Animals

All experiments were performed with intact, adult male crickets, Gryllus bimaculatus, obtained from our laboratory colonies, which were maintained at 25 ± 0.5°C in a 12 hr light and 12 hr dark cycle (LD12:12; light: 06:00–18:00, Japanese Standard Time, JST). To prevent any interindividual sound communication, one forewing was removed. One optic lobe of the crickets was also removed just before the activity recording was started, because, in crickets, one optic lobe is enough to create the rhythm (Okada et al., 1991). The method for the surgery has been described elsewhere (Tomioka and Chiba, 1989).

Activity recording

The animals were kept individually in an activity chamber with rocking substratum whose movement caused by a moving animal was sensed by a magnetic reed-switch connected to a computer for data storage. The signals of the movement were summed every 6 min. Food and water were available ad libitum and the temperature was kept constant at 25 ± 0.5°C. Light dark cycles and constant light were given by a 15 W cool white fluorescent lamp controlled by an electric timer. The light intensity at the chamber was about 250 lux.

Injection

Male crickets with one optic lobe removed and with a clear nocturnal rhythm were fixed on a platform and anesthetized by a continuous flow of CO₂ to prevent high efflux of hemolymph. With a fine razor knife, a small window was cut in the head capsule to expose the optic lobe. 32.2 nl of 5,7-DHT (creatinin-sulfate salt, Sigma) solution or insect Ringer's solution (Fielden, 1960) was then injected into the medulla of the optic lobe with a glass micropipette, broken to a tip diameter of 5–10 μm, using a micromanipulator (Narishige, M-3333) and a nanoliter injector (WPI, A203XVY). The concentration of 5,7-DHT was 0.5 mM and dissolved in Ringer's solution containing 0.1% ascorbic acid. The injection was performed between 7:00 and 9:00 (JST).

Data analysis

Locomotor records of individuals were double plotted in a conventional manner by computer with a resolution of 6 min. The freerunning period of individuals was determined by the chi-square periodogram (Sokolove and Bushell, 1978). Values are expressed as mean ± SEM.

Measurements of biogenic amines

By means of high pressure liquid chromatography (HPLC; Eicom, EP-10) with electrochemical detection (ECD; Eicom, ECD 300) the levels of serotonin, dopamine and their metabolites (Nacetyldopamine, N-acetylserotonin, 5-hydroxyindole-3-acetic acid: 5-HIAA), in a single optic lobe were measured at the end of experiments. The injected optic lobes were dissected quickly and placed in 30 μl of ice-cold 0.1 M perchloric acid containing 100 ng/ml 3,4-dihydroxybenzylamide (DHBA) as an internal standard. The sample was homogenized and centrifuged at 12,000 × g for 30 min at 4°C. The supernatant was directly injected to the HPLC column or stored at −80°C until use. All chemicals were obtained from Sigma Chemical Co (St. Louis, Mo, USA). Standard solution of serotonin sulfate complex and other biogenic amines as well as the mobile phase were made as described in Nagao and Tanimura (1988). In brief, the mobile phase contained 0.18 M monochloroacetic acid, 0.16 M NaOH, 50 μM ethylenediaminetetraacetic acid (EDTA) disodium, with 1.85 mM sodium-1-octanesulfonic acid (SOS) as the ion pair reagent and 8.5% (v/v) CH₃CN as the organic modifier. The pH was adjusted to 3.6 by an addition of NaOH. The HPLC system consisted of a solvent delivery pump, an injection valve (Rheodyne), a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm average particle size) placed in a column oven (Sugai, U-620). An electrochemical detector with a carbon graphite electrode (Eicom, WE-3G) was used. The detector potential was set at 0.7 V versus an Ag/AgCl reference electrode. Signals from the electrochemical detector were recorded and integrated by computer (Waters, Data station 805). Comparing with standards the concentration of biogenic amines in the optic lobes were measured for 5,7-DHT injected animals and Ringer's-solution injected animals. The results were calculated in pmol/optic lobes (mean ± SD).

Statistical analysis

The difference between mean values was statistically analyzed with two-tailed Student's t-test or by χ²-analysis. Significance was considered at P < 0.05.

Period under DD and LL

The locomotor activity of 12 5,7-DHT injected and 11 Ringer's injected animals was recorded. They were held in LD12:12 for the first three or four days, then transferred to constant darkness (DD) and subsequently to LL at 6:00 on day 17 or 18. Figure 1 shows representative activity records for Ringer's injected (Fig. 1A) and 5,7-DHT injected (Fig. 1B) animals. Under DD, the freerunning period of 5,7-DHT injected animals was 23.55 ± 0.11 hr, which was slightly shorter, but not significantly different from that of Ringer's-injected animals (23.79 ± 0.21 hr). Under LL, 80% of 5,7-DHT injected animals showed arrhythmicity: the ratio was significantly greater as compared with the control, Ringer's injected animals (18%, P < 0.01, χ²-test). In 5,7-DHT injected animals, the duration of initial intense nocturnal activity became longer in a few days after the injection (Fig. 1B); The average duration of the first nocturnal bout was 4.05 ± 0.35 hr which was significantly longer than that of the control animals (1.63 ± 0.07 hr; P < 0.001, t-test).

Fig. 1

Locomotor rhythms of crickets injected with Ringer's-solution (A) or 5,7-DHT (B) on the first day and transferred to constant darkness (DD) and subsequently to constant light (LL). Under DD both animals freeran with a period shorter than 24 hr. Under LL the Ringer's injected animal (A) freeran with a period longer than 24 hr, while the 5,7-DHT injected animal (B) showed no clear rhythm. Black and white bars indicate light (white) and dark (black) portions, respectively.

Resychronization and masking effect

The resynchronizability to a 6 hr shifted LD was examined in 21 animals injected with 5,7-DHT and 16 animals with Ringer's-solution. Three days after the injection, the LD cycle was phase advanced by 6 hr (lights-on 0:00, lights-off 12:00), by shortening the dark phase. Figure 2 shows representative activity records for a Ringer's injected animal (Fig. 2A) and a 5,7-DHT injected animal (Fig. 2B). After the 6 hr advance shift, the Ringer's solution injected animals needed 3.17 ± 0.15 days until resynchronization to the new LD cycle. The animals injected with 5,7-DHT, however, needed 6.55 ± 0.33 days and the entrainment was not so robust in comparison with the control animals. The difference in numbers of transient cycles was statistically significant (P < 0.001, t-test). Two 5,7-DHT injected animals needed more than 12 days for the reentrainment to the new cycle.

Fig. 2

Locomotor rhythms of crickets injected with Ringer's solution (A) or 5,7-DHT (B) on the day indicate by x. Three days after the injection, indicated by PS the animals were transferred to a 6 hr advanced LD cycle. The Ringer's injected animal (A) needed 3 days until entrained to the new cycle, while the 5,7-DHT injected animal (B) needed 7 days. The lights-on induced masking effect was more prominent in the 5,7-DHT injected animal. For further explanations see Fig. 1 and text.

During the transient cycles the masking effect was clearly induced at lights-on, where the late subjective night phase was exposed to light. The light induced masking effect was significantly greater in the 5,7-DHT injected animals: The duration of masking activity in the injected animals were significantly longer than that of Ringer's injected controls (P < 0.001, t-test) from the first day of phase-shift until the fourth day when the phase shifts almost completed in the control animals (Fig. 3). In the steady state entrainment to the shifted LD, the initial bout of nocturnal activity persisted for 1.29 ± 0.3 hr in the Ringer's solution injected animals, whereas in the 5,7-DHT injected animals it continued for 3.34 ± 0.21 hr, being significantly longer (P < 0.01, t-test).

Fig. 3

The daily duration (mean ± SD) of the light induced activity (masking effect) after the 6 hr phase advance for Ringer's injected crickets (white columns) and for 5,7-DHT injected crickets (black columns). n = 16–21; ***, P < 0.001.

Biogenic amine concentration

The biogenic amine concentration in the optic lobes of 5,7-DHT injected and Ringer's-injected animals was significantly different only for serotonin. As shown in Fig. 4, the concentration of serotonin was 2.85 ± 0.88 pmol/optic lobe in the 5,7-DHT injected animals, which was significantly lower than that of Ringer's injected animals (6.43 ± 1.07 pmol/optic lobe; P < 0.001, t-test). The concentrations of N-acetyldopamine, dopamine, 5-HIAA, N-acetylserotonin were similar in both groups.

Fig. 4

The concentration (mean ± SD) of biogenic amines measured by HPLC-ECD in the optic lobes of Ringer's solution injected animals (white columns) and 5,7-DHT injected animals (black columns). NADA, N-acetyldopamine; DA, dopamine; 5-HIAA, 5-hydroxyindole-3-acetic acid; NA-5HT, N-acetylserotonin; 5-HT, 5-hydroxytryptamine, serotonin. n = 14-18; ***, P < 0.001.

