatulids. It is accepted as a generality (Buck, 1955; Nicol, i960)that luminescent responses of metazoan animals are mediated through the action of thenervous system and that light production can be used as an indicator of the activity ofneural elements. Results of experiments on the enteropneust Ptychodera demonstrateluminescent responses varying with the characteristics of the electrical stimulation. Onthe basis of the information available now some attempt at synthesis and comparisonwith other luminescent groups may be made.

The luminescent responses of Ptychodera seem to correspond closely in many waysto those of pennatulids (sea pens belonging to the soft corals) a group in whichluminescence is also under the control of a nerve net. Luminescence in the sea penshas been extensively studied (Parker, 1919; Nicol, 1955a, b, 1958; Davenport &Nicol, 1956). The response to single shocks is variable within and between species.Renilla usually requires two or more shocks to evoke luminescence while Pennatulaand Virgularia usually respond to the first. In Ptychodera a single shock of moderatestrength will elicit luminescence, while at lower intensity facilitation of several stimuliis required. The single shock of moderate strength really uses facilitation in producingthe luminous response since it acts to evoke repetitive discharge in the nerve net andthe response begins on one of the subsequent impulses. For Ptychodera, the maximuminterval still preserving some facilitation is about 5 sec. at 22° C , for Renilla it isaround 5 sec. at 15-170 C. For Ptychodera, the interval producing maximum facilitationis about 0-15 sec.; for Renilla it is 0-33 sec. (Nicol, 1955a).

If the strength of single shocks is increased above threshold, the enteropneust willexhibit an increase in luminous intensity as well as some spread. A gradation ofintensity with stimulus strength does not occur in pennatulids (Nicol, 1955 a) exceptin certain cases where intense shocks elicit a bright flash to the first shock with adecreased response to the second which starts the normal facilitating series; Nicolattributes this to repetitive firing in the nerve net. The bright flash is conducted overthe whole rachis and cannot be duplicated at lower stimulus intensities by using higher

Control of luminescence in hemichordates 13

frequencies of stimulation. The optimal frequency for facilitating responses is 3/sec.and the refractory period is 0-2 sec. so that repetitive firing to the first shock shouldnot be more effective than stimuli at 3/sec.

In both groups raising the frequency of stimulation results in an increase in the levelof luminosity and in both this is due to facilitation. In Remlla the site of facilitationis considered to be at the neuro-effector junction (Buck, 1954; Nicol, 1955a). InPtychodera facilitation of intensity occurs by increase in density of photocytes as afunction of frequency.

Interneural facilitation occurs where spread is obtained. In Renilla (Nicol, 1955 a),where the net is normally through-conducting, it can be converted to a facilitating netby fatigue or transections to produce a partial block. The same is true of Ptychoderawhere the extensive rapid conducting system is converted into a local facilitatingsystem by physiological or post-stimulatory depression or by section of the ventral cord.

In Leioptilus the net is normally through-conducting but this may be abolished byfatigue. In a fatigued individual light spreads longitudinally along the dorsal surfaceand does not cross the rachis to the contralateral side as in fresh preparations. Thisindicates a longitudinal polarization of conduction pathways which become functionalfor the luminescent response in the condition of fatigue (Davenport & Nicol, 1956).In Ptychodera pathways for conduction of the luminescent wave are normally polarizedso that conduction is more readily obtained posteriorly. In the fatigued or physio-logically depressed condition this polarization is exaggerated.

In both groups there is a lability of the response even to low-frequency stimulation.The luminescence of pennatulids varies greatly in this lability; Remlla on the one handgives responses to hundreds of stimuli at moderate frequencies and Virgularia andStylatula on the other respond only to several stimuli before reaching a refractorycondition. Ptychodera would lie somewhere between these extremes in this respect.

In both groups the ability to luminesce is dependent upon previous dark-adaptation.The photogenic material is not destroyed by light but its release to adequate stimuliis inhibited.

SUMMARY

1. Luminescence in Ptychodera and Balanoglossus is normally neurally mediated.2. The main luminescence appears to be intracellular; in addition a luminous

slime is secreted.3. The response of individual photocytes is all-or-none, but requires neuroeffector

facilitation which grades intensity by recruitment (in contrast to crustacean muscle).4. Repetitive discharge, both to mechanical and single electrical stimuli, is con-

sidered to be a prime factor together with interneural and neuro-effector facilitation,in determining spread of excitation in the general plexus.

5. Four types of spread of luminescence occur, differing in conduction velocitypolarization of spread, area of spread, necessary conditions for elicitation and adequatestimulus. At least three different neural pathways are involved.

6. Prolonged autoexcitatory displays produce luminous waves arising repetitivelyfrom the same locus and from multiple loci; the general plexus is implicated.

