Optic lobe circadian pacemaker sends its information to the contralateral optic lobe in the cricket Gryllus bimaculatus

The bilaterally paired optic lobe pacemakers of the cricket Gryllus bimaculatus are mutually coupled. In the present study we recorded the neural activity conveyed from the brain toward the optic lobe with a suction electrode to examine the coupling signals. The results demonstrated that the brain efferents to the optic lobe encode the circadian information: Both in constant light (LL) and constant darkness (DD), the neural activity of brain efferents showed a clear circadian rhythm with a nocturnal peak. Since the rhythm survived the severance of the contralateral optic nerve but disappeared when the contralateral optic lobe was removed, it is apparent that the rhythm originates from the contralateral optic lobe. The amplitude of the rhythm was greater in LL than in DD, suggesting that light affects the amplitude of the rhythm. This was confirmed by the fact that the light-induced response was under circadian control, being greater during the subjective night. These data suggest that the bilaterally paired optic lobe pacemakers exchange circadian information as well as light information. The data are also consistent with the results of previous behavioral experiment.Abbreviations DD constant darkness - LD light dark cycle - LL constant light     

Mutual interactions between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between the bilaterally paired optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording locomotor activity, under constant light or constant red light, after the optic nerve was unilaterally severed.

Analysis of coupling between optic lobe circadian pacemakers in the cricket Gryllus bimaculatus

The coupling mechanism between weakly coupled two optic lobe circadian pacemakers in the cricket Gryllus bimaculatus was investigated by recording the locomotor activity, under light-dark cycles with various lengths, after the optic nerve was unilaterally severed. The activity rhythm split into two components under the light cycles different from 24 h: one was readily entrained to the light cycle and the other only loosely entrained or freeran. Additional removal of the optic lobe on the intact side resulted in a loss of the entrained component and that on the blinded side caused the reverse effect, indicating that the entrained component was driven by the pacemaker on the intact side and the other by the one on the blinded side. The synchronization between the two components was achieved only in light cycles with a limited length between 23 and 25 h. Without this range, the desynchronization of the components occurred. In the split rhythm, the phase-dependent modulation of the period of freerunning component and the mutual suppression of locomotor activity during the subjective day phase were clearly observed. The suppression was also evident in the lights-on peak that was the masking effect of light. The light cycle with dim light significantly reduced the ratio of animals with the pacemaker coupling as well as the magnitude of the period modulation. These results suggest (1) that the mutual coupling is achieved only when the difference in the periods between the two pacemakers is within an allowable range, (2) that the photic information is also involved in the mechanism of mutual coupling, and (3) that the suppression of activity occurs at the regulatory center for locomotion.Abbreviations CT circadian time - DD constant darkness - LL constant light - LD light to dark cycle - T length of light to dark cycle - freerunning period     

Distribution of circadian photoreceptors in the compound eye of the cricket Gryllus bimaculatus

Adult male crickets (Gryllus bimaculatus) show a nocturnal circadian locomotor rhythm, which is driven by the pacemaker in the optic lamina-medulla complex and synchronizes to the light-dark (LD) cycle received by the compound eye. To see whether there was any specially differentiated circadian photoreceptor area in the eye, we examined the effect of a partial reduction of various areas of the compound eye, in addition to a removal of the contralateral optic lamina-medulla-compound eye complex, on entrainability of the locomotor rhythm. All operated animals showed a response to the LD cycle in their locomotor rhythm, no matter which area of the eye was left intact: They either stably entrained to an LD cycle or showed a sign of weak entrainment. The capacity for stable entrainment was still retained when only 262 ommatidia were left. Transient cycles needed for re-entrainment, following a 6-hr phase advance of the LD cycle, were measured in 20 reduced-eye animals showing clear stable entrainment. They were in inverse proportion to the number of ommatidia in the reduced eye: The fewer ommatidia there were, the more transient cycles were observed (r = -0.76, p less than 0.001). These results suggest that almost the whole area of the compound eye may contain circadian photoreceptors, and that the photic information from each ommatidium may additively affect the circadian clock to entrain via neural integration mechanisms.     

