Orcokinin immunoreactivity in the accessory medulla of the cockroach Leucophaea maderae

The accessory medulla is the master circadian clock in the brain of the cockroach Leucophaea maderae and controls circadian locomotor activity. Previous studies have demonstrated that a variety of neuropeptides are prominent neuromediators in this brain area. Recently, members of the orcokinin family of crustacean neuropeptides have been identified in several insect species and shown to be widely distributed in the brain, including the accessory medulla. To investigate the possible involvement of orcokinins in circadian clock function, we have analyzed the distribution of orcokinin immunostaining in the accessory medulla of L. maderae in detail. The accessory medulla is densely innervated by approximately 30 orcokinin-immunoreactive neurons with cell bodies distributed in five of six established cell groups in the accessory medulla. Immunostaining is particularly prominent in three ventromedian neurons. These neurons have processes in a median layer of the medulla and in the internodular neuropil of the accessory medulla and send axonal fibers via the posterior optic commissure to their contralateral counterparts. Double-labeling experiments have revealed the colocalization of orcokinin immunostaining with immunoreactivity for pigment-dispersing hormone, FMRFamide, Mas-allatotropin, and γ-aminobutyric acid in two cell groups of the accessory medulla, but not in the ventromedian neurons or in the anterior and posterior optic commissure. Immunostaining in the ventromedian neurons suggests that orcokinin-related peptides play a role in the heterolateral transmission of photic input to the pacemaker and/or in the coupling of the bilateral pacemakers of the cockroach.This study was supported by the Deutsche Forschungsgemeinschaft, grant HO 950/9.     

Immunocytochemical characterization of the accessory medulla in the cockroach Leucophaea maderae

Several lines of evidence suggest that pigment-dispersing hormone-immunoreactive neurons with ramifications in the accessory medulla are involved in the circadian system of insects. The present study provides a detailed analysis of the anatomical and neurochemical organization of the accessory medulla in the brain of the cockroach Leucophaea maderae. We show that the accessory medulla is compartmentalized into central dense nodular neuropil surrounded by a shell of coarse fibers. It is innervated by neurons immunoreactive to antisera against serotonin and the neuropeptides allatostatin 7, allatotropin, corazonin, gastrin/cholecystokinin, FMRFamide, leucokinin I, and pigment-dispersing hormone. Some of the immunostained neurons appear to be local neurons of the accessory medulla, whereas others connect this neuropil to various brain areas, including the lamina, the contralateral optic lobe, the posterior optic tubercles, and the superior protocerebrum. Double-label experiments show the colocalization of immunoreactivity against pigment-dispersing hormone with compounds related to FMRFamide, serotonin, and leucokinin I. The neuronal and neurochemical organization of the accessory medulla is consistent with the current hypothesis for a role of this brain area as a circadian pacemaking center in the insect 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     

Candidates for the light entrainment pathway to the circadian clock of the Madeira cockroach Rhyparobia maderae

The circadian pacemaker controlling locomotor activity rhythms in the Madeira cockroach is located at the accessory medulla (AMe). The ipsi- and contralateral compound eyes provide light input to the AMe, possibly via the γ-aminobutyric acid (GABA)-immunoreactive (-ir) distal tract, which connects the glomeruli of the AMe to the ipsilateral medulla and lamina. To identify possible light-entrainment pathways, double-label immunocytochemistry was performed employing antibodies against GABA, myoinhibitory peptide (MIP), allatotropin (AT) and orcokinin (ORC). While all antisera tested, except the anti-ORC, prominently stained the glomeruli of the AMe, colocalization with anti-GABA was detected neither in the glomeruli nor in the distal tract. However, one median neuron that colocalized GABA-, AT- and MIP-immunoreactivity appeared to connect all glomeruli of the AMe to the medulla and lamina. Furthermore, one distal–frontoventral local neuron with arborizations in all glomeruli of the AMe colocalized anti-AT- and anti-MIP immunoreactivity. As candidates for contralateral light entrainment pathways, one ventromedian and one ventral neuron colocalized MIP- and ORC immunoreactivity, projecting via posterior and anterior commissures. Both branched in the interglomerular region of the AMe, where arborizations co-labeled with anti-ORC- and anti-MIP antisera. A possible role for MIP in light entrainment is supported also by injections of Rhyparobia maderae-specific MIP-2, which generated an all-advance phase-response curve late at night. Future experiments will challenge our hypothesis that GABA-, MIP- and AT-ir neurons provide ipsilateral light entrainment to all glomeruli, while MIP- and ORC-ir neurons carry contralateral light entrainment to the AMe’s interglomerular region, either delaying or advancing AMe neurons light-dependently.     