Selective reduction of serotonin

Animals with 5,7-DHT injected into the optic lobe showed the locomotor rhythm clearly different from that of Ringer's injected animals. Direct injection of the chemical into the medulla region of cricket's optic lobe resulted in significant reduction of serotonin level in the tissue without apparent damage to the dopaminergic system. The serotonin level was reduced to 50% of that in the Ringer's injected animals when examined 4 weeks after the injection. Preliminary results showed that it was even reduced to nearly 20% of that of the control 2 days after the injection (Germ, unpublished data). However, the content of serotonin in the brain did not change significantly after the injection of 5,7-DHT into the optic lobe (Germ, unpublished data). Since the content of dopamine as well as metabolites of dopamine and serotonin were at the similar level, the change in locomotor rhythms would be attributable to the reduced level of serotonin in the optic lobe, although we should carefully examine other biogenic amines in the future study.

Role of serotonin in the circadian system

The fact that the freerunning period under DD is not significantly different between 5,7-DHT injected animals and Ringer's injected controls suggests that serotonin is not a necessary component for the generation of circadian rhythm. However, it seems to have important roles in the circadian system. The present study revealed that reduction of serotonin in the optic lobe resulted in 4 major effects: the longer transient cycles for resynchronization, the greater masking effects of light, the longer duration of the initial bout of nocturnal activity, and frequent arrhythmicity in LL.

The 5,7-DHT injected animals showed light-induced masking effects significantly longer than the control animals. This intensified masking effect may be attributable to the reduced level of serotonin in the optic lobe. There are lines of evidence showing that serotonin is a neuromodulator in the insect visual system (Nässel, 1988). The administration of serotonin reportedly reduces the sensitivity of visual interneurons not only in the cricket (Tomioka et al., 1993) but also in the honey bee (Kloppenburg and Erber, 1995). It is thus likely that the reduced level of serotonin releases the visual inter-neurons from serotonergic inhibition to cause a higher sensitivity to light which would eventually result in the higher light-induced masking effect. The higher percentage of arrhythmicity in 5,7-DHT injected animals under LL also suggests the increased sensitivity of the visual system, since the cricket often became arrhythmic when the illumination level of LL was increased (Tomioka and Chiba, 1982).

The 5,7-DHT injected animals showed the longer initial bout of nocturnal activity both under LD and DD (Figs. 1, 2). In the cricket, the center for generating the locomotor activity is thought to reside in the central brain and it produces the rhythm under the regulation of the optic lobe pacemaker (Tomioka and Chiba, 1989). Since the 5,7-DHT injection seems to affect the serotonin content only in the optic lobe, it is thus likely that the prolonged first nocturnal activity bout is caused by the increased output from the optic lobe pacemaker. This hypothesis is also supported by the following facts. First, when serotonin is injected into the haemoceal, the activity of the injected animal is substantially reduced (Okada and Tomioka, unpublished observation). Second, the application of exogenous serotonin to the optic lobe compound eye system in vitro reduces the neural activity in the optic stalk (Tomioka, 1994). Since similar relationship between the serotonin level and the locomotor activity has also been reported for 5,7-DHT injected hamsters (Morin and Blanchard, 1991), the role of serotonergic system in regulation of locomotor activity may be common among insects and vertebrates.

One of our most significant findings is that the 5,7-DHT injected animals needed significantly longer transient cycles until their locomotor rhythms were entrained to a 6 hr advanced LD. The longer transients seem to contradict to our hypothesis that the reduced level of serotonin increases the sensitivity of photoreceptive system. One possibe explanation for this is that serotonin may act more directly on the circadian pacemaker through a pathway distinct from the one for photic entrainment. There are numbers of reports showing that the serotonergic system is a phase shifting agent inducing a phase response curve different from the one for light in the marine gastropod Aplysia californica (Corrent and Eskin, 1982), in the cockroach Leucophaea maderae (Page, 1987), in the cricket Gryllus bimaculatus (Tomioka, 1994) and in the rat (Prosser et al., 1993).

Masking activity may also contribute to the longer transient cycles. It is reported in mammals that the forced activity often causes the phase shifts in a phase dependent manner and that it is the morning phase during which the phase delay is induced (Reebs and Mrosovsky, 1989). Although there is no report available for the feedback of activity to the pace-making system in insects, the fact that, in 5,7-DHT injected animals, the longer masking effects of lights-on always accompanied the longer transient cycles may be also interpreted by the phase-delay or deceleration of the circadian rhythm. The serotonergic system may reduces the sensitivity of the photoreceptive system to suppress the light-induced untimely activity and facilitate the reentrainment.