AMER. ZOOL., 25:265-274 (1985) Neuroendocrine Correlates of Circadian Rhythmicity in Crustaceans 1 HUGO ARECHIGA, JOSE LUIS CORTES, UBALDO GARCIA, AND LEONARDO RODRIGUEZ-SOSA Departamento de Fisiologia y Biofisica, Centro de Investigation y de Estudios Avanzados del IPN, 07000 Mexico, D.F. SYNOPSIS. The secretion of neurohormones from the crustacean X-organ - sinus gland system is controlled by environmental influences, light being the most conspicuous. Two sets of photoreceptors appear to mediate the influence of light on neurosecretion based on intracellular recordings from X-organ neurons and estimations of hormone release. Extra-retinal photoreceptors can initiate neurohormonal release from the eyestalk. Neurosecretory activity is also influenced by putative neurotransmitters. GABA is found in high concentrations in the medulla temninalis of the eyestalk and is released by stimu- lation, in a calcium-dependent manner. Diurnal variations occur in the amounts of eyestalk neurohormones, either those present in the eyestalk or released by electrical stimulation of the isolated sinus gland. Rhythm phases vary from one hormone to another. Neurohormones secreted in the eyestalk are also found in other regions of the central nervous system. Rhythms of neurosecretion are present both in the secretion in the isolated eyestalk and in eyestalkless animals, thus indicating that rhythmicity is a distributed property of the neurosecretory system. INTRODUCTION Since its inception, the study of circadian rhythmicity in crustaceans has been linked to the knowledge of the neurosecretory system. The early discovery that integu- mentary chromatophores were unrespon- sive to nerve stimulation but reactive to the injection of eyestalk extracts (Perkins, 1928) opened the way to the study of neu- rosecretion. Three decades ago the X-or- gan-sinus gland system in the eyestalk was characterized as a set of neurosecre- tory cells with their somata in the X-organ and their endings in the sinus gland (Bliss and Welsh, 1954). Before the true nature of the neurosecretory system in the eye- stalk was disclosed, the relationship between the eyestalk and circadian rhyth- micity had been adumbrated by the rec- ognition that eyestalk ablation led either to loss or to profound alterations of diurnal rhythmicity in a variety of functions, most conspicuously integumentary pigment position and locomotion (Brown, 1961). In fact, the major physiological func- tions known to be affected by eyestalk abla- tion or eyestalk extract injection, also dis- play diurnal rhythmicity. Such is the case of the integumentary chromatophores, 1 From the Symposium on Advances in Crustacean Endocrinology presented at the Annual Meeting of the American Society of Zoologists, 27—30 December 1983, at Philadelphia, Pennsylvania. whose position undergoes a diurnal rhyth- micity, and is known to be influenced by hormones mediating both the concentra- tion and dispersion of pigment granules in the chromatophores. Such hormones are known to be present in the eyestalk (Klein- holz, 1976). The distal retinal pigment dis- plays a diurnal rhythm of position (see Are- chiga, 1977) and the hormone responsible for driving the migration to the light- adapted position is mostly located in the eyestalk (Kleinholz, 1966). The blood sugar levels are also known to vary along the 24- hr cycle (Hamann, 1974), and the hyper- glycaemic hormone (CHH) has been shown to be in greatest amounts in the sinus gland of the eyestalk (Keller et al., 1985). Loco- motion is also a rhythmic function, affected by eyestalk ablation and extract injection (Arechiga and Naylor, 1976; Arechiga, 1979). The basic notion which emerged from the early studies was that of the neurose- cretory cell in the X-organ - sinus gland system as the final common path integrat- ing, not only neuroendocrine reflexes ini- tiated by environmental stimuli of which the light-dark changes are the most con- spicuous, but also receiving (or generating) time signals of a circadian nature. Little however is known on the nature of the receptors and afferent pathways conveying the information about illumination to the neurosecretory cells. The role of the eyestalk in the integra- 265 at Universidad Nacional Autonoma de Mexico on February 17, 2015 http://icb.oxfordjournals.org/ Downloaded from
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AMER. ZOOL., 25:265-274 (1985)
Neuroendocrine Correlates of Circadian Rhythmicity in Crustaceans1
HUGO ARECHIGA, JOSE LUIS CORTES, UBALDO GARCIA, AND LEONARDO RODRIGUEZ-SOSA
Departamento de Fisiologia y Biofisica, Centro de Investigation y de Estudios Avanzados del IPN, 07000 Mexico, D.F.
