Animals

Adults and nymphs of the cricket Gryllus bimaculatus were used. They were obtained from a laboratory colony maintained at 25°C in a 12 hr light and 12 hr dark cycle (LD12:12, L: 0600–1800 Japanese Standard Time). They were fed laboratory chow and water via damped absorbent cotton.

Activity recording and analysis

Animals were kept individually in an activity chamber of a transparent plastic box (180 × 90 × 50 mm) with a rocking substratum. Before housing the animal into the chamber, either of the forewings was removed to prevent any sound communication between individuals. The rocking movement of the substratum caused by a moving insect was sensed by a magnetic reed switch placed on the bottom of the box. A signal from the switch was collected by a computer (NEC, PC-9801NS/A), which summed the signal every 6 min. The total count of every 6 min was stored on a floppy diskette. The activity chambers were placed in an environment controlled room in which the temperature was kept constant at 25 ±1°C. Light and dark cycles were given by a 20W cool white fluorescent lamp controlled by an electric timer. The light intensity within actographs were about 400 lux at the animal's level.

At the end of an experiment, the raw data from individual cricket were double plotted in conventional manner to facilitate the visual inspection of the rhythmicity. Daily activity profiles were obtained by averaging the activity for 7 to 20 days. Data for the first 5 days after the imaginal molt were included for nymphal activity since the nymphal activity pattern persisted until this period.

Measurements of biogenic amines

The concentrations of 5-HT and its derivatives in the brain were measured by means of high pressure liquid chromatography (HPLC; Eicom, EP-10) with an electrochemical detector (ECD; Eicom, ECD300). The brains were dissected quickly and placed in 50 μl of ice-cold 0.1M perchloric acid containing 100 ng/ml 3,4-dihydro-xybenzylamine (DHBA) as an internal standard. The sample was homogenized and centrifuged at 12,000 g for 30 min at 4°C. 10μl of supernatant was directly injected to the HPLC column using the autosampler (Toso, AS-8021). The mobile phase was composed of 0.18 M monochloroacetic acid, 0.16 M NaOH, 50 μM ethyl-enediaminetetraacetic acid (EDTA) disodium, with 1.85 mM sodium-1-octanesulfonic acid (SOS) as the ion pair reagent and 8.5% (v/v) acetonitrile as the organic modifier. The pH was adjusted to 3.6 by the addition of NaOH. The HPLC system consisted of a solvent delivery pump, an injection valve (Rheodyne), a C18 reversed-phase column (150 mm × 4.6 mm) 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 0.7V versus an Ag/AgCl reference electrode. Signals from the detector were recorded and integrated by computer (Waters, Data Station 805). Quantifications, which were based on the peak area of the chromatograms, were obtained by calculating the ratio of the peak area of the substances to the peak area of the internal standard. Concentrations were obtained by a comparison of ratios between the sample and standard chromatograms. The results were calculated in pmol/mg of brain wet weight.

Injection

Animals were fixed on a platform and anesthetized by a continuous flow of CO₂. The brain was exposed by cutting a small square piece of head capsule with a razor knife. 32.2 nl of 25 μM or 250 μM 5,7-DHT (Sigma) was injected into near center of the brain with a glass micropipette using an injector (WPI, A203XVY) mounted on a micromanipulator (Narishige, M-3333). 5,7-DHT was dissolved in insect Ringer's solution (Fielden, 1960) containing 1% ascorbic acid. The Ringer's solution was injected as a control.

Rhythm reversal from diurnal to nocturnal

The locomotor activity was assayed from the last (8th) instar to adult in 14 male crickets. A representative record is shown in Figure 1. Under LD12:12, the nymphal cricket was diurnally active with a peak in the middle of day. In this particular animal, 7 days after the imaginal molt, an activity bout suddenly appeared in the night, quickly advanced to synchronize to the lights-off in a few days. In contrast, the diurnal component gradually decreased; a nocturnal pattern was thus established, as previously reported (Tomioka and Chiba, 1982). Once the nocturnal pattern was established, it persisted throughout the adulthood. The rhythm reversal from diurnal to nocturnal was observed in all the crickets, with the establishment of nocturnality in 6.0±0.9 days after the imaginal molt (n=14).

Fig. 1

A: A representative double plotted actogram of a male cricket G. bimaculatus under a 12 hr light and 12 hr dark cycle. The cricket was diurnally active in nymphal stage and became nocturnal within 7 days after the imaginal molt. White and black bars indicate light (white) and dark (black) cycle. IM, imaginal molt. During the blank area indicated by a star, data were lost by a failure of recording system. B, C: Average daily activity profiles of locomotor activity in 14 male crickets. Plotted are means and SEM. Dotted areas indicate the dark phase.

Change in 5-HT level during the late post-embryonic development

5-HT levels in the brain during the rhythm reversal were examined in a total of 72 animals. Sampling was performed with 6th to 8th (last) instar nymphs, adults of day 0-9 and completely nocturnal adults that had undergone more than 10 days after the imaginal molt. Sampling was carried out at the middle of the light phase (12:00) and at least 5 animals were used for each point.

Figure 2 shows the concentration of 5-HT in the brain. 5-HT level in the brain was rather constant throughout the late nymphal stages and the mean value was 6.2 pmol/mg (WW). After the imaginal molt, however, it increased gradually until day 5 and then rapidly increased at day 6. Statistically significant increase was observed when compared values of day 0 with day 5 (t-test, p<0.05). In mature crickets, the concentration reached over 12.0 pmol/mg (WW). It was about 2 times that in nymphal brain and very close to the value previously reported by Nagao and Tanimura (1988).

Fig. 2

The change in 5-HT concentration in the cerebral lobe of the cricket G. bimaculatus during the late post-embryonic development. Samples were collected at 12:00 JST. Data from 5–6 animals were pooled. Vertical bars indicate standard deviation.

Effect of 5,7-DHT on the locomotor rhythm

We then examined the effects of 5-HT reduction on the rhythm reversal by injecting 5,7-DHT, a neurotoxine specific to the serotonergic neurons, into the brain. The chemical was dissolved in the Ringer's solution at a concentration of 25 μM or 250 μM. Four and 6 crickets at early 8th instar nymphal stage were injected with 32.2 nl 5,7-DHT of 25 μM and 250 μM, respectively. Same amount of Ringer's solution was injected in 5 crickets as control. Figure 3 shows representative activity records after the injection. All animals injected with Ringer's solution exhibited the normal rhythm reversal as exemplified in Figure 3A. The average daily activity pattern was very similar to that of intact animals both in nymphal and adult stages (Fig. 4A, B). In 5,7-DHT injected animals, however, considerable abnormality in their locomotor activity pattern was observed (Figs. 3B-D, 4C-F). In nymphs injected with 25 μM 5,7-DHT (Figs. 3B, 4C), slight but significant increase was induced in nighttime activity (t-test, P<0.01). Daytime activity often increased with a sharp lights-on peak, but the increase was not statistically significant (t-test, P>0.3). After the imaginal molt, the dense activity occurred in the latter half of the dark phase to early light phase with the development of nighttime activity (example: Fig. 3B), resulting in significant increase of the daytime activity compared with Ringer's solution injected controls (Fig. 4D, t-test, P<0.05). The daytime activity tended to decrease after two weeks of imaginal molt, whereas the heavy activity persisted throughout the adult life. Animals injected with 250 μM of 5,7-DHT reproduced these tendencies more clearly (example: Fig. 3C). The nocturnal activity, which started with an intense light-off peak, significantly increased even in the nymphal stage compared with Ringer's solution injected control animals (t-test, P<0.05, Fig. 4E), forming, in some cases, a rather nocturnal pattern (Fig. 3D). At lights-on a sharp peak occurred (Fig. 4E). In adults, activity level during the daytime was again significantly increased (t-test, P<0.05) because of the occurrence of the dense activity from the late night to early day (Figs. 3C, D and 4F). It appeared that lights-on peak still existed but was obscured by the gradual increase of the activity in the late night (Fig. 4F). The activity in the nighttime was not significantly affected (t-test, P>0.1). The maximal activity at lights-off occurred earlier in the 250 μM 5,7-DHT injected adults than those injected with Ringer's solution and with 25 μM 5,7-DHT. The 5-HT level in the brain injected with 250 μM 5,7-DHT was 8.5±1.4 (SD) pmol/mg (n=5), being significantly lower than the control value of 12.1±1.0 (SD) pmol/mg (n=5, t-test, p<0.001).

Fig. 3

Four double plotted actograms of crickets injected with either 32.2 nl of Ringer's solution (A), 25 μM (B) or 250 μM 5,7-DHT (C, D). In A, data were missed during days 17–19 because of the failure of recording system. Note that heavy activity from late night to early day occurred in the 5,7-DHT injected animals. For further explanations see text and Fig. 1.

Fig. 4

Average daily activity profiles of Ringer's solution injected and 5,7-DHT injected nymphal and adult crickets. Dotted areas indicate the dark phase. Vertical bars indicate SEM. For further explanations see text.