Light affects the branching pattern of peptidergic circadian pacemaker neurons in the brain of the cockroach Leucophaea maderae

Pigment-dispersing factor-immunoreactive neurons anterior to the accessory medulla (aPDFMes) in the optic lobes of insects are circadian pacemaker neurons in cockroaches and fruit flies. The authors examined whether any of the aPDFMes of the cockroach Leucophaea maderae are sensitive to changes in period and photoperiod of light/dark (LD) cycles as a prerequisite to adapt to changes in external rhythms. Cockroaches were raised in LD cycles of 11:11, 13:13, 12:12, 6:18, or 18:6 h, and the brains of the adults were examined with immunocytochemistry employing antisera against PDF and orcokinin. Indeed, in 11:11 LD cycles, only the number of medium-sized aPDFMes specifically decreased, while it increased in 13:13. In addition, 18:6 LD cycles increased the number of large- and medium-sized aPDFMes, as well as the posterior pPDFMes, while 6:18 LD cycles only decreased the number of medium-sized aPDFMes. Furthermore, PDF-immunoreactive fibers in the anterior optic commissure and orcokinin-immunoreactive fibers in both the anterior and posterior optic commissures were affected by different lengths of light cycles. Thus, apparently different groups of the PDFMes, most of all the medium-sized aPDFMes, which colocalize orcokinin, respond to changes in period and photoperiod and could possibly allow for the adjustment to different photoperiods.     

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)     

Antibodies against the PER protein of Drosophila label neurons in the optic lobe,central brain,and thoracic ganglia of the crickets Teleogryllus commodus and Teleogryllus oceanicus

We describe labeling of neurons in the central nervous system of two cricket species, Teleogryllus commodus and T. oceanicus, with both mono- and polyclonal antibodies against the PER protein. Western blots reveal that the monoclonal antibodies recognize a single protein with a molecular weight of approximately 94 kDa, i.e., similar to that of the PER protein of the moth, Anterea pernii. Neurons and their processes are labeled both in the optic lobes and in the central brain. Processes occur in the accessory medulla, the medulla, and proximal lamina, in the central complex, in the non-glomerular neuropil, and in the retrocerebral complex, suggesting that PER-containing neurons form a widely distributed network. Neurons and processes were also labeled in the meso- and metathoracic ganglia. Four to six PER-immunoreactive (ir) neurons with processes in the accessory medulla were double labeled by an antibody against pigment-dispersion factor (PDF), a peptide that is implicated in circadian rhythmicity in Drosophila. In the central brain, projections of fibers labeled by the anti-PER and anti-PDF antibodies were mainly distinct, with overlap only in a few restricted regions. In most neurons, including those projecting into the accessory medulla, PER labeling was restricted to the cytoplasm and there was no indication of circadian variation in the intensity of staining.     