双斑蟋复眼和视叶的显微结构

【目的】为探索昆虫视觉信号处理的重要神经结构,详细观察和描述了直翅目(Orthoptera)蟋蟀科(Gryllidae)代表性昆虫双斑蟋Gryllus bimaculatus De Geer复眼和视叶的组织学结构特征。【方法】利用扫描电镜技术和组织学切片技术,观察分析了30只双斑蟋的复眼和视叶组织学结构。【结果】双斑蟋复眼约有3400个小眼,均为六边形结构,小眼间隙内分布有机械感受器——感觉毛和钟形感受器。每个小眼均由角膜、晶锥、感杆束、6个网膜细胞及基膜等构成。视叶呈两个扇形结构,由三大神经纤维网构成,分别为神经节层、外髓、内髓。【结论】双斑蟋复眼表面具有少量感觉毛和钟形感受器,每个小眼均由角膜、晶锥、感杆束、6个网膜细胞及基膜等构成,属并列像眼,视叶由三大神经纤维网构成。     

Postembryonic changes in circadian photo-responsiveness rhythms of optic lobe interneurons in the cricket Gryllus bimaculatus

The photo-responsiveness of 2 groups of interneurons responding to light in the protocerebrum was investigated at 2 developmental stages, the last instar nymphs and adults, in the cricket Gryllus bimaculatus. The cricket is diurnally active during the nymphal stage but becomes nocturnal as an adult. In both adults and nymphs, light-induced responses of optic lobe light-responding interneurons that conduct light information from the optic medulla to the lobula and the cerebral lobe showed a circadian rhythm peaking during the subjective night. Amplitudes of the rhythms were not significantly different between adults and nymphs, but adults showed more stable day and night states than did nymphs. The medulla bilateral neurons that interconnect the bilateral medulla areas of the optic lobe also showed circadian rhythms in their light-induced responses in both adults and nymphs. The rhythm had a clear peak and a trough in adults, and its amplitude was significantly greater than that of nymphs. These results suggest that the 2 classes of interneurons are differentially controlled by the circadian clock. The difference might be related to their functional roles in the animal's circadian behavioral organization.     

Characterization of an optic lobe circadian pacemaker by in situ and in vitro recording of neural activity in the cricket,Gryllus bimaculatus

Summary The nature of the circadian rhythms of the optic lamina-medulla compound eye complex was examined in male crickets Gryllus bimaculatus by recording the multiple unit activity from the optic lobe in situ and in vitro. In most in situ preparations, the neural activity of the complex was higher during the subjective night than during the subjective day, both under constant light and dark. The same pattern was also obtained from nymphal crickets, suggesting that the properties of the pacemaker are common to both nymphs and adults. In a few cases, both diurnal and nocturnal increments in the activity were simultaneously observed, indicating there are two neuronal groups conveying different circadian information. The circadian rhythm was also demonstrated in the optic lobes in vitro, providing evidence that the optic lobe contains the circadian pacemaker that is capable of generating the rhythmicity without any neural or humoral factors from the rest of the animal.Abbreviations DD constant darkness - JST Japanese standard time - LD light to dark cycle - LL constant light     

Protein synthesis is a required process for the optic lobe circadian clock in the cricket Gryllus bimaculatus

The effects of a translation inhibitor, cycloheximide (CHX), on the circadian neuronal activity rhythm of the optic lamina-medulla compound eye complex cultured in vitro were investigated in the cricket Gryllus bimaculatus. When the complex was treated with 10(-5) M CHX for 6 h, the rhythm exhibited a marked phase shift. The magnitude and direction of the phase shift were dependent on the phase at which the complex was treated with CHX; phase delays occurred during the late subjective day to early subjective night, whereas phase advances occurred around the late subjective night. Continuous application of CHX abolished circadian rhythms of both the spontaneous neuronal activity and the visually evoked response. However, it abolished neither the spontaneous activity nor the visually evoked response. As washed with fresh medium after CHX treatment, the rhythm soon reappeared and the subsequent phase was clearly correlated to the termination time of the treatment. These results suggest that protein synthesis is also involved in the cricket optic lobe circadian clock, and that the clock-related protein synthesis may be active during the late subjective day to subjective night.     

Characterization of PDF-immunoreactive neurons in the optic lobe and cerebral lobe of the cricket, Gryllus bimaculatus

Pigment-dispersing factor (PDF) is a neuropeptide playing important roles in insect circadian systems. In this study, we morphologically and physiologically characterized PDF-immunoreactive neurons in the optic lobe and the brain of the cricket Gryllus bimaculatus. PDF-immunoreactivity was detected in cells located in the proximal medulla (PDFMe cells) and those in the dorsal and ventral regions of the outer chiasma (PDFLa cells). The PDFMe cells had varicose processes spread over the frontal surface of the medulla and the PDFLa cells had varicose mesh-like innervations in almost whole lamina, suggesting their modulatory role in the optic lobe. Some of PDFMe cells had a hairpin-shaped axonal process running toward the lamina then turning back to project into the brain where they terminated at various protocerebral areas. The PDFMe cells had a low frequency spontaneous spike activity that was higher during the night and was often slightly increased by light pulses. Six pairs of PDF-immunoreactive neurons were also found in the frontal ganglion. Competitive ELISA with anti-PDF antibodies revealed daily cycling of PDF both in the optic lobe and cerebral lobe with an increase during the night that persisted in constant darkness. The physiological role of PDF is discussed based on these results.     