SYNOPSIS. The secretion of neurohormones from the crustacean X-organ - sinus glandsystem is controlled by environmental influences, light being the most conspicuous. Twosets of photoreceptors appear to mediate the influence of light on neurosecretion basedon intracellular recordings from X-organ neurons and estimations of hormone release.Extra-retinal photoreceptors can initiate neurohormonal release from the eyestalk.
Neurosecretory activity is also influenced by putative neurotransmitters. GABA is foundin high concentrations in the medulla temninalis of the eyestalk and is released by stimu-lation, in a calcium-dependent manner.
Diurnal variations occur in the amounts of eyestalk neurohormones, either those presentin the eyestalk or released by electrical stimulation of the isolated sinus gland. Rhythmphases vary from one hormone to another. Neurohormones secreted in the eyestalk arealso found in other regions of the central nervous system. Rhythms of neurosecretion arepresent both in the secretion in the isolated eyestalk and in eyestalkless animals, thusindicating that rhythmicity is a distributed property of the neurosecretory system.
INTRODUCTION
Since its inception, the study of circadianrhythmicity in crustaceans has been linkedto the knowledge of the neurosecretorysystem. The early discovery that integu-mentary chromatophores were unrespon-sive to nerve stimulation but reactive to theinjection of eyestalk extracts (Perkins,1928) opened the way to the study of neu-rosecretion. Three decades ago the X-or-gan-sinus gland system in the eyestalkwas characterized as a set of neurosecre-tory cells with their somata in the X-organand their endings in the sinus gland (Blissand Welsh, 1954). Before the true natureof the neurosecretory system in the eye-stalk was disclosed, the relationshipbetween the eyestalk and circadian rhyth-micity had been adumbrated by the rec-ognition that eyestalk ablation led eitherto loss or to profound alterations of diurnalrhythmicity in a variety of functions, mostconspicuously integumentary pigmentposition and locomotion (Brown, 1961).
In fact, the major physiological func-tions known to be affected by eyestalk abla-tion or eyestalk extract injection, also dis-play diurnal rhythmicity. Such is the caseof the integumentary chromatophores,
1 From the Symposium on Advances in CrustaceanEndocrinology presented at the Annual Meeting of theAmerican Society of Zoologists, 27—30 December1983, at Philadelphia, Pennsylvania.
whose position undergoes a diurnal rhyth-micity, and is known to be influenced byhormones mediating both the concentra-tion and dispersion of pigment granules inthe chromatophores. Such hormones areknown to be present in the eyestalk (Klein-holz, 1976). The distal retinal pigment dis-plays a diurnal rhythm of position (see Are-chiga, 1977) and the hormone responsiblefor driving the migration to the light-adapted position is mostly located in theeyestalk (Kleinholz, 1966). The blood sugarlevels are also known to vary along the 24-hr cycle (Hamann, 1974), and the hyper-glycaemic hormone (CHH) has been shownto be in greatest amounts in the sinus glandof the eyestalk (Keller et al., 1985). Loco-motion is also a rhythmic function, affectedby eyestalk ablation and extract injection(Arechiga and Naylor, 1976; Arechiga,1979).
The basic notion which emerged fromthe early studies was that of the neurose-cretory cell in the X-organ - sinus glandsystem as the final common path integrat-ing, not only neuroendocrine reflexes ini-tiated by environmental stimuli of whichthe light-dark changes are the most con-spicuous, but also receiving (or generating)time signals of a circadian nature. Littlehowever is known on the nature of thereceptors and afferent pathways conveyingthe information about illumination to theneurosecretory cells.
tion of circadian rhythmicity, either as thesite of a self-sustained circadian oscillatoror as a part of a more complex system inte-grating rhythmicity has not been clearlyestablished either. Ablation experimentshave led to equivocal results. The diurnalrhythm of spontaneous locomotion in Orco-nectes, Carcinus and Nephrops appears to besuppressed by bilateral eyestalk ablation,whereas the light-driven rhythm persistsafter such operation in Procambarus clarkii(Page and Larimer, 1976). Eyestalk abla-tion has been reported to abolish therhythm in Gecarcinus only in some speci-mens of a group, while rhythmicity persistsin others (Bliss, 1962). Diurnal rhythmicityof integumentary pigment position hasbeen found to persist after bilateral eye-stalk ablation (Webb et al., 1954).