A circadian neuropeptide,pigment-dispersing factor-PDF,in the last-summer cicada Meimuna opalifera: cDNA cloning and immunocytochemistry

Pigment-dispersing factor (PDF), an 18-amino acid neuropeptide, is a principal circadian neuromodulator functioning downstream of the insect brain's circadian clock, modulating daily rhythms of locomotor activity. Recently, we found that PDF precursors of the cricket Gryllus bimaculatus comprise a nuclear localization signal (NLS). Moreover, the nuclear localization of PDF immunoreactivity and the translocation of GFP-fused PDF precursor into the nucleus have both been demonstrated. These suggest a fundamental role for PDF peptide in the circadian clock system within the nucleus, in addition to its role in downstream neural events. In the present study, we carried out the cDNA cloning of PDF from adult brains of the last-summer cicada Meimuna opalifera, and found that an isolated clone (545 bp) encodes an ordinary PDF precursor protein. PDF peptide itself shows a high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to the crustacean beta-PDH peptides. The computer-assisted sequence analysis of PDF precursor revealed a possible translocation into the nucleus, despite the lack of a definite NLS-like sequence. Using immunocytochemistry, the optic lobes of M. opalifera revealed PDF-immunoreactive neurons in both the medulla and lamina neuropiles. All these PDF cells exhibited prominent immunolabeling of both their perikarya and axons, but not their nuclei. Our results provide the first structural and immunocytochemical identification of PDF neurons in Hemiptera.     

Extracellular long-term recordings of the isolated accessory medulla,the circadian pacemaker center of the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>, reveal ultradian and hint circadian rhythms

In the cockroach Leucophaea maderae transplantation studies located the circadian pacemaker center, which controls locomotor activity rhythms, to the accessory medulla (AMe), ventromedially to the medulla of the brain’s optic lobes. The AMe is densely innervated via GABA- and manyfold peptide-immunoreactive neurons. They express ultradian action potential oscillations in the gamma frequency range and form phase-locked assemblies of synchronously spiking cells. Peptide application resulted in transient rises of extracellularly recorded activity. It remained unknown whether transient rises in spontaneous electrical activity as a possible indication of peptide release occur in the isolated circadian clock in a rhythmic manner. In extracellular glass electrode recordings of the isolated AMe in constant darkness, which lasted at least 12 h, the distribution of daytime-dependent changes in activity independently of the absolute action potential frequency was examined. Rapid, transient changes in activity preferentially occurred at the mid-subjective night, with a minimum at the middle of the subjective day, hinting the presence of circadian rhythms in the isolated circadian clock. Additionally, ultradian rhythms in activity change that are multiples of a fundamental 2 h period were observed. We hypothesize that circadian rhythms might originate from coupled ultradian oscillations, possibly already at the single cell level.     

Signaling of Pigment-Dispersing Factor (PDF) in the Madeira Cockroach Rhyparobia maderae

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF''s importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²⁺ imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²⁺ baseline concentration and frequency of oscillating Ca²⁺ transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²⁺ levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1–4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²⁺ imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.     

Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab,Cancer borealis

Mass spectrometric methods were applied to determine the peptidome of the brain and thoracic ganglion of the Jonah crab (Cancer borealis). Fractions obtained by high performance liquid chromatography were characterized using MALDI-TOF MS and ESI-Q-TOF MS/MS. In total, 28 peptides were identified within the molecular mass range 750-3000Da. Comparison of the molecular masses obtained with MALDI-TOF MS with the calculated molecular masses of known crustacean peptides revealed the presence of at least nine allatostatins, three orcokinin precursor derived peptides, namely FDAFTTGFGHS, [Ala(13)]-orcokinin, and [Val(13)]-orcokinin, and two kinins, a tachykinin-related peptide and four FMRFamide-related peptides. Eight other peptides were de novo sequenced by collision induced dissociation on the Q-TOF system and yielded AYNRSFLRFamide, PELDHVFLRFamide or EPLDHVFLRFamide, APQRNFLRFamide, LNPFLRFamide, DVRTPALRLRFamide, and LRNLRFamide, which belong to the FMRFamide related peptide family, as well as NFDEIDRSGFA and NFDEIDRSSFGFV, which display high sequence similarity to peptide sequences within the orcokinin precursor of Orconectes limosus. Our paper is the first (neuro)peptidomic analysis of the crustacean nervous system.     

Presumptive insect circadian pacemakers in vitro: immunocytochemical characterization of cultured pigment-dispersing hormone-immunoreactive neurons of Leucophaea maderae

The accessory medulla with its associated pigment-dispersing hormone-immunoreactive neurons appears to be the pacemaker that controls the circadian locomotor activity rhythm of the cockroach Leucophaea maderae. To permit studies at the level of individual, identified, pacemaker neurons, we developed specific long-term primary cell cultures of fully differentiated adult neurons of the accessory medulla. As judged from soma diameter distribution, the cultures contain an unbiased representation of apparently all neuronal types of the accessory medulla. The cultured cells survive and grow processes for more than 2 months with or without additional hemocyte coculturing. However, a strong positive effect on initial outgrowth was observed with hemocyte coculturing. At least six different morphological cell types of the accessory medulla could be distinguished in vitro. Among these only one cell type, the monopolar type C cell, was recognized in vitro with an antiserum against the neuropeptide pigment-dispersing hormone. Thus, the identifiable monopolar type C cells are candidates for circadian pacemaker neurons and will be the focus of further physiological characterizations.     

Localization of leucomyosuppressin in the brain and circadian clock of the cockroach <Emphasis Type="Italic">Leucophaea maderae</Emphasis>

The myosuppressins (X1DVX2HX3FLRFamide), which reduce the frequency of insect muscle contractions, constitute a subgroup of the FMRFamide-related peptides. In the cockroach Leucophaea maderae, we have examined whether leucomyosuppressin (pQDVDHVFLRFamide) is present in the accessory medulla, viz., the circadian clock, which governs circadian locomotor activity rhythms. Antisera that specifically recognize leucomyosuppressin stain one to three neurons near the accessory medulla. MALDI-TOF mass spectrometry has confirmed the presence of leucomyosuppressin in the isolated accessory medulla. Injections of 1.15 pmol leucomyosuppressin into the vicinity of the accessory medulla at various circadian times have revealed no statistically significant effects on the phase of circadian locomotor activity rhythms. This is consistent with the morphology of the myosuppressin-immunoreactive neurons, which restrict their arborizations to the circadian clock and other optic lobe neuropils. Thus, leucomyosuppressin might play a role in the circadian system other than in the control of locomotor activity rhythms.     

Pigment-dispersing factor (PDF) has different effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.     

cDNA cloning and nuclear localization of the circadian neuropeptide designated as pigment-dispersing factor PDF in the cricket Gryllus bimaculatus

Pigment-dispersing factor (PDF) was recently reported to be a principal circadian neuromodulator involved in transmitting circadian rhythms of daily locomotion in insects. In Drosophila, PDF functions in some of the neurons expressing the clock genes period, timeless, Clock, and cycle, and those clock genes in turn regulate pdf gene expression. In the present study, we cloned a cDNA encoding PDF in the brain of a nocturnal insect, the cricket Gryllus bimaculatus, and found that an isolated clone (310 bp) codes for an extraordinarily short precursor protein with no definite signal sequence, but a nuclear localization signal (NLS)-like sequence instead. The cricket PDF exhibits high sequence identity (78-94%) and similarity (89-100%) to insect PDFs and also to crustacean beta-PDH peptides. In the optic lobes of G. bimaculatus there are PDF-immunoreactive neurons in both the medulla and lamina neuropiles. Among the strongly immunoreactive lamina PDF neurons, on electron microscopy we identified cells exhibiting distinct staining that is not only cytoplasmic but also nuclear. When GFP-fused PDF precursor proteins were expressed in COS-7 cells, distinct translocation of the fusion protein into the nucleus was observed. This is the first finding of PDF peptide in the nucleus, which suggests a fundamental role of PDF peptide per se in the circadian clock system.     

Pigment-dispersing hormone-immunoreactive fibers persist in crickets which remain rhythmic after bilateral transection of the optic stalks

Lesion studies combined with immunocytochemical experiments were used to examine whether pigment-dispersing hormone-immunoreactive processes in the midbrain of crickets correlate with the presence of circadian activity. After interruption of both optic stalks in the crickets Teleogryllus commodus and Gryllus bimaculatus animals retained circadian rhythms in their activity patterns. This suggests that there is another circadian oscillator in the midbrain. The pigment-dispersing hormone-immunoreactive fibers in the midbrain of both operated species appeared not to degenerate although they were separated from their somata in the optic lobes. These data could suggest that surviving fibers, separated from the somata of the circadian pacemaker in the optic lobes, contribute to the persistance of circadian activity in operated crickets.Abbreviations DD constant darkness - LD light dark cycles - PDH pigment-dispersing hormone - PDHLI pigment-dispersing hormone-like immunoreactivity - PDH Me pigment-dispersing hormone-immunoreactive cells of the accessory medulla - QV Quantification value     

Phase response curves of a molecular model oscillator: implications for mutual coupling of paired oscillators

Increasing evidence indicates that the accessory medulla is the circadian pacemaker controlling locomotor activity rhythms in insects. A prominent group of neurons of this neuropil shows immunoreactivity to the peptide pigment-dispersing hormone (PDH). In Drosophila melanogaster, the PDH-immunoreactive (PDH-ir) lateral neurons, which also express the clock genes period and timeless, are assumed to be circadian pacemaker cells themselves. In other insects, such as Leucophaea maderae, a subset of apparently homologue PDH-ir cells is a candidate for the circadian coupling pathway of the bilaterally symmetric clocks. Although knowledge about molecular mechanisms of the circadian clockwork is increasing rapidly, very little is known about mechanisms of circadian coupling. The authors used a computer model, based on the molecular feedback loop of the clock genes in D. melanogaster, to test the hypothesis that release of PDH is involved in the coupling between bilaterally paired oscillators. They can show that a combination of all-delay- and all-advance-type interactions between two model oscillators matches best the experimental findings on mutual pacemaker coupling in L. maderae. The model predicts that PDH affects the phosphorylation rate of clock genes and that in addition to PDH, another neuroactive substance is involved in the coupling pathway, via an all-advance type of interaction. The model suggests that PDH and light pulses, represented by two distinct classes of phase response curves, have different targets in the oscillatory feedback loop and are, therefore, likely to act in separate input pathways to the clock.     

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.     

Examination of the role of FMRFamide-related peptides in the circadian clock of the cockroach Leucophaea maderae

The accessory medulla, the circadian clock of the cockroach Leucophaea maderae, is abundant in neuropeptides. Among these neuropeptides are the FMRFamide-related peptides (FaRPs), which generally share the C-terminal RFamide. As a first step toward understanding the functional role of FaRPs in the circadian clock of the cockroach, immunocytochemistry with antisera against various FaRPs, MALDI-TOF mass spectrometry, and injections of two FaRPs combined with running-wheel assays were performed. Prominent FMRFamide-like immunoreactivity was found in maximally four soma clusters associated with the accessory medulla and in most neuropils of the protocerebrum. By MALDI-TOF mass spectrometry, various extended FMRFamides of the cockroach L. maderae were partially identified in thoracic perisympathetic organs, structures known to accumulate extended FMRFamides in insects. By mass match, several of these peptides were also detected in the accessory medulla. Injections of FMRFamide and Pea-FMRFa-7 (DRSDNFIRF-NH₂) into the vicinity of the accessory medulla caused time-dependent phase-shifts of locomotor activity rhythms at circadian times 8, 18, and 4. Thus, our data suggest a role for the different FaRPs in the control of circadian locomotor activity rhythms in L. maderae.     

Neuropeptide Y-immunoreactive neuronal system and colocalization with FMRFamide in the optic lobe and peduncle complex of the octopus (Octopus vulgaris)

The distribution of neuropeptide Y