7. There is a diurnal rhythm of responsiveness to stimuli. This is attributed to thephotocyte rather than to the nervous system.

14 CHARLES H. BAXTER AND PETER E. PICKENS

The work was assisted by a grant from the National Institute of Health to Prof.Theodore H. Bullock.

REFERENCES

BUCK, J. B. (1955). Some reflections on the control of bioluminescence. In F. H. Johnson (Ed.), TheLuminescence of Biological Systems. Washington, D.C.: American Association for the Advancementof Science.

BULLOCK, T. H. (1940). Functional organization of the nervous system of Enteropneusts. Biol. Bull.,Woods Hole, 79, 91-113.

BULLOCK, T. H. (1944). The giant nerve fiber system in balanoglossids. J. Comp. Neurol. 80, 355-67.BULLOCK, T. H. (1945). The anatomical organisation of the nervous system of Enteropneusta. Quart.

J. Micr. Sci. 85, 55-n 1.CBOZIER, W. J. (1915). The behavior of an enteropneust. Science, 41, 471-2.CBOZIER, W. J. (1917). Photic sensitivity of Balanoglossus. J. Exp. Zool. 34, 211, 217.CROZIER, W. J. (1920). Rhythm of light production in balanoglossids. Anat. Rec. 30, 186.DAHLGREN, U. (1917). Production of light by animals. Enteropneusta. J. Franklin Inst. 183, 620-4.DAVENPORT, D. & NICOL, J. A. C. (1956). Observations on luminescence in sea pens (Pennatulacea).

Proc. Roy. Soc. B, 144, 480-96.DELAGE, Y. & HEROUARD, E. (1898). Traiti de Zoologie Concrite. Vol. vin. Lesprocordis, p. 53.HARVEY, E. N. (1926). On the inhibition of animal luminescence by light. Biol. Bull., Woods Hole, 51,

85-8.HARVEY, E. N. (1952). Bioluminescence. New York: Academic Press.HORRIDGE, G. A. (1957). The co-ordination of the protective retraction of coral polyps. Phil. Trans. B,

*4°. 495-529-HYMAN, L. H. (1959). The Invertebrates: Smaller Codomate Groups. Vol. V. New York: McGraw-Hill.JOSEPHSON, R. K. (1961). Repetitive potentials following brief electrical stimuli in a hydroid. J. Exp.

Biol. 38, 579-94-JOSEPHSON, R. K., REISS, R. F. & WORTHY, R. M. (1961). A simulation study of a diffuse conducting

system based on coelenterate nerve nets. J. Theoret. Biol. j , 460-87.KNIGHT-JONES, E. W. (1952). On the nervous system of Saccoglostus cambrensis (Enteropneusta).

Phil. Trans. B, 336, 315-54-KtJWANO, H. (1902). New enteropneust from Misaki. Anttot. Zool. Jap. 4, 77-84.NICOL, J. A, C. (1952). Studies on Chaetopterus variopedatus (Renier). I. Nervous control of light

production. J. mar. Biol. Ass. UJC. 30, 433-52.NICOL, J. A. C. (1954). The nervous control of luminescent responses in polynoid worms. J. mar. Biol.

Ass. U.K. 33, 225-55.NICOL, J. A. C. (1955 a). Observations on luminescence in Renilla (Pennatulacea). J. Exp. Biol. 3a,

299-320.NICOL, J. A. C. (1955 ft). Nervous regulation of luminescence in the sea pansy Renilla koUiheri. J. Exp.

Biol. 3a, 619-35.NICOL, J. A. C. (1958). Observations on the luminescence of Permatula phosphorea, with a note on the

luminescence of Virgularia nurabilis. J. mar. Biol. Ass. U.K. 37, 551-63.NICOL, J. A. C. (i960). Biology of Marine Animals. London: Sir Isaac Pitman and Sons Ltd.PANCERI, P. (1875). La m c e e gli organi luminosi di alcuni annelidi. R.C. Accad. Napoli, 14.PANTIN, C. F. A. (1935 a). The nerve net of Actinozoa. I. Facilitation. J. Exp. Biol. 13, 119-37.PANTTN, C. F. A. (19356). The nerve net of Actinozoa. III. Polarity and after-discharge. J. Exp. Biol.

13, 156-64.PARKER, G. H. (1919). The Elementary Nervous System. Philadelphia and London: J. B. Lippincott Co.RAO, K. (1954). Bionomics of Ptychodera flava. J. Madras Univ. B, 34, 1-5.SILBN, L. (1950). On the nervous system of Glouobalanus marginatus Meek (Enteropneusta). Acta

Zool, Stockh., 31, 149-̂ 75.