In this article, we shall review recent workin our laboratory on the light input to theneurosecretory system and on the func-tional organization of circadian rhythmic-ity of neurosecretory activity. The inter-play of the neurosecretory system and otherpossible mechanisms of coupling in theintegration of circadian rhythms will alsobe discussed.
LIGHT INPUT TO THE EYESTALKNEUROSECRETORY SYSTEM
Two of the identified neurohormonessecreted in the eyestalk are released as aresponse to changes of illumination. Lightunder natural conditions elicits the releaseof the light-adapting distal retinal pigmenthormone (DRPH), an octadecapeptide.DRPH (Fernlund, 1976) causes the disper-sion of pigment granules within the distalretinal cells in a number of crustaceanspecies (see Kleinholz and Keller, 1979).Conversely, the second identified neuro-peptide from the eyestalk, the red pigmentconcentrating hormone (RPCH), an octa-peptide (Fernlund, 1974), induces the cen-tripetal migration of pigment granules inintegumentary chromatophores, thus pro-ducing a blanching effect. This neuroen-docrine reflex occurs as a response to dark-ness. No information is available indicatingthe reflex release of other eyestalk neu-rohormones as a response to changes inillumination. Although light is also known
to promote the migration of proximal ret-inal pigments, such a response does notappear to be mediated by a neurohormonerelease, but it is rather a direct responseof the retinula cells to illumination in thecrayfish Procambarus (Olivo and Larsen,1978; Frixione et al., 1979). Light alsoaffects spontaneous locomotor activity onshort term basis in Nephrops norvegicus(Arechiga and Atkinson, 1975) but thehormonal involvement in this response hasnot been clarified. Thus more than onechannel is likely to participate in the inte-gration of these responses.
The photoreceptors triggering DRPH release
As far as is known, DRPH release is theonly influence promoting distal pigmentdispersion. Consequently, the neurosecre-tory cells producing DRPH are the finalcommon pathway for a light-induced neu-roendocrine reflex with a circadian rhythmof secretion. In a search of the location ofthe photoreceptors triggering DRPHrelease in crayfish Procambarus bouvieri andProcambarus clarkii, we developed a prep-aration in which a segment of eyestalk con-taining the compound eye was implantedon the carapace. We found that diffuse illu-mination with similar characteristics tothose necessary to trigger the migration ofdistal pigment in the natural retina (whitelight, 300 lux for 30 min) induces the dis-persion of distal pigment in the implantedeye (Arechiga, 1977). The photoreceptorstriggering this response must be locatedoutside the retina, since bilateral retinalablation leaving intact the rest of the eye-stalk fails to suppress the light-induced dis-tal pigment dispersion. We established thatneither the time course nor the intensityfunction of the photomotor response in theimplanted eye are affected by the retinalremoval (Fig. 1). Bilateral ablation of wholeeyestalk does prevent the light-induced dis-tal pigment migration, which indicates thatthis distal pigment migration is not a directresponse to light in the implanted eye. Theextra-retinal phototransduction triggeringDRPH release in Procambarus appears toinvolve at least two pigments, one with apeak response at 480 nm and the otherresponding maximally at 540 nm (Cortes
FIG. 1. Response of implanted eye in carapace ofcrayfish to diffuse illumination. Upper graph com-pares the intensity functions (Abscissa, light intensity)in control animals with those obtained after bilateralretinal excision. Lower graph presents a similar com-parison for the time course of the photomechanicalresponse (Abscissa, time course of response). Eachpoint represents the average of 7 preparations. Ver-tical bars indicate the standard error of the mean.Ordinate (AA) reduction in area of glow induced bylight reflected on the retina. The area is proportionalto the retraction of distal pigment.
and Arechiga, 1984). Pigments with suchabsorption peaks have been described alsofor retinal photoreceptors (see Shaw andStowe, 1982).