Photoperiodic modulation of circadian rhythms in the cricket Gryllus bimaculatus

The waveform and the free-running period of circadian rhythms in constant conditions are often modulated by preceding lighting conditions. We have examined the modulatory effect of variable length of light phase of a 24h light cycle on the ratio of activity (alpha) and rest phase (rho) as well as on the free-running period of the locomotor rhythm in the cricket Gryllus bimaculatus. When experienced the longer light phases, the alpha/rho-ratio was smaller and the free-running period was shorter. The magnitude of changes in alpha/rho-ratio was dependent on the number of cycles exposed, while the free-running period was changed by a single exposure, suggesting that there are separate regulatory mechanisms for the waveform and the free-running period. The neuronal activity of the optic lobe showed the alpha/rho-ratio changing with the preceding photoperiod. When different photoperiodic conditions were given to each of the two optic lobe pacemakers, the alpha/rho-ratio of a single pacemaker was rather intermediate between those of animals treated with either of the two conditions. These results suggest that the storage of the photoperiodic information occurs at least in part in the optic lobe pacemaker, and that the mutual interaction between the bilateral optic lobe pacemakers is involved in the photoperiodic modulation.     

Diurnal and circadian rhythm in compound eye of cricket (Gryllus bimaculatus): changes in structure and photon capture efficiency

Day-night changes in rhabdom size of compound eyes were investigated in three groups of crickets (Gryllus bimaculatus): nymphs and adult males and females. In both adults and nymphs, the rhabdoms were larger at night than during a day. In adults, the mean rhabdom occupation ratios (RORs) of ommatidial retinulae at midnight were about two times greater than the values at midday. This change contributes to control of the photon capture efficiency (PCE) of the eye according to photic environment. The RORs of adult males at midnight were higher than those of both adult females and nymphs. This suggests that the PCE of the compound eye of adult males is the greatest of all groups. Under constant darkness, day-night changes in ROR were detected only in adult males, but neither in adult females nor in nymphs. On the other hand, no day-night changes were detected in any experimental group under constant light. These results suggest that the change in rhabdom size between day and night is an adaptation to the photic environment that is controlled mainly by the light-dark (day-night) cycle. However, the change in male adults is induced by an endogenous circadian clock.     

Spectral and polarized light sensitivity of photoreceptors in the compound eye of the cricket (Gryllus bimaculatus)

Summary Retinula cells in the compound eye of the cricket (Gryllus bimaculatus) were recorded intracellularly and stained with Lucifer yellow. Two different methods were used to determine the spectral sensitivity of these cells: a) the spectral scanning method, and b) the conventional flash method. Three spectral types, with S()-curves close to the rhodopsin-absorption functions, were found with _max at 332 nm (UV), 445 nm (blue) and 515 nm (green), respectively.Blue receptors were only recorded in the anatomically specialized dorsal rim area (DRA), and UV and green receptors in the dorsal region of the pigmented part of the eye, whereby green receptors were only found in the ventral eye. On the basis of these results, model calculations are presented for di- and trichromatic colour vision in the cricket.The fluorescence markings revealed green receptors whose axons project with short visual fibres to the lamina, and a UV receptor with a long visual fibre which projects through the lamina to the medulla. The blue receptors send their axons either to the lamina and medulla (long visual fibres) or only to the lamina (short visual fibres).The temporal dynamics of the three receptor types were examined. The blue receptors lack a phasic component of the receptor potential, and the time from stimulus on-set to peak potential is strongly increased compared to the UV and green receptors. Light adaptation reduces the latency to less than half of the dark adapted state.Spectral adaptation experiments revealed an unidirectional coupling between UV and green receptors, and it was found that polarization sensitivity (PS) in blue cells was much higher (PS= 6.5±1.5) than that of UV (PS=1.76±0.05) and green (2.26±0.57) receptors. The functional aspects of the three receptor types are discussed with respect to the presented physiological and morphological data.Abbreviations DA dorsal area - DRA dorsal rim area - PS polarization sensitivity     

