Analysis of the Mechanism underlying the Rhythm Reversal from Diurnal to Nocturnal in the Cricket Gryllus bimaculatus, with Special Reference to the Role of Serotonin

The cricket, Gryllus bimaculatus, shows a rhythm reversal from diurnal to nocturnal in about a week after the imaginal molt. In the present study, we investigated the role of serotonin (5-HT) in the rhythm reversal. The 5-HT content in the brain measured by HPLC equipped with an electrochemical detector gradually increased after the imaginal molt, and in fully nocturnal adults it was about 2 times of nymphal level. We then examined the effects of 5,7-dihydroxytryptamine (5,7-DHT), a selective neurotoxine to serotonergic neurons, on the locomotor rhythm. In most animals with 5,7-DHT (25 muM or 250 muM, 32.2 nl) injected into the brain, daytime activity significantly increased even after the rhythm reversal, while nighttime activity was not significantly affected, forming rather diurnal pattern. The serotonin content in the brain of animals injected with 250 muM 5,7-DHT was reduced by about 30%. On the basis of these results, possible involvement of 5-HT in the neural mechanism controlling the locomotor rhythm is discussed.     

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     

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.     

A specific area of the compound eye in the cricket Gryllus bimaculatus sends photic information to the circadian pacemaker in the contralateral optic lobe

The circadian locomotor rhythm of the cricket Gryllus bimaculatus is primarily regulated by a pair of interacting optic lobe circadian pacemaker systems. The interaction involves phase-dependent modulation of the free-running period and phase-dependent suppression of activity. Since photic information has been shown to be involved in the interaction, we examined the regional difference in photoreception for the interaction within cricket compound eyes. The activity rhythm of animals receiving partial reduction of one compound eye combined with severance of the contralateral optic nerve split into entrained and free-running components under a 13-h light to 13-h dark cycle. All the animals operated on showed a phase-dependent suppression of activity, and most animals showed a phase-dependent modulation of the period of the free-running component. However, removal of the dorsocaudal area of the compound eye resulted in a severe reduction of the amplitude of the phase-dependent-period modulation. These results suggest that the dorsocaudal portion of the compound eye is a specific region receiving photic signals that are transmitted to the circadian pacemaker in the contralateral optic lobe and that the phase-dependent suppression of activity is caused by a mechanism separate from that for the period modulation.     

Intrastriatal Injection of 5,7-Dihydroxytryptamine Decreased 5-HT Levels in the Striatum and Suppressed Locomotor Activity in C57BL/6 Mice

Effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 5,7-dihydroxytryptamine (5,7-DHT) on striatal levels of dopamine (DA), 5-hydroxytryptamine (5-HT), and their metabolites, as well as on locomotor activity were investigated in C57BL/6 mice. The results showed that MPTP significantly increased locomotor activity and decreased striatal DA levels. However, injection of the serotonergic neurotoxin 5,7-DHT in the striatum, either alone or following high doses of MPTP, significantly decreased locomotor activity, and concomitantly decreased striatal levels of 5-HT and 5-HIAA. This study suggests that the increased locomotor activity may be due to increased striatal serotonergic activity which overcompensates for the DA deficiency. The locomotor hypoactivity, induced by 5,7-DHT, might be due to the decreased striatal levels of 5-HT and 5-HIAA.     

The serotonergic somatosensory projection to the tectum of normal and eyeless salamanders

The spinotectal somatosensory projection was compared in normal, genetically eyeless, and embryonically manipulated salamanders. In normal animals, serotonin fluorescence was restricted to the intermediate tectalneuropil. This same region showed both high levels of serotonin uptake and somatosensory single unit electrical activity. In mutant eyeless salamanders and in normal animals enucleated early in development, serotonin fluorescence, serotonin uptake, and somatosensory activity were present in the superficial tectal neuropil. One-eyed animals, either genetically normal axolotls with one eye enucleated embryonically or genetically eyeless animals in which a normal eye had been transplanted, showed normal intermediate serotonin fluroescence and somatosensory physiology in the visually innervated half-tectum. In the visually uninnervated half-tectum, they showed superficial serotonin fluorescence and somatosensory physiology. In normal animals, 5,7-dihydroxytryptamine (5,7-DHT), a specific poison for serotonergic fibers, eliminated physiological responses in the contralateral somatosensory tectal region. The 5,7-DHT poisoning also abolished U.V.-induced serotonin fluorescence in the intermediate tectal neuropil. These results are discussed in terms of (1) evidence for serotonin as a central neurotransmitter for somatosensory information in the tectum, (2) the effects of eyelessness on tectal organization, and (3) related results in other animals.     