FIG. 2. Intracellular record from the neuropil seg-ment of an X-organ neuron. Lucifer yellow stain. Leftrecord, burst of action potentials induced by light.Right record, epsp's recorded in the same lead, aftersuppressing the spike firing by hyperpolarizing theneuron at two different membrane potentials (10 mVfor upper trace and 20 mV for lower trace). Cali-brations, 20 mV, 100 ms for left trace. 5 mV, 200msec for right trace. (Modified from Glantz el al.,1983.)
Extra-retinal photoreceptors have beenpostulated as parts of the entrainmentpathway of circadian locomotor rhythm inthe crayfish Procambarus clarkii (Page andLarimer, 1976). The position of theseextra-retinal photoreceptors has not beenidentified with precision, and the spectralsensitivity of the various crustacean neu-roendocrine reflexes, has not been ana-lysed. There is no information as to thephotoreceptive mechanisms involved in therelease of RPCH.
Light input to the neurosecretory cells
Another strategy for analysis of the lightinput to the neurosecretory cells is to recordtheir electrical activity. Using intracellularrecordings from neurons in the crayfishX-organ, combined with injection of luci-fer yellow, we have been able to detect elec-trical responses of single neurons to lightpulses. Recordings made from the distalsegment of the X-organ - sinus gland axons,or from the sinus gland terminals showedthat retinal illumination resulted either inchanges in ongoing spontaneous activity,or in trains of action potentials in otherwisesilent cells (Glantz et al, 1983; also see Fig.2). When recordings were made from theaxons in the neuropil area, light stimula-
tion resulted in post-synaptic potentials,accompanied by changes in the firing rate.
Figure 2 illustrates an excitatory post-synaptic potential (epsp), intracellularlyrecorded from the neuropil area in thecrayfish medulla terminate, as a response toillumination. The recording microelec-trode was filled with the dye lucifer yellow,which was injected into the impaled cell.The recording site was located in the prox-imal branching area of an X-organ neuron,as illustrated in Figure 2. Under restingpotential light results in a burst of spikes.After suppressing spiking by hyperpolar-izing the cell, epsp's are conspicuous. Byfocusing the light beam on different partsof the eyestalk from the cornea to the opticnerve, it was observed that stimulationthrough the cornea was the most effectiveon the X-organ cells (Glantz and Arechiga,unpublished). Most of the responses wereof excitatory nature, and only occasionally,inhibitory responses were evoked by light.This behavior of the X-organ cells in themedulla terminate is in interesting contrastwith that reported by Kirk and Glantz(1982) for a group of neurons, presumablyof neurosecretory nature in the medulla ter-minate of the crayfish Pacifastacus lenius-culus which are inhibited by illuminatingthe cornea.
From these results, it seems clear thatlight exerts a trans-synapic influence onX-organ neurosecretory cells. Retinal pho-toreceptors appear to initiate the responsein the X-organ cells. This would implicatetwo sets of photoreceptors influencing thesecretion of neurohormones in theX-organ - sinus gland system. The func-tional significance of an extra-retinal set ofphotoreceptors in the control of DRPHrelease becomes clear by considering thatdistal pigment dispersion is triggered byintensities of illumination higher than thelevel which saturates the retinal photore-ceptors (Arechiga, 1977).
Light is not the only environmentalinfluence affecting the release of eyestalkneurohormones. Asphyxia was shown toinduce reflex hyperglycaemia (Kleinholzand Little, 1949) presumably actingthrough the release of the hyperglycaemichormone, and stress induced by different
means is known since long ago to promotethe migration of the distal retinal pigmentto the light-adapted position (Kleinholz,1961). There is no information availableas to the sensory structures involved in thispossible multichannel influence on neu-rosecretory activity. However, as pre-sented in the next section, the medulla ter-minate X-organ cells do receive efferentinfluences.