Post-embryonic development of circadian rhythm in the cricket,Gryllus bimaculatus: A rhythm reversal

Summary Locomotor activity of the male cricketGryllus bimaculatus DeGeer was recorded from the 7th or last (8th) instar nymph. The nymph showed a diurnal rhythm (nymphal rhythm = NR), while the adult, on the contrary, was nocturnal (adult rhythm = AR) (Fig. 1). This rhythm reversal occurred suddenly 3 to 5 days after the imaginal molt, almost simultaneously with the first spermatophore formation and the start of stridulation (calling song) (Fig. 2). In addition to the antiphase relationship, both rhythms also differed in the freerunning period (tau) and wave form. Tau_scdd was significantly longer in NR (24.33 h) than in AR (23.91 h) (Fig. 3). AR was characterized by a sharp activity peak in each cycle, which NR, however, lacked (Fig. 1, 3, 6). On the basis of these differences, two possibilities are discussed; one is that NR and AR are separate oscillations and the other is that both are coupled to different phase points of one oscillation.Abbreviations LD light dark - DD constant darkness - LL constant light - NR nymphal rhythm - AR adult rhythm     

Pigment-dispersing factor sets the night state of the medulla bilateral neurons in the optic lobe of the cricket,Gryllus bimaculatus

Pigment-dispersing factor (PDF) is an octadeca-neuropeptide widely distributed in the insect brain and suggested to be involved in the insect circadian systems. We have examined its effects on the neuronal activity of the brain efferents in the optic stalk including medulla bilateral neurons (MBNs) in the cricket, Gryllus bimaculatus. The MBNs are visually responding interneurons connecting the bilateral medulla, which show a clear day/night change in their light responsiveness that is greater during the night. Microinjection of PDF into the optic lobe induced a significant increase in the spontaneous activity of the brain efferents and the photo-responsiveness of the MBNs during the day, while little change was induced during the night. The enhancing effects began to occur about 20 min after the injection and another 10 min was necessary to reach the maximal level. The effects of PDF were dose-dependent. When 22 nl of anti-Gryllus-PDF (1:200) IgG was injected into the medulla, the photo-responsiveness of the MBNs was suppressed in both the day and the night with greater magnitude during the night. No significant suppression was induced by injection of the same amount of IgG from normal rabbit serum. These results suggest that in the cricket optic lobe, PDF is released during the night and enhances MBNs' photo-responsiveness to set their night state.     

RNA interference of the clock gene period disrupts circadian rhythms in the cricket Gryllus bimaculatus

Periodic expression of so-called clock genes is an essential part of the circadian clock. In Drosophila melanogaster the cyclic expression of per and tim through an autoregulatory feedback loop is believed to play a central role in circadian rhythm generation. However, it is still elusive whether this hypothesis is applicable to other insect species. Here it is shown that per gene plays a key role in the rhythm generation in the cricket Gryllus bimaculatus. Measurement of per mRNA levels in the optic lobe revealed the rhythmic expression of per in light cycles with a peak in the late day to early night, persisting in constant darkness. A single injection of per double-stranded RNA (dsRNA) into the abdomen of the final instar nymphs effectively knocked down the mRNA levels as adult to about 50% of control animals. Most of the per dsRNA-injected crickets completely lost the circadian locomotor activity rhythm in constant darkness up to 50 days after the injection, whereas those injected with DsRed2 dsRNA as a negative control clearly maintained it. The electrical activity of optic lobe efferents also became arrhythmic in the per dsRNA-injected crickets. These results not only suggest that per plays an important role in the circadian rhythm generation also in the cricket but also show that RNA interference is a powerful tool to dissect the molecular machinery of the cricket circadian clock.     

Pigment‐dispersing factor affects circadian molecular oscillations in the cricket Gryllus bimaculatus

Pigment‐dispersing factor (PDF) is an important neurotransmitter in insect circadian systems. In the cricket Gryllus bimaculatus, it affects nocturnal activity, the free‐running period and photic entrainment. In this study, to investigate whether these effects of PDF occur through a circadian molecular machinery, we measured mRNA levels of clock genes period (per) and timeless (tim) in crickets with pdf expression knocked‐down by pdf RNAi. The pdf RNAi decreased per and tim mRNA levels during the night to reduce the amplitude of their oscillation. The phase of the rhythm advanced by about 4 h in terms of trough and/or peak phases. On the other hand, pdf mRNA levels were little affected by per and tim RNAi treatment. These results suggest that PDF affects the circadian rhythm at least in part through the circadian molecular oscillation while the circadian clock has little effect on the pdf expression.     

Physics of directional hearing in the cricket Gryllus bimaculatus

In the cricket ear, sound acts on the external surface of the tympanum and also reaches the inner surface after travelling in at least three pathways in the tracheal system. We have determined the transmission gain of the three internal sound pathways; that is, the change of amplitude and phase angle from the entrances of the tracheal system to the inner surface of the tympanum. In addition, we have measured the diffraction and time of arrival of sound at the ear and at the three entrances at various directions of sound incidence. By combining these data we have calculated how the total driving force at the tympanum depends on the direction of sound. The results are in reasonable agreement with the directionality of the tympanal vibrations as determined with laser vibrometry.At the frequency of the calling song (4.7 kHz), the direction of the sound has little effect on the amplitudes of the sounds acting on the tympanum, but large effects on their phase angles, especially of the sound waves entering the tracheal system at the contralateral side of the body. The master parameter for causing the directionality of the ear in the forward direction is the sound wave entering the contralateral thoracic spiracle. The phase of this sound component may change by 130–140° with sound direction. The transmission of sound from the contralateral inputs is dominated by a very selective high-pass filter, and large changes in amplitude and phase are seen in the transmitted sounds when the sound frequency changes from 4 to 5 kHz. The directionality is therefore very dependent on sound frequency.The transmission gains vary considerably in different individuals, and much variation was also found in the directional patterns of the ears, especially in the effects of sounds from contralateral directions. However, the directional pattern in the frontal direction is quite robust (at least 5 dB difference between the 330° and 30° directions), so these variations have only little effect on how well the individual animals can approach singing conspecifics.Abbreviations CS contralateral spiracle - CT contralateral tympanum - IS ipsilateral spiracle - IT ipsilateral tympanum - P the vectorial sum of the sounds acting on the tympanum     

Phase shifts of the circadian locomotor rhythm induced by pigment-dispersing factor in the cricket Gryllus bimaculatus

Pigment-dispersing factors (PDFs) are octadeca-peptides widely distributed in insect optic lobes and brain. In this study, we have purified PDF and determined its amino acid sequence in the cricket Gryllus bimaculatus. Its primary structure was NSEIINSLLGLPKVLNDA-NH(2), homologous to other PDH family members so far reported. When injected into the optic lobe of experimentally blinded adult male crickets, Gryllus-PDF induced phase shifts in their activity rhythms in a phase dependent and dose dependent manner. The resulted phase response curve (PRC) showed delays during the late subjective night to early subjective day and advances during the mid subjective day to mid subjective night. The PRC was different in shape from those for light, serotonin and temperature. These results suggest that PDF plays a role in phase regulation of the circadian clock through a separate pathway from those of other known phase regulating agents.     

Dioptrics of the facet lenses in the dorsal rim area of the cricket Gryllus bimaculatus

1. The optics of the corneal facet lenses from the dorsal rim area (DRA) and from the dorso-lateral areas (DA) of the compound eye of the cricket Gryllus bimaculatus were studied.
2. The DRA of the cricket eye contains quite normally shaped facet lenses. The diameter of the facet lens in the DA is 2-fold larger compared to that in the DRA. The radius of curvature of the front surface is distinctly less in the DA facet lenses, as the surface of the facet lenses in the DRA are virtually flat.
3. The averaged axial refractive index of the facet lenses of Gryllus bimaculatus, measured by interference microscopy, was 1.496 ± 0.008 (n = 42) in the DRA and 1.469 ± 0.004 (n = 39) in the DA. The geometrical thickness of the lenses was calculated to be 77 ± 3 μm (n = 42) in the DRA and 56 ± 1 μm (n = 39) in the DA.
4. Analysis of the diffraction pattern obtained with a point light source revealed distinct focusing properties of both the DRA and the DA facet lenses; striking Airy-like diffraction patterns were obtained in both cases.
5. Focal distances measured directly at the backfocal plane were 40 ± 8 μm (n = 84) in the DRA of all the animals studied, and 60–90 μm (n = 62) in DA depending on the animal. Analysis of the diffraction of the point light source yielded very similar focal distances: 40 ± 5 μm (n = 10) in DRA and 81 ± 8 μm (n = 11) in DA. In the DRA, focal distance of the facet lenses was smaller than the cone length, 58 ± 3 μm (n = 9) while in the DA the focal distance matched the effective cone length, 71 ± 5 μm (n = 16).