Neural pathways involved in mutual interactions between optic lobe circadian pacemakers in the cricketGryllus bimaculatus

The circadian locomotor rhythm of the cricketGryllus bimaculatus is primarily generated by a pair of optic lobe circadian pacemakers. The two pacemakers mutually interact to keep a stable temporal structure in the locomotor activity. The interaction has two principal effects on the activity rhythm, i.e., phase-dependent modulation of the freerunning period and phase-dependent suppression of activity driven by the partner pacemaker. Both effects were mediated by neural pathways, since they were immediately abolished after the optic stalk connecting the optic medulla to the lobula was unilaterally severed. The neural pathways were examined by recording locomotor activity, under a 13 h light to 13 h dark cycle, after the optic nerves were unilaterally severed and the contralateral optic stalk was partially destroyed near the lobula. When the dorsal half of the optic stalk was severed, locomotor rhythm mostly split into two components: one was readily entrained to the given light-dark cycle and the other freeran with a marked fluctuation in freerunning period, where the period of the freerunning component was lengthened or shortened when the onset of the entrained component occurred during its subjective night or day, respectively. The phase-dependent modulation of activity was also observed in both components. However, severance of the ventral half of the optic stalk resulted in appearance only of the freerunning component; neither the phase-dependent modulation of its freerunning period nor the change in activity level was observed. These results suggest that neurons driving the mutual interaction and the overt activity rhythm run in the ventral half of the proximal optic stalk that includes axons of large medulla neurons projecting to the cerebral lobe and the contralateral medulla.Abbreviations LD light dark cycle - freerunning period     

Nonphotic enhancement of adjustment to new light-dark cycles: masking interpretation discounted

The adjustment of hamsters to advanced light-dark (LD) cycles can be greatly accelerated by scheduling a single 3-hr bout of extra activity in a novel running wheel, starting about 7 hr before the time when the animals become active in the preceding LD cycle. The present experiments were designed to provide stronger evidence that this effect depends on a shift in the pacemaker rather than on masking. It was shown that when hamsters were put into continuous darkness (DD) 1 day after the exercise-accelerated phase shift, their free-running rhythms took off from a time nearer to the onset of darkness in the new LD cycle than in the preceding LD cycle. An incidental finding was that in DD the free-running period of the hamsters with the accelerated phase shifts was longer than that of the control animals. Further evidence that the 3-hr exercise pulse had produced a greater phase advance than that occurring in undisturbed control animals was obtained by giving a light pulse at the same clock time to all animals after they had been in DD for 8 days. The animals that had previously exercised for the additional 3-hr phase-advanced in response to the light pulse, while the undisturbed control animals phase-delayed.     

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.     

Entrainment of the circatidal swimming activity rhythm in the cumacean Dimorphostylis asiatica (Crustacea) to 12.5-hour hydrostatic pressure cycles

The cumacean Dimorphostylis asiatica (Crustacea) exhibits a circatidal swimming activity rhythm. The animals were exposed to a 12.5 hr sinusoidal change of hydrostatic pressure of 0.3 atm amplitude in the laboratory. Under constant dark conditions, most of the specimens were entrained to a daily bimodal swimming activity rhythm by the hydrostatic pressure cycle. A small number of individuals exhibited a unimodal daily rhythm, with no apparent entraining from the administered cycles. A marked feature was a flexible phase relationship between the entrained daily bimodal rhythm and the hydrostatic pressure cycles: the swimming activity of most of the specimens occurred around the pressure-decreasing phase, but for a small number of individuals it coincided with the pressure-increasing phase. Such flexibility suggests a weak entraining effect of hydrostatic pressure on the circatidal rhythm of this species. When exposed to 24 hr light-dark cycles and a hydrostatic pressure cycle simultaneously, the specimens exhibited a rhythmic activity entrained by the hydrostatic pressure cycle during the dark period, which closely resembles the temporal activity pattern of this species in the field. The light cycles entrained the swimming activity via direct inhibition and induction of activity (i.e., masking). Under light-dark conditions, the specimens exhibited activity on the pressure-increasing phase more frequently compared with specimens kept in constant darkness.     

5,7-Dihydroxytryptamine: Regional Brain Concentrations Following Intraventricular Administration to Rats

The concentrations of 5,7-dihydroxytryptamine (5,7-DHT) and serotonin (5-HT) were measured in brainstem, hypothalamus and cerebral cortex 0, 2, 6, 12, and 24 hours following the bilateral, lateral ventricular injection of 5,7-DHT (100 g/each ventricle) into adult male rats. At 6 hours, 5,7-DHT levels had decreased 99% from 0 hr values in all brain regions. Thereafter, 5,7-DHT levels continued to decline in cortex, but not in hypothalamus or brainstem; at 24 hr, but not 48 hr, 5,7-DHT peaks were still measurable in each brain region examined. Serotonin levels in all three regions also fell markedly by 2-6 hours after 5,7-DHT administration. At 24 hours, hypothalamus and brainstem 5HT levels had declined >70% and cerebral cortex 50% below control values. The relevance of these findings to the protective action of monoamine reuptake blockers is discussed.     

Glutamic acid decarboxylase in different areas of the developing chick central nervous system

The temporal course of the development of GAD activity in GABAergic neurons was studied in the chick retina, optic lobe and cerebellum. The developmental pattern of GAD activity was similar in the three areas studied, showing typical sigmoideal curves, which reached a maximal value at the 3^rd post-hatching day. Kinetic studies during development revealed that K_m remained unchanged while V_max increased 3-fold in the retina (48.99±0.84 nmol/hr/mg protein), almost 4-fold in the optic lobe (162.77±4.32 nmol/hr/mg protein) and 3.5 fold in the cerebellum (69.30±1.26 nmol/hr/mg protein). The developmental pattern of GAD activity in homogenates of the three areas studied from dark-reared and light-reared chicks with respect to normal light-dark cycle animals showed no significant differences. These results indicate that the increase in GAD activity during development are not due to a change in the affinity for its substrate but rather to changes in the concentration of the enzyme. The developmental pattern of GAD activity in the chick visual system was not affected by environmental conditions suggesting that the developmental profile is lightindependent.     

Effects of the intraventricular administration of 5,7-dihydroxytryptamine on FSH, LH and prolactin secretion in neonatally estrogenized male rats

The role of the serotoninergic system in the control of LH, FSH and prolactin secretion was analyzed in control and neonatally estrogenized male rats. Animals injected s.c. with 500 micrograms of estradiol benzoate (EB) on day 1 of life, or their corresponding sham-treated controls, were divided on day 75 into the following groups: (1) orchidectomized; (2) injected intraventricularly with 5,7-dihydroxytryptamine (5,7-DHT); (3) orchidectomized and treated with 5,7-DHT, and (4) sham operated. 15 days later, the animals were decapitated and their FHS, LH and prolactin plasma values measured by specific RIA systems. After the treatment with 5,7-DHT, control animals showed a decline in basal prolactin levels but no modification in basal LH and FSH values. After castration, 5,7-DHT-treated animals showed a reduced LH increase and a more marked prolactin decrease. In neonatal estrogen-treated animals, the 5,7-DHT injection did not change FSH, LH or prolactin levels but did partially or completely abolish the post-castration rise in FSH and LH levels, respectively. These data seem to indicate that neonatal estrogenization induced a modification of the serotoninergic role in the control of LH, FSH and prolactin.     

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.

Orcadian Rhythm of Serotonin Binding in Rat Brain—I. Effect of the Light-Dark Cycle

High affinity serotonin binding to rat brain membranes showed a circadian rhythm with minimal binding at 1000 and a maximal binding at 0000. Brain serotonin levels were almost inverse to the rhythm of serotonin binding. Under reverse light-dark conditions, lights on from 1900 to 0700, a significant phase shift in serotonin binding and concentration of about 8-10 hr was found. The adaptation of the rats to the inverse light-dark cycle was ascertained by plasma ACTH and corticosterone assay.     

The 5,7-DHT-induced anticonflict effect is dependent on intact adrenocortical function.

We have recently reported that the anxiolytic-like effect observed in rats severely depleted of brain serotonin (5-HT) by means of 5,7-DHT is indirect and probably involves the GABA(A)/benzodiazepine chloride ionophore receptor complex (GABAA/BDZ-RC). One tentative explanation for this effect considered the involvement of corticosteroids. In the present series of experiments we have therefore investigated the effect of adrenalectomy (ADX) on the 5,7-DHT-induced anxiolytic-like effect displayed by rats in Vogel's conflict test. ADX totally abolished the anticonflict effect of the 5,7-DHT lesion. Replacement treatment with corticosterone, but not with dexamethasone, reinstated the anticonflict effect. These results indicate that an intact adrenocortical function, possibly via brain steroid type I receptors, is required for the expression of the 5,7-DHT-induced anxiolytic-like effect. It is postulated that ADX lowers the concentration of endogenous positive modulators at the GABAA/BDZ-RC to a level no longer sufficient to produce anxiolytic-like effects in 5,7-DHT-lesioned animals. The finding that 5,7-DHT-lesioned animals were more sensitive than sham-lesioned controls to the anticonflict effect of the barbiturate-like corticosteroid THDOC provides further support for the contention that an increased endogenous activity at the GABAA/BDZ-RCes is involved in the anxiolytic-like effect observed in rats with a severe depletion of brain 5-HT.     

Circadian Period Modulation and Masking Effects Induced by Repetitive Light Pulses in Locomotor Rhythms of the Cricket, Gryllus bimaculatus

Effects of 15 min light pulses given at various intervals (every 1, 2, 4, 6, 8 and 12 hr) under constant darkness on the locomotor rhythm were investigated in the adult male cricket, Gryllus bimaculatus. A single pulse per 24 hr induced period modulation in a circadian phase dependent manner, yielding a period modulation curve (PMC): the 15 min light pulse lengthened the period in the early subjective night (CT11-16) and shortened it during the late subjective night to the early subjective day (CT20-5). Frequent light pulses modulated the freerunning period of the rhythm dependent on the interval of the pulses: when compared with the freerunning period in DD (23.74 +/- 0.03 hr) the period was significantly shorter in intervals of 2 and 4 hr, but lengthened when the interval was 1 and 12 hr. Frequent light pulses also resulted in entrainment of the rhythm to run with the period of 24 hr and the ratio of the entrained animals varied from 12% to 72% depending on the interval of the light pulses. The period modulation and the entrainment by the repetitive light pulses could be interpreted according to the PMC. In about 15% of animals, the light pulses induced a rhythm dissociation, suggesting that the bilaterally paired circadian pacemakers have their own sensitivity to the entraining photic information. The light pulse caused a masking effect, i.e., an intense burst of activity. The magnitude of the light induced responses was dependent on the circadian phase. The strongest masking effect was observed in the subjective night. The phase of the prominent period modulation and of the marked masking effects well coincides with the previously reported sensitive phase of the photoreceptive system.     

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.     

Dissociation between the circadian rhythm of locomotor activity and the pineal clock in the Japanese newt

The circadian locomotor activity rhythm of the Japanese newt has been thought to be driven by a putative brain oscillator(s) subordinate to the pineal clock. The existence of mutual coupling between the pineal clock and the brain oscillator(s) in vivo was examined. We covered the newt's skull with aluminum foil and simultaneously reversed the light-dark cycle, thereby allowing the pineal organ to be exposed to constant darkness while the rest of the animal was exposed to the reversed light-dark cycle. In control animals, whose heads were covered with transparent plastic, the rhythm of synaptic ribbon number in the pineal photoreceptor cells was entrained to the reversed light-dark cycle. Rhythms from newts whose heads were shielded, however, were similar to those observed in the unoperated newts kept under constant darkness. The locomotor activity rhythms of both head-covered animals and control animals were entrained to the reversed light-dark cycle. These data suggest that extrapineal photoreception can entrain the putative brain oscillator(s), but not the pineal clock. Thus, at least in an aspect of photic entrainment, there seems to be little or no mutual coupling between the pineal clock and the putative brain oscillator(s) in the circadian system of the Japanese newt.Abbreviations LD light-dark - DD constant darkness - SCN suprachiasmatic nucleus - SR synaptic ribbon     

The short term effects of 5,7-dihydroxytryptamine on peripheral serotonin stores in Rhodnius prolixus and their long-term recovery

The serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) appears to affect invertebrate systems differently from vertebrate ones. The basis for toxicity in vertebrates appears to involve the intraneuronal actions of monoamine oxidase (MAO) upon the toxin. In insects, MAO is not present in appreciable amounts. In this study, we demonstrate that in vitro 5.7-DHT competitively inhibits the uptake of [³H]serotonin by serotonergic neurohaemal areas. The apparent K_M increases from 4.9 × 10⁻⁷ to 1.7 × 10⁻⁶ M. This neurotoxin also causes a significant release of previously accumulated [³H]serotonin in nominally Ca²⁺-free saline. While 5,7-DHT does not affect the uptake of [³H]tryptophan, it reduces the subsequent synthesis of [³H]serotonin. In vivo, the tissues appear to have recovered 2 weeks after toxin treatment, as determined by immunohistochemistry. At 24 h, 1 week and 2 weeks after injection, the tissues are able to take up and release [³H]serotonin normally. 1 and 2 weeks after injection, insects ingest a normal-sized blood meal, a behaviour acutely disrupted by 5,7-DHT treatment. The results of this and other invertebrate studies suggest that 5,7-DHT does not destroy serotonergic neurons, as it does in vertebrates. 5,7-DHT may be a more useful tool to study the functions of serotonin in invertebrates as one may transiently affect serotonin stores.