The Cyclic Production of 5-Hydroxytryptamine in the Opilionid. Western Michigan University

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1 AM. ZOOLOGIST, 6: (1966). The Cyclic Production of 5-Hydroxytryptamine in the Opilionid DONA J. FOWLER AND CLARENCE J. Western Michigan University GOODNIGHT SYNOPSIS. Techniques were developed for the measurement o two cyclic responses in the opilionid, Leiobunum longipes. These responses were an overt locomotor behavior and an internal secretory activity, both measured over a 24-hour period. An activity platform was designed to measure and to record continuously the movements of the opilionid. The procedure of fluorometric analysis, used to quantitate indoles, was adapted for use with the opilionid and the presence of the neurosecretory compound, 5-hydroxytryptamine, was established. A method was developed for maintaining tissues in culture over extended periods. Adult males and females of Leiobunum longipes showed a unimodal pattern of locomotor activity. The pattern of secretion of 5-HT was also shown to be unimodal in character. There was an interesting parallel between the patterns of activity and secretion of 5-HT, with greatest peak at 2:00 AM, which suggested a relationship between the two. The secretion, of 5-HT from tissues in vitro showed a bimodal cyclic output with a primary peak at 2:00 AM. A secondary peak occurred at midmorning, when the secretion in intact animals was at a low point. Most organisms exhibit cyclic behavioral responses which coincide with cyclic environmental events. Many of the daily cyclic biological activities, thus far described, are quite regular, but the mechanisms that control them are not known. In some cases, control seems to be independent and spontaneous as though intrinsic to cells, and is said to be endogenous; in other cases, the data indicate an organismal dependence upon external stimuli, such as cycles of light and dark, fluctuations in temperature, tidal rhythms, etc. Whether or not the cyclic controls of biological activity are exogenous or endogenous, the resultant behavior is certainly controlled by nervous and endocrine responses. The present study concerns the interactions between the arachnid, Leiobunum longipes, and its environment. Measurements were made of the relationship of the animal's rhythmic activity to natural periods of light and dark in the environment. Responses of the whole organism to normal cycles of day and night were compared with physiological responses to the same cycles in isolated organs (brain and gut) maintained in tissue cultures. The isolated tissues were held over extended periods of time, and their cyclic activity was established by a measurement of their 24-hour production of 5-hydroxytryptamine (5- HT). This substance was chosen for measurement because it has been described as being both an endocrine substance and produced by nervous tissue. Erspamer (1963) extensively reviewed the literature describing the occurrence and distribution of 5-HT. It is established that this substance is widely dispersed throughout the plant and animal kingdoms and is found in the nervous and secretory tissues of both invertebrates and vertebrates. There is a rapid rate of turnover of 5-HT in the organism, and there are diverse responses of different tissues to this substance (cf. Erspamer). The location of 5-HT in nervous tissue and neurosecretory cells, where the secretions could be released at some distance from the point of synthesis, has suggested an endocrine function for this substance (Grobman and Bern, 1962). These authors suggested the involvement of serotonin in the autonomic nervous system, probably as a neurotransmitter with parasympathetic function, with noradrenalin serving similarly for sympathetic function. The original investigations were supported by a grant from the National Science Foundation (G ). (187) MATERIALS AND METHODS The opilionid, Leiobunum longipes, was

2 188 DONA J. FOWLER AND CLARENCE J. GOODNIGHT taken from its natural habitat in a nearby deciduous woodland during midsummer and kept in the normal light cycle for that time of the year, (Light:Day= 14:10), in an air-conditioned environment of 25 C and 65% relative humidity within terraria over moistened soil. They were used for experiments within 0-2 days of captivity. The measurement of locomolor activity The measurements of organismal activity required apparatus especially designed for individuals with some unique characteristics lightness in weight (less than 0.02 g), ability to climb over a variety of surfaces, and distribution of weight over a relatively large area due to the long, widely-spread legs. Also, it was necessary to record continuously over long periods of time. These requirements were fulfilled by constructing an activity platform of plexiglass and cheesecloth, which was connected to a linear pressure-displacement transducer, which in turn was connected to an E & M six-channel physiograph recorder (Fowler and Goodnight, 1965). The animals were not fed during the 24-hour run, but a water source was available. The activity apparatus was held in the same air-conditioned environment, with light and dark cycle as previously described. Eight animals (four of each "sex) were placed within the activity chamber and used for no longer than 24 hours, since they exhibited stress behavior after that period of time. The runs were all begun and terminated in the late afternoon and evening hours, but the actual initial timing of the trials was varied deliberately in order to eliminate a possible source of error due to the animal's reaction to the apparatus. The Measurement of 5-HT The presence of 5-hydroxytryptamine in the opilionid was verified, using the technique of fluorometric analysis (Vanable, 1963). A quantitative measurement was established by the following procedure: The brain and gut from two animals were separated by microdissection and pooled on Whatman #1 filter paper. Solutions of 5-HT were placed alone and in combination with the two kinds of tissue. The solutions of 5-HT were alternated with the tissues and thus served as standards for establishment of a range of 5-HT in the photofluorometer and for localization of the movement of the compound on paper after solvent separation. A combination of butanol:acetic acid:water was used to separate 5-HT from the tissues over a 4-hour period. The compound was eluted from the paper and the fluorescent complex with ninhydrin was formed in solution. The compound was excited at 380 m/t and the fluorescence was transmitted at 515 m/x, using Corning filters, 3-71 and The percentage amounts of 5-HT, as compared to standard units, were converted to /JL/XM. (This method allows a measurement of 5-HT in the range of 12 ^JVI/ml.) In a study of a 24-hour secretion pattern, six animals were taken every four hours for 24 consecutive hours. They were killed simultaneously and quickly and the brains and guts from two individuals were pooled for a sample. Each sample was run in triplicate. The same procedures were used for a measurement of 5-HT in cultured tissue. The brains and guts were dissected and placed in culture media in Falcon tissue culture flasks. The flasks were held in an atmosphere saturated with COj and water within transparent desiccators, and the desiccators were placed in the. same environment as were the intact animals. For separation of 5-HT from these tissues, the cultured masses were removed from the flasks and treated by the same procedures as the freshly-dissected tissues. RESULTS All data on activity are shown in Tables 1-3 and in Figures 1-3. There is an interesting parallel between locomotor activity and the secretion of 5-HT. Each has a unimodal peak that occurs in the middle of the night and each shows minimal activity during daylight hours. Every two hours, the average locomotor activity of eight opilionids was recorded for a 24-hour period. The data shown in

3 CYCLIC PRODUCTION OF 5-HYDROXYTRYPTAMINE 189 TABLE 1. Data on locomolor activity for the opilionid. [Expressed as the means of the average number of minutes of activity (8 opilionids) per two hours.] Time (2400 cycle) Means Since these are pooled and averaged values that were computed because the assumption of homogeneity of variance was accepted, there is a single standard error for all: S.E. = Table 1 and Figure 1 are pooled data from five 24-hour trials. A homogeneity of variances was shown between days, and a test of interaction of time and days by analysis of variances was not significant. An attempt was made to fit a mathematical probability curve to these data, and a quadratic equation was found to account for 75% of all the data. As has been reported elsewhere (Schmitt, 1960), a harmonic equation may be more- appropriate for describing a biological function in a time sequence, but the time sequence was not long enough in this study to investigate that possibility. The amount of 5-HT produced in gut and brain tissues over a 24-hour period was expressed in ju.ju.m and is shown in Table 2 and Figure 2. Since variations were found between individuals, particularly at peak periods, and a test of homogeneity was unacceptable, the data for all individuals per time period were summed. The data in Table 2 and Figure 2 represent a total of TABLE 2. Sums of 5-HT from all individual tissues. (Measured as in vivo secretion, expressed in Time Days The individual high value at 0200 was approximately 150 n/im of 5-HT per 2 brains and 2 guts. Each sample represents a pooling of six brains and six guts. six individuals per time period per 24 hours. The data for individuals could not be pooled or averaged without a homogeneity of variances, because the probability of an error in rejecting the hypothesis, when it should be accepted, would be questionable for the tests on the means in an analysis of variance. The data in Table 2 and Figure 2 show the secretory activity of 96 individuals over four separate 24-hour periods. The in vitro data (Table 3 and Figure 3) showed a bimodal pattern of secretion of 5-HT over a 24-hour period. They were treated mathematically like the in vivo data. 40,30 ~ /o V 18 TIME IN : : 22 HOURS f 02 FIG. 1. Daily rhythmicity of activity of the opilionid, Leiobunum longipes, plotted over a quadratic curve. The activity exhibits the unimodal pattern, with an activity plateau between 10:00 PM and 2:00 AM. The stippled area indicates the night-time period of the 24-hour cycle. O O Observed data. X X The calculated polynomial curve derived from the observed data. The "closeness of fit" of opilionid activity to a polynomial equation is shown. As can be seen, the deviation of observed activity data from this curve is quite large at some points. DISCUSSION Generalized Movement The laboratory measurement of locomotor activity confirmed the report of different field investigators that the greatest activity of the opilionid is at night (Todd, 1949). The increase in activity was seen to begin before dark, about 6:00 PM in a midwestern, midsummer's evening, and to climb rapidly to a plateau by 10:00 PM

4 190 DONA J. FOWI.ER AND CLARENCE J. GOODNIGHT which lasted until 2:00 AM. After this time, total activity dropped off to almost zero before dawn. The pattern appeared to be strictly unimodal. Some investigators have thought that the beginning of activity was correlated with the rise in evening humidity as well as the decrease in light intensity. However, there was no variation of the light or humidity in the laboratory, and still the movement pattern began an hour or two before the lights went off, at the same time it would have occurred in the natural habitat , TIME FIG. 2. Daily rhythmicity of the secretion of 5-HT in the opilionid, Leiobunum longipes, in vivo. The data, summed for individuals over four different days (1-4), are shown graphically. It can be seen that there is variation between days as to the slopes of the curves and the magnitude of the peaks. However, it can also be seen that the rise in secretory activity is unimodal throughout. No investigation was made of possible "Zeitgebers" of movements. Some preliminary investigations suggested that darkness had very little effect upon the rhythm of these nocturnal animals while continuous light presented a significant stress, which may even be lethal. However, these results need further verification. Neurosecretion The abrupt behavioral change from the diurnal, lethargic, resting animal to the aggressive, very active, nocturnal animal must IN HOURS beginning in the early evening (1800) and.peaking in the middle of the night (0200), after which it drops sharply to a daytime low at dawn. This is a characteristic pattern for each day tested. A, B, C, and D are data for Days 1, 2, 3, and 4, respectively. The stippled areas indicate the night-time period of the 24-hour cycle. i 10

5 CYCLIC PRODUCTION OF 5-HYDROXYTRYPTAMINE TIME 2 IN FIG. 3. Daily rhythmicity o the secretion of 5-HT in tissues of the opilionid, Leiobunum longipes, in vitro. The secretion pattern in vitro (A) is rhythmic and has its greatest peak at 0200 as does the secretion pattern in vivo (B). However, the overall pattern in 24 hours is bimodal in character, with a second peak at 1000 (midmorning) at a time when the pattern in vivo is at its lowest point. The curve shown in B is representative o all data obtained in vivo and is used for comparative purposes with the in vitro data. The stippled areas indicate the nighttime period of the 24-hour cycle. involve a whole series of neurophysiological events. The content of 5-hydroxytryptamine in intact animals over a 24-hour period followed the same unimodal pattern as the activity, with a peak at 2:00 AM. Although there was a variation in the slope and amplitude of the peaks between individuals, the overall pattern between days was quite similar. The rise in production of 5-HT began before dark, around 6:00 PM (1800), as did locomotion. However, there was a single sharp peak of production of 5-HT as opposed to the plateau in the peak of locomotor activity (Figures 1 and 2). The decline in amount of 5-HT is sharp from that time to the daytime low value that occurs before dawn. - Biological cyclic changes have been defined as regularly-occurring quantitative or qualitative successive changes in state, with the distinctive feature that the sequence of occurrence rather than the duration tends to be constant (Cloudsley-Thompson, 1960). This is an adequate description of the production of 5-HT between sampling days. The sequence of occurrence remained constant while the quantitative measurement of duration was variable. Therefore, a pattern could be described or predicted very well, even though a mathematical measurement of change could not be developed. 5-Hydroxytryptamine has been found in nearly all plants and animals in which it has been sought. In animals it appears to be a neuro-secretory compound. Its cyclic production in mammals was described by Quay, 1964, and Synder, et al., 1964, who used the laboratory white rat, which is subject only to man-made environmental photoperiods. The white rat had a higher amount of 5-HT during lighted periods, a time when this nocturnal animal was not active. In a later study of a pigeon (Quay, 1965) the amount of 5-HT was found to increase during the daylight hours, when the activity of the animal was highest. The measurements were made on tissues which have a nervous connection to the eye. The invertebrate, the opilionid, showed a higher production of 5-HT during the dark period, which corresponded to the time of greatest activity of the animal. Since both brain and gut tissues were used in the determination of the amount of 5- HT, the data would represent a broader or more complete sampling of individual production of the compound than would TABLE 3. Sums of 5-HT from all individual tissues, in vitro for approximately SO days. Time Amount Total A total of 72 brains and guts were removed from Falcon tissue culture flasks during a 24-hour period, after 80 days in culture. Each sample represents a pooling of two brains and two guts. The total values are the sums of 5-HT (/i/tm) found in 6 brains and 6 guts at a single time period.

6 192 DONA J. FOWLER AND CLARENCE J. GOODNIGHT the measurement of 5-HT from a single tissue of a larger animal. The similarity of the cyclic secretion pattern to the cyclic activity pattern suggests that heightened motor activity in these forms could be a feedback factor to control production of 5-HT. Such a possibility was examined by the development of tissue culture techniques for opilionid tissues, so that organismal feedback controls would be removed (Fowler and Goodnight, 1965). Fortunately, these tissues were relatively easy to maintain over extended periods. They were held in the same photoperiod as the intact animals. Even after 80 days the isolated tissues exhibited a cyclic production of 5-HT which had one of the two peaks at the time of the single peak in the tissues from the intact opilionid (Figure 3). The second peak occurred at a time of low organismal production of 5-HT, which suggested that feedback controls in intact animals might be inhibiting an endogenous bimodal pattern of secretion in the productive tissues. The indication of an intrinsic rhythmic output of 5-HT by certain tissues is under further investigation. Confirmation of a cyclic production of 5-HT in vitro would aid the biologist in his search for an understanding of the nature of the biological clock. The presence of the highly versatile compound, 5-HT, in the opilionid is of interest to the investigator of environmental physiological responses. Vertebrate investigators have suggested that 5-HT may be involved in neural transmission, in the awake center of the brain, and in the control of migration of pigment. Further investigations of the metabolic activities of 5-HT in the invertebrates as well as the vertebrates will provide the biologist with more information concerning the physiological adjustments of the individual to its environment. SUMMARY 1. An activity platform designed to measure the locomotor movements of opilionids, Leiobunum longipes, established the nocturnalism of the species. The activity followed a unimodal pattern, with a gradual increase before dark to a plateau level by 10:00 PM. After 2:00 AM, the activity declined rapidly to a daytime low that stabilized before dawn. 2. The presence of 5-hydroxytryptamine in the opilionid was established by use of the fluorometric method for indoles. Measurement of the secretion for 24 hours revealed a unimodal output of 5-HT which corresponded to the locomotor activity pattern. Thus, production of 5-HT rose before dark and reached a peak value at 2:00 AM, after which it dropped off sharply to the daytime low value that occurred before dawn. 3. A method for culturing.opilionid tissues was developed and the ability of isolated tissues to secrete 5-HT was confirmed. These cultured tissues produced 5-HT in a bimodal cycle, with the time of the highest amount of secretion coinciding with the nocturnal peak in intact animals. 4. These results were discussed in relationship to other data concerning the circadian output of 5-HT. REFERENCES Cloudsley-Thompson, J Adaptive functions of circadian rhythms. Cold Spring Harbor Symposium on Quantitative Biol. 25: Erspamer, V Hydroxytryptamine, p In U. S. von Euler and H. Heller, [ed.]. Comparative endocrinology, vol. 2. Academic Press, New York. Fowler, D. J., and C. J. Goodnight An apparatus for recording the activity of opilionids. Ann. Entomol. Soc. Am. 58: Fowler, D. J., and C. J. Goodnight. The maintenance of adult opilionid tissues in vitro. Trans. Am. Microscop. Soc. (In press) Grobman, A., and H. A. Bern A textbook of comparative endocrinology. John Wiley and Sons, Inc., New York and London. 468 pp. Quay, W. B Circadian and estrous rhythms in pineal melatonin and 5-hydroxyindole-3-acetic acid. Proc. Soc. Exptl. Biol. Med. 115: Quay, W. B Daily rhythms and photic responses of pineal hydroxyindole derivatives in the pigeon (Columba livia). (Abstr.) Am. Zoologist 5:218. Schmitt, O. H Biophysical and mathematical models of circadian rhythms. Cold Spring Harbor Symposium on Quantitative Biol. 25: Synder, S. H., J. Axelrod, J. Fischer, and R. Wurt-

7 CYCLIC PRODUCTION OF 5-HYDROXYTRYPTAMINE 193 man Neural and photic regulation o 5- special reference to those of the Oxford district. HTP carboxylase in the rat pineal gland. Nature J. Animal Ecol. 18: : Variable, J. W., Jr A ninhydrin reaction giv- Todd, V The habits and ecology of the ing a sensitive quantitative fluorescence assay for British harvestmen (Arachnida, Opiliones) with 5-HT. Anal. Biochem. 6: