Localization of the circadian pacemakers of Hemideina thoracica (Orthoptera; Stenopelmatidae)

The location of the circadian pacemakers of the orthopteran Hemideina thoracica (White) has been investigated through observation of the effects of surgical removal of brain tissues (principally optic lobes and tracts) on free-running and entrained locomotor rhythms. Bilobectomy and severance of optic tracts invariably resulted in arrhythmicity, whereas rhythmicity was sustained following unilateral lobectomy, generally with increases in the free-running period (FRP) and decreases in both the active-phase lengths and activity-to-rest ratios of the rhythm. Bilobectomized subjects could be entrained by temperature cycles, but exhibited no transients or residual rhythmicity, indicating that temperature brought about a direct response or masking effect. These results support the hypothesis that the circadian locomotor pacemakers of Hemideina are located within each optic lobe, and that there are no extraoptic centers for the control of the timing of locomotor activity. Although confirmation of the pacemaker role of the optic lobes requires transplantation of the tissues, the conclusion may be drawn by inference from other studies (e.g., Leucophaea maderae--Page, 1983; Gryllus bimaculatus--Tomioka and Chiba, 1986). Light entrainment continued after surgical binding and blackening of the compound eyes and ocelli, supporting the view that direct illumination of neural tissue through the cuticle may be one possible pathway for light entrainment.     

Unequal coupling between locomotor pacemakers of the German cockroach,Blattella germanica (L.)

Male adult German cockroaches, Blattella germanica (L.), expressed robust locomotor circadian rhythmicity under 28 degrees C and constant darkness (DD) conditions. By surgically severing the connections between the optic lobes and midbrain and their subsequent regeneration, we demonstrated that the locomotor circadian pacemaker was located in the optic lobes and that it controlled the locomotor circadian rhythm through neural pathways. From the results that unilaterally optic tract severed males still showed locomotor circadian rhythmicity (93.1%, n=29) without significantly changing the circadian period (tau) or level of motor activity, we concluded that the right and left optic lobes each contain a circadian pacemaker competent to drive the locomotor circadian rhythm. These two pacemakers were strongly coupled since only one rhythm was expressed when the pacemakers were independently exposed to opposite lighting conditions (DD or LL) at the same time. However, an unequal contribution of each pacemaker in determining the overt circadian period was found under constant dim light (10 lux) conditions, revealing a major-minor coupling relationship between these two pacemakers, so that the unilaterally blinded male expressed either a LL-rhythm with a circadian period of 24.27+/-0.21 h (41.7%, n=24) or a DD-rhythm with a circadian period of 23.43+/-0.19 h (58.3%, n=24). However, higher intensity of photic information (200-300 lux) could overpower this relationship and cause the minor pacemaker to lead the rhythm.     

Neural organization of the circadian system of the cockroach Leucophaea maderae

The cockroach Leucophaea maderae was the first animal in which lesion experiments localized an endogenous circadian clock to a particular brain area, the optic lobe. The neural organization of the circadian system, however, including entrainment pathways, coupling elements of the bilaterally distributed internal clock, and output pathways controlling circadian locomotor rhythms are only recently beginning to be elucidated. As in flies and other insect species, pigment-dispersing hormone (PDH)-immunoreac- tive neurons of the accessory medulla of the cockroach are crucial elements of the circadian system. Lesions and transplantation experiments showed that the endogeneous circadian clock of the brain resides in neurons associated with the accessory medulla. The accessory medulla is organized into a nodular core receiving photic input, and into internodular and peripheral neuropil involved in efferent output and coupling input. Photic entrainment of the clock through compound eye photoreceptors appears to occur via parallel, indirect pathways through the medulla. Light-like phase shifts in circadian locomotor activity after injections of γ-aminobutyric acid (GABA)- or Mas-allatotropin into the vicinity of the accessory medulla suggest that both substances are involved in photic entrainment. Extraocular, cryptochrome-based photoreceptors appear to be present in the optic lobe, but their role in photic entrainment has not been examined. Pigment-dispersing hormone-immunoreactive neurons provide efferent output from the accessory medulla to several brain areas and to the peripheral visual system. Pigment-dispersing hormone-immunoreactive neurons, and additional heterolateral neurons are, furthermore, involved in bilateral coupling of the two pacemakers. The neuronal organization, as well as the prominent involvement of GABA and neuropeptides, shows striking similarities to the organization of the suprachiasmatic nucleus, the circadian clock of the mammalian brain.     

Pigment-dispersing hormone (PDH)-immunoreactive neurons form a direct coupling pathway between the bilaterally symmetric circadian pacemakers of the cockroach Leucophaea maderae

Circadian locomotor activity rhythms of the cockroach Leucophaea maderae are driven by two bilaterally paired and mutually coupled pacemakers that reside in the optic lobes of the brain. Transplantation studies have shown that this circadian pacemaker is located in the accessory medulla (AMe), a small neuropil of the medulla of the optic lobe. The AMe is densely innervated by about 12 anterior pigment-dispersing-hormone-immunoreactive (PDH-ir) medulla (PDHMe) neurons. PDH-ir neurons are circadian pacemaker candidates in the fruitfly and cockroach. A subpopulation of these neurons also appears to connect both optic lobes and may constitute at least one of the circadian coupling pathways. To determine whether PDHMe neurons directly connect both accessory medullae, we injected rhodamine-labeled dextran as neuronal tracer into one AMe and performed PDH immunocytochemistry. Double-labeled fibers in the anterior, shell, and internodular neuropil of the AMe contralaterally to the injection site showed that PDH-ir fibers directly connect both accessory medullae. This connection is formed by three anterior PDHMe neurons of each optic lobe, which, thus, fulfill morphological criteria for a direct circadian coupling pathway. Our double-label studies also showed that all except one of the midbrain projection areas of anterior PDHMe neurons were innervated ipsilaterally and contralaterally. Thus, anterior PDHMe neurons seem to play multiple roles in generating circadian rhythms. They also deliver timing information output and perform mutual pacemaker coupling in L. maderae. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

Rôle of eyes,optic lobes,and pars intercerebralis in locomotory and stridulatory circadian rhythms of Teleogryllus commodus

Locomotory and stridulatory activity rhythms of male crickets (Teleogryllus commodus) were assayed simultaneously following various experimental procedures. These included (a) severing the pathways between the ocelli and the brain, between the ommatidia and the optic lobes, or between the optic lobes and the brain, and (b) RF cauterization of the pars intercerebralis. The results indicate that (1) light-dark cycles which entrain both rhythms are perceived by the compound eyes and not by the ocelli; (2) loss of connexion between the brain and the optic lobes leads to arrhythmicity in both behaviours, but a single optic lobe can maintain rhythmicity; (3) absence of neurosecretory cells in the pars intercerebralis is correlated with loss of stridulatory activity and arrhythmicity in locomotory behaviour. It is suggested that the pars intercerebralis serves as a site of coupling between a circadian pacemaker and various overt behaviours. However, intermediate control by the pars intercerebralis is assumed to be exerted via channels that can be either hormonal or purely neuronal in nature.     

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     

Organization of the Circadian System in Insects

The circadian systems of different insect groups are summarized and compared. Emphasis is placed on the anatomical identification and characterization of circadian pacemakers, as well as on their entrainment, coupling, and output pathways. Cockroaches, crickets, beetles, and flies possess bilaterally organized pacemakers in the optic lobes that appear to be located in the accessory medulla, a small neuropil between the medulla and the lobula. Neurons that are immunoreactive for the peptide pigment-dispersing hormone (PDH) arborize in the accessory medulla and appear to be important components of the optic lobe pacemakers. The neuronal architecture of the accessory medulla with associated PDH-immunoreactive neurons is best characterized in cockroaches, while the molecular machinery of rhythm generation is best understood in fruit flies. One essential component of the circadian clock is the period protein (PER), which colocalizes with PDH in about half of the fruit fly's presumptive pacemaker neurons. PER is also found in the presumptive pacemaker neurons of beetles and moths, but appears to have different functions in these insects. In moths, the pacemakers are situated in the central brain and are closely associated with neuroendocrine functions. In the other insects, neurons associated with neuroendocrine functions also appear to be closely coupled to the optic lobe pacemakers. Some crickets and flies seem to possess central brain pacemakers in addition to their optic lobe pacemakers. With respect to neuronal organization, the circadian systems of insects show striking similarities to the vertebrate circadian system. (Chronobiology International, 15(6), 567-594, 1998)     

Manipulating the light/dark cycle: effects on dopamine levels in optic lobes of the honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) brain

This study examines the relationship between cyclical variations in optic-lobe dopamine levels and the circadian behavioural rhythmicity exhibited by forager bees. Our results show that changing the light–dark regimen to which bees are exposed has a significant impact not only on forager behaviour, but also on the levels of dopamine that can be detected in the optic lobes of the brain. Consistent with earlier reports, we show that foraging behaviour exhibits properties characteristic of a circadian rhythm. Foraging activity is entrained by daily light cycles to periods close to 24 h, it changes predictably in response to phase shifts in light, and it is able to free-run under constant conditions. Dopamine levels in the optic lobes also undergo cyclical variations, and fluctuations in endogenous dopamine levels are influenced significantly by alterations to the light/dark cycle. However, the time course of these changes is markedly different from changes observed at a behavioural level. No direct correlation could be identified between levels of dopamine in the optic lobes and circadian rhythmic activity of the honey bee.     

A circadian rhythm in neural activity can be recorded from the central nervous system of the cockroach

Summary Evidence presented in this paper indicates that a robust circadian rhythm in the frequency of neural activity can be recorded from the central nervous system of intact cockroaches, Leucophaea maderae. This rhythmicity was abolished by optic lobe removal. Spontaneous neural activity was then used as an assay to demonstrate that the optic lobe is able to generate circadian oscillations in vitro. These results provide direct evidence that the cockroach optic lobe is a self-sustained circadian oscillator capable of generating daily rhythms in the absence of neural or hormonal communications with the rest of the organism.Abbreviations CNS central nervous system - DD constant dark - LD light/dark cycle - SCN suprachiasmatic nucleus - ZT Zeitgeber time     

The nervous system of Loligo. II. Suboesophageal centres.

A well-marked hierarchy of centres can be recognized within the suboesophageal lobes and ganglia of the arms. The inputs and outputs of each lobe are described. There are sets of motoneurons and intermediate motor centres, which can be activated either from the periphery or from above. They mostly do not send fibres up to the optic or higher motor centres. However, there is a large set of fibres running from the magnocellular lobe to all the basal supraoesophageal lobes. The centre for control of the four eye-muscle nerves in the anterior lateral pedal lobe receives many fibres direct from the statocyst and from the peduncle and basal lobes, but none direct from the optic lobe. The posterior lateral pedal is a backward continuation of the oculomotor centre, containing large cells that may be concerned in initiating attacks by the tentacles. An intermediate motor centre in the posterior pedal lobe probably controls steering. It sends fibres to the funned and head retractors, and by both direct and interrupted pathways to the fin lobe. It receives fibres from the crista nerve and basal lobes, but none direct from the optic lobe. The jet control centre of the ventral magnocellular lobe receives fibres from the statocyst and skin and also from the optic and basal lobes. Some of these last also give extensive branches throughout the palliovisceral lobes. The branching patterns of the dendritic collaterals differ in the various lobes. Some estimates are given of the numbers of synaptic points. The dendritic collaterals of the motoneurons spread through large volumes of neuropil and they overlap. The incoming fibres spread widely and each presumably activates many motoneurons either together or serially. Many of the lobes contain numerous microneurons with short trunks restricted to the lobe, but there are none of these cells in the chromatophore lobes or fin lobes. The microneurons have only few dendritic collaterals, in contrast to the numerous ones on the nearby motoneurons.     

Connections between wide-field monocular and binocular movement detectors in the brain of a hawk moth

Summary Recordings were made in the brain of Sphinx ligustri of pairs of directionally selective movement detectors, and the spike trains analysed with a computer for possible synaptic connections between two classes of movement detector. (1) Neurones with large binocular fields which arise in the medial protocerebrum and project to the medulla or lobula of one optic lobe, or to the ventral nerve cord. (2) Movement detectors which project from the lobula complex of one optic lobe to the opposite medial protocerebrum. The majority of the second group had back-to-front preferred directions over the ipsilateral eye, and of these many were weakly sensitive to stimuli to the opposite eye. The ipsilateral receptive field covered most of the eye.Optic lobe output cells with the appropriate preferred direction provide a powerful excitatory input to the binocular movement detectors centrifugal to the medulla. Each centrifugal movement detector probably receives excitatory inputs from no more than two optic lobe output cells with back-to-front preferred direction. The same set of optic lobe output neurones probably feeds several cells projecting to the medulla and lobula of both optic lobes, and, possibly, to the ventral nerve cord.Evidence was obtained that the optic lobe output cells themselves receive few excitatory inputs, and that therefore the receptive fields of their input cells are large.Two moving stimuli were presented in different areas of the receptive field. Movement through the null direction in one area inhibited the response to movement in the preferred direction in another area. This suppression was stronger in optic lobe output cells with front-to-back preferred direction than in units with back-to-front preferred direction. Thus the optic lobe output cells, or wide-field units feeding them, receive inhibitory inputs from wide-field units with the opposite preferred direction.Similar tests in which moving stimuli were presented to both eyes gave results indicating that the binocular centrifugal movement detectors may receive inhibitory inputs from movement detectors with back-to-front preferred direction. The possible functional significance of these inhibitory inputs is discussed.I am very greatful to F. A. Miles for helpful discussion and criticism. Financial support came from the U. K. Science Research Council.     

A genetic analysis of visual system development in Drosophilia melanogaster.

The eyes and optic lobes of adult Drosophila melanogaster comprise a highly organized system of interconnected neurons. The eye and optic lobe primordia are physically separate during the embryonic and larval stages of development, and these tissues do not come into contact until the third larval instar, as a consequence of axons growing from the receptor cells of the developing eyes to the primordial optic lobes. After this contact, the axons of the eyes arrange themselves into their complex and orderly adult pattern. Simultaneously, the optic lobe cells begin elaborating axons which organize into their precise adult array. One question posed by this system is: Does cellular pattern formation in either the eyes or optic lobes depend on eye-brain interactions, or do the two tissues organize autonomously? To answer this question, mutations were found which cause abnormal ommatidial array in the eyes and which also perturb the normal adult axon array in the optic lobes. By means of X ray-induced somatic recombination and by genetically controlled mitotic chromosome loss (gynandromorph formation), flies mosaic for genotypically mutant and normal tissue were constructed. Analysis of the neuronal array in mosaic flies in which eye and optic lobe tissue differed genotypically showed that the axon array phenotype of the optic lobe depends on the genotype of the eye tissue innervating that lobe, while the eye phenotype does not depend on optic lobe genotype. Thus, the axonal organization of the D. melanogaster optic lobe has been shown to depend on the transmission of information from the eyes to the optic lobes.     

Expression of serotonin (5-HT) during CNS development of the cephalopod mollusk, Idiosepius notoides

Cephalopods are unique among mollusks in exhibiting an elaborate central nervous system (CNS) and remarkable cognitive abilities. Despite a profound knowledge of the neuroanatomy and neurotransmitter distribution in their adult CNS, little is known about the expression of neurotransmitters during cephalopod development. Here, we identify the first serotonin-immunoreactive (5-HT-ir) neurons during ontogeny and describe the establishment of the 5-HT system in the pygmy squid, Idiosepius notoides. Neurons that are located dorsally to each optic lobe are the first to express 5-HT, albeit only when the lobular neuropils are already quite elaborated. Later, 5-HT is expressed in almost all lobes, with most 5-HT-ir cell somata appearing in the subesophageal mass. Further lobes with numerous 5-HT-ir cell somata are the subvertical and posterior basal lobes and the optic and superior buccal lobes. Hatching squids possess more 5-HT-ir neurons, although the proportions between the individual brain lobes remain the same. The majority of 5-HT-ir cell somata appears to be retained in the adult CNS. The overall distribution of 5-HT-ir elements within the CNS of adult I. notoides resembles that of adult Octopus vulgaris and Sepia officinalis. The superior frontal lobe of all three species possesses few or no 5-HT-ir cell somata, whereas the superior buccal lobe comprises many cell somata. The absence of 5-HT-ir cell somata in the inferior buccal lobes of cephalopods and the buccal ganglia of gastropods may constitute immunochemical evidence of their homology. This integrative work forms the basis for future studies comparing molluscan, lophotrochozoan, ecdysozoan, and vertebrate brains.     

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.     

Neurobiology of the scallop. II. structure of the parietovisceral ganglion lateral lobes in relation to afferent projections from the mantle eyes

The lateral lobes of the scallop parietovisceral ganglion have been examined morphologically with respect to their functional role as optic lobes. The gross morphology of the lateral lobe and projections of optic nerve fibers within it were investigated by 1) supravital methylene blue staining, and 2) autoradiography using tritiated proline injected intraocularly for incorporation and transport by the optic fibers. Ultrastruc‐turally, the lateral lobe was examined using standard electron microscopic techniques. The lateral lobe is composed of a cortical rind of cells, 8–15 μm in diameter at the ventral surface and 15–20 μm in diameter at the ventral surface, surrounding a central neuropil. The neuropil contains three distinct regions: 1) the glomerular neuropil, a series of densely staining spherical subunits associated with the eyes and pallial nerves, 2) the subcellular neuropil, a synaptic region adjacent to the ventral cell layer also having a visual function, and 3) the subglomerular neuropil, the remaining, rather unspecialized neuropil of the lateral lobe. Synaptic profiles with symmetrical membrane thickenings, a 32 nm synaptic cleft, and three types of vesicles are seen throughout the neuropil, although the density of synapses is greater in the glomerular region. Clear, dense core and neurosecretory vesicles are seen individually or as mixed populations in the presynaptic terminals. Autoradiographic experiments have revealed that optic fibers enter the lateral lobe and project directly to the subcellular neuropil where they synapse with cells located on the ventral surface of the lateral lobe cells. These cells in turn form the dense glomerular structures previously identified as visual association centers and send efferent fibers into the pallial nerves. The projection of optic fibers to the ventral surface of the lobe is consistent with previous electrophysiological recordings of visual activity at this site.     

Circadian Activity Rhythm of the House Fly Continues after Optic Tract Severance and Lobectomy

Under constant conditions, locomotor activity in about 50% of 63 adult Musca domestica continued to be rhythmic after bilateral severance of optic tracts or bilateral lobectomy. Apparently, the optic lobes of Musca do not contain the oscillator for rhythmic control of locomotor activity as has been proposed for other insects. In 20% of the individuals, several circadian components of activity rhythms were found after operation indicating a role of the optic lobes in the coupling of oscillators. The remaining 30% of the flies with severed optic tracts appeared to be arrhythmic. Most of these flies had vacuolized tissue in the central brain. However, disruption of rhythmicity did not correlate with a common pattern of degeneration. Therefore no conclusions can be drawn as to the localization of the circadian control of locomotor activity in the brain. Flies showing an arrhythmic activity pattern could still be synchronized by LD cycles. Activity did not occur solely during the light period as is the case in controls; but was phase delayed by about 6 hr towards the dark period. Since all flies with severed optic tracts could be synchronized by LD cycles, Musca domestica must possess extraocular photoreceptors.     

Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants

Mutations at the disconnected (disco) locus of Drosophila melanogaster disrupt neural cell patterning in the visual system, leading to the loss of many optic lobe neurons. Drosophila's presumptive circadian pacemaker neurons – the dorsal and ventral lateral neurons – are usually among the missing cells, and most disco flies are behaviorally arrhythmic. In this study, I show that ventral lateral neurons (LN_vs) are occasionally present and provoke robust circadian rhythmicity in disco mutants. Of 357 individual disco flies four animals with robust circadian rhythmicity were found. All four retained LN_vs together with terminals in the superior protocerebrum. Residual or bi-circadian rhythmicity was found in about 20% of all flies; the remaining flies were completely arrhythmic. One of the flies with residual rhythmicity and two of the arrhythmic flies also had some LN_vs stained. However, these flies lacked the LN_v fibers in the superior protocerebrum. The results suggest that the presence of single LN_vs is sufficient to provoke robust circadian rhythmicity in locomotor activity if the LN_v terminals reach the superior protocerebrum. The presence of residual or bi-circadian rhythmicity in 20% of the flies without LN_vs indicates that also other cells contribute to the rhythmic control of locomotor activity. Accepted: 17 September 1997     

The role of ocelli in circadian singing rhythms of crickets

ABSTRACT. . There is a direct quantitative relationship between the free-running period (r) of the circadian stridulation rhythm of male Australian field crickets, Teleogryllus commodus (Walker), and the intensity of the constant light conditions. Both T. commodus and the house cricket, Acheta domesticus (L.), show free-running periods of the singing rhythm of c. 24 h when the light intensity is 0.00025 lux. In both species the severance of the three ocellar nerves significantly slows the circadian period which is indicative of a reduced perception of the available light intensity. To test whether this period reduction is a peripheral or a central effect, electro-retinograms (ERGs) were recorded from compound eyes of male T. commodus with the ocelli fully functional, then occluded, and then uncovered. The size of the compound eye ERG is reduced by 20% with ocellar occlusion and can subsequently be fully restored to the intact level, which indicates that the ocellar effect is a peripheral one. Intensified CoCl₂ fills reveal one neurone in A. domesticus and two in T. commodus which travel from the lateral ocellar nerve out into the ipsilateral optic lobe of the compound eye. These neurones all terminate in or distal to the lobular neuropile. The data are interpreted to indicate a role for ocelli in modulating the light intensity perception of the compound eye. The final effect of the ocellar afferents is at a peripheral level prior to the input of the visual information to the optic lobe circadian pacemaker. Hence ocelli play an indirect role in circadian rhythmicity, augmenting the sensitivity of the primary photoreceptors to better perceive photic entrainment signals.     

Hofbauer-Buchner eyelet affects circadian photosensitivity and coordinates TIM and PER expression in Drosophila clock neurons

Extraretinal photoreception is a common input route for light resetting signals into the circadian clock of animals. In Drosophila melanogaster, substantial circadian light inputs are mediated via the blue light photoreceptor CRYPTOCHROME (CRY) expressed in clock neurons within the brain. The current model predicts that, upon light activation, CRY interacts with the clock proteins TIMELESS (TIM) and PERIOD (PER), thereby inducing their degradation, which in turn leads to a resetting of the molecular oscillations within the circadian clock. Here the authors investigate the function of another putative extraretinal circadian photoreceptor, the Hofbauer-Buchner eyelet (H-B eyelet), located between the retina and the medulla in the fly optic lobes. Blocking synaptic transmission between the H-B eyelet and its potential target cells, the ventral circadian pacemaker neurons, impaired the flies' ability to resynchronize their behavior under jet-lag conditions in the context of nonfunctional retinal photoreception and a mutation in the CRY-encoding gene. The same manipulation also affected synchronized expression of the clock proteins TIM and PER in different subsets of the clock neurons. This shows that synaptic communication between the H-B eyelet and clock neurons contributes to synchronization of molecular and behavioral rhythms and confirms that the H-B eyelet functions as a circadian photoreceptor. Blockage of synaptic transmission from the H-B eyelet in the presence of functional compound eyes and the absence of CRY also results in increased numbers of flies that are unable to synchronize to extreme photoperiods, supplying independent proof for the role of the H-B eyelet as a circadian photoreceptor.     

The optic lobes of Lepidoptera

Variants of the Golgi-Colonnier (1964) selective silver procedure have been used to show up neurons in insect brains. Neural elements are particularly clearly impregnated in the optic lobes. Three classes of nerve cells can be distinguished; perpendicular (class I), tangential (class II) and amacrine cells (class III). There are many types of neurons in each class which together have a very wide variety of form. Their components are related to specific strata in the optic lobe regions. Short visual cells from the retina terminate in the lamina in discrete groups of endings (optic cartridges). Pairs of long visual fibres from ommatidia pass through the lamina and end in the medulla. Class I cells link these two regions in parallel with the long visual fibres and groups of these elements define columns in the medulla. These in turn give rise to small-field fibres that project to the lobula complex. Tangential processes intersect the parallel arrays of class I cells at characteristic levels. Some are complex in form and may invade up to three regions. Another type provides a direct link between the ipsi- and contralateral optic lobe. Amacrine cells are intrinsic to single lobe regions and have processes situated at the same levels as those of classes I and II cells. A fifth optic lobe region, the optic tubercle, is connected to the medulla and lobula and also receives a set of processes from the mid-brain. There are at least six separate types of small-field relays which could represent the retina mosaic arrangement in the lobula.