Efferent influences on the eyestalk
The composition of the crustacean opticnerve was investigated by Nunnemacher etal. (1962), who counted in crayfish about17,000 axons. By single unit analysis of sen-sory input, Wiersma and Yamaguchi (1966)identified that over 90% of the axons inthe optic nerve of Procambarus clarkii werenon-visual fibers, running efferently to theeyestalk. As a matter of fact, the notion ofnon-visual efferent fibers in the optic nerveof several crustacean species was put for-ward by Bliss and Welsh (1954) by postu-lating the presence of neurosecretoryaxons, running from the supraoesophagealganglion and the thoracic ganglia to endin the sinus gland. More recently, Andrewet al. (1978) did not detect such neurose-cretory axons reaching the sinus gland, butdid identify efferent fibers, presumablynon-peptidergic in the X-organ - sinusgland tract of the crayfish. Larimer andSmith in Procambarus clarkii (1980)observed axons containing dense-coregranules of 132 nm diameter in the opticnerve of Procambarus, but their site of ter-mination was not located.
Gamma-aminobutyric acid (GABA) hasbeen known since long ago to be presentin the crustacean central nervous system(Dudel et al., 1963), and its role as an inhib-itory neurotransmitter in the neuromus-cular junction of the lobster Homarus andthe crayfish Procambarus is well docu-mented (see Atwood, 1977). It is also likelythat it exerts a similar role in the inhibitorysynapses on the abdominal stretch receptor(see Craelius and Fricke, 1981). Its possiblerole in the crustacean central nervous sys-tem is much less clear. On electrophysio-logical grounds, Iwasaki and Satow (1971,1973) postulated that the neurosecretory
RETENTION TIME 4.64 minyGABA STANDARD BASAL RELEASE ELECTRICAL STIMULATION
FIG. 3. Release of endogenous GABA from eyestalk of the crayfish. GABA was determined by reverse-phaseliquid chromatography. Left trace, calibration of the column with exogenous GABA. Right trace, perfusateobtained after electrical stimulation of the eyestalk with pulses of 100 msec duration, applied at 2 Hz, during10 min.
cells in the eyestalk of the crayfish Procam-barus clarkii receive an inhibitory efferentinfluence from axons in the optic nerve.These authors recorded inhibitory post-synaptic potentials (ipsp's) from cell bodiesin the crayfish medulla terminalis, whichwere blocked by picrotoxin, thus suggest-ing the possibility that they could bemediated by GABA. Kirk et al. (1982) havealso proposed GABA as an inhibitorytransmitter acting on the medulla externaneurosecretory cells. Recently, Aramburo(1983) has determined the presence ofGABA in the eyestalk of the shrimp Pen-aeus vannamei. In a search of the distribu-tion of GABA in the eyestalk of Procam-barus clarkii, we have been able to estimateby reverse phase HPLC, in 1-1.5 nmol theGABA content of the medulla terminalis.The incubation in high potassium solutions(20-60 nm) either of the whole eyestalk orisolated medullae terminalis results in therelease of GABA (see Fig. 3) in a calcium-dependent manner (Garcia et al., unpub-lished).
By recording extracellularly from theneurosecretory endings in the sinus gland,in isolated eyestalks of Procambarus (seeArechiga et al., 1985) a depression of spon-taneous activity has been observed in thecrayfish, concurrently with a reduction ofrelease of the neurodepressing hormone(NDH). Quackenbush and Fingerman(1983) have reported a dose-dependentinhibition of release of melanophore con-centrating hormone and erythrophoreconcentrating hormone in the isolatedeyestalk of Uca pugilator. These findings,consistent in the various crustaceans so farstudied, suggest that GABA acts as a trans-mitter inhibiting neurosecretory cells in thecrustacean eyestalk.
Another putative neurotransmitterwhich has been postulated to influence theactivity of neurosecretory cells in the eye-stalk is 5-hydroxytryptamine (5-HT). Theinjection of 5-HT was found to induce incrayfish a rise in blood sugar, and theresponse was prevented by eyestalk abla-tion, thus suggesting that 5-HT acts by
releasing CHH (Keller and Beyer, 1968).Bauchau and Mengeot (1966) reported that5-HT injection results in dispersion ofmelanophores in intact Carcinus maenas anderythrophores in Uca pugilator. This effectappears also to be mediated by hormonalrelease from the eyestalk (Rao and Finger-man, 1970). 5-HT, as well as norepineph-rine and dopamine have been found toaffect the release of various chromatopho-rotropins in Uca (Rao and Fingerman, 1983;Quackenbush and Fingerman, 1983; Fin-german, 1985). In the crayfish Procamba-rus, 5-HT affects both electrical activity ofneurosecretory cells and release of neu-rodepressing hormone (Arechiga et al.,1984). However, the electrophysiologicaleffects of 5-HT on eyestalk neurons havenot been totally clarified (see Cooke andSullivan, 1983). 5-HT has been identifiedin the eyestalk of Uca (Fingerman et al,1974) in the haemolymph of the lobster(Livingstone et al., 1980) and of the cray-fish Pacifastacus leniusculus (Elofsson et al.,1982). A role as a neurohormone has beenconsequently proposed. Whether 5-HT actson the neurosecretory cells of the crusta-cean eyestalk as a blood-borne agent or asa synaptic transmitter is a matter yet to bedefined.
ORCADIAN RHYTHMS OF NEUROSECRETION
As stated above, rhythms of neurosecre-tory activity have been proposed as under-lying the rhythmicity observed in the effec-tors of the various eyestalk neurohormones.Actually, the involvement of the sinus glandin the control of rhythmicity antedated theconcept of neurosecretion (Welsh, 1941).Fifty years ago, Pyle (1943) reported his-tological changes of eyestalk neurons alongthe molt cycle; Williams et al. (1979a) pos-tulated a tidal rhythm of secretory activityrestricted to a particular neurosecretorycell type in the X-organ of Carcinus maenas.In physiological experiments, the diurnalrhythm of blood sugar concentration hasbeen ascribed to a rhythm of (CHH) secre-tion (Hammann, 1974). Circadian varia-tions have been reported in the concen-trations of DRPH (Arechiga and Mena,1975) and NDH (Arechiga and Huber-man, 1980a) in the crayfish eyestalk. For
both hormones, the maxima are at day-time. Aoto (1966) described in various partsof the nervous system of Palaemon pauci-dens, diurnal variations in potency for sev-eral chromatophorotropins, which hefound to be out of phase among them-selves, some with higher potencies at night,and others at daytime. Fingerman and Fin-german (1977) reported a diurnal varia-tion of RPDH in the eyestalk of Uca, andestablished its correlation with the rhythmof 5-HT content in the eyestalk. We haveexplored the hormonal content of the cray-fish sinus gland at different times of day.Two hormones were studied, RPCH andNDH. Both were separated from othersinus gland peptides by successive stages ofsephadex G-25 and G-15 columns and highvoltage paper electrophoresis as describedby Huberman et al. (1979), and determinedby bioassay on Procambarus clarkii. RPCHwas bioassayed on erythrophores in the iso-lated abdominal epidermis, and NDH onthe isolated abdominal tonic stretch recep-tor. As seen in Figure 4, there is an ampledifference of activity in the perfusates fromisolated, electrically stimulated sinus glands,when comparing samples taken at daytimehours with those at night; both are in anti-phase, and whereas NDH release is greaterin daytime, the yield of RPCH is higher atnight. Since NDH is present both in theeyestalk and in various other parts of thecentral nervous system (Arechiga et al.,1979; Arechiga and Huberman, 19806), ithas been a convenient system to use todefine whether the control of rhythmicityis a property of a single region of the ner-vous system, or is a more distributed func-tion. Isolated crayfish eyestalks were keptin organ culture, determining NDH activ-ity by bioassaying its effect either on thespontaneous discharge of motoneurons inisolated abdominal ganglia, or on isolatedabdominal stretch receptors. The eyestalkmaintains a self-sustained rhythm in dark-ness with a period of ~22 hr (see Fig. 5).NDH activity was also determined in theeyestalkless animals, by sampling variouscentral nervous structures, and again, adiurnal rhythm of NDH was found(Cabrera-Peralta and Arechiga, unpub-lished). This indicates that the control of
NEUROHORMONES RELEASED BY ELECTRICAL STIMULATIONOF ISOLATED SINUS GLAND AT DIFFERENT TIMES
ERYTHROPHORE CONCENTRATING HORMONE
BASAL STIMi IIHRS 1
BASAL STIM
NEURODEPRESSING HORMONE
v//9in l I lBASAL STIMI IIHRS 1
BASAL STIM
FIG. 4. Electrically induced release of neurohor-mones from the crayfish sinus gland at different timesof day. Upper graph, erythrophore concentratingactivity in the perfusate of a sinus gland, comparingthe basal yield of a 10 min control period with thatobtained after stimulating at 1 Hz for 10 min. Ordi-nate, concentration index, as an arbitrary estimationof the erythrophore concentration. Lower graph, asimilar comparison for the release of neurodepressinghormone. Ordinate, NDH activity, as determined bythe lowering of tonic activity in the slow adaptingstretch receptor in arbitrary units.
neurosecretory rhythmicity is not an exclu-sive feature of a restricted area in the cen-tral nervous system. This observation of adistributed circadian neurosecretory sys-tem is consistent with early experimentsshowing that eyestalkless animals retaindiurnal chromatophore rhythmicity (seeBrown, 1961).
The persistence of diurnal rhythmicityin eyestalkless animals also indicates theparticipation of extra-retinal photorecep-tors in the control of neurosecretory rhyth-micity in crustaceans. It appears therefore,that the circadian rhythmicity of neurose-cretory activity is a function of multiple
ISOLATED EYESTALK
15 °C
HOUR OF DAY
Fic. 5. Rhythmic changes of neurodepressing hor-mone content in isolated crayfish eyestalks. Each pointis the average value of 5 samples. Ordinate, NDHcontent in arbitrary units. Abscissa, hour of day. Upperbar indicates light (clear segments) and dark (blacksegments) periods.
oscillators. Evidence from other zoologicalgroups suggests that a similar organizationcould support the expression of a wide vari-ety of circadian rhythms (see Block andPage, 1978; Rusak and Zucker, 1979; Ta-kahashi and Menaker, 1982).
In crustaceans, the existence of severalcircadian oscillators is a tenable hypothesisfrom available evidence (see Arechiga andHuberman, 1980a; Larimer and Smith,1980). For rhythms of neurosecretion itremains to be defined whether the rhyth-micity is endogenous to the neurosecretorycells. It is clear that in the integration ofrhythms in complex functions, the tem-poral variations of a given input interactwith those in the other variables affectingthe function under study. One exampleillustrating this point is the circadianrhythm of amplitude in the crayfish elec-troretinogram (ERG). It depends on at leastthree different rhythms. 1) The rhythm ofdistal retinal pigment position, which inturn is a function of the secretion of DRPHand perhaps other neurohormones. 2) Therhythm of proximal retinal pigment posi-tion, which is not known to be affected byany neurohormone from the X-organ -sinus gland system. 3) The rhythm of sen-sitivity of the photoreceptors, which is
present in animals devoid of retinal shield-ing pigments (see Arechiga and Huber-man, 1980a).
The rhythm of retinal input in turn,interacts at the higher levels of visual inte-gration with that of locomotor activity,which is conveyed by efferent fibers to theoptic peduncle (Arechiga, 1977). The loco-motory rhythm in turn, is modulated bythe neurohormonal secretion from theeyestalk (Williams etal., 19796).
From this evidence, it becomes clear thatone major task ahead is to define the path-ways and mechanisms of internal couplingof the various rhythms.
ACKNOWLEDGMENTS
The authors are grateful to V. Anayafor his assistance during the experimentalwork. The experiments were partly sup-ported by grant PCCBNAL 790004 fromConsejo Nacional de Ciencia y Tecnologia(CONACyT) de Mexico to H.A. J.L.C.,U.G. and L.R.S. held CONACyT predoc-toral fellowships during their participationin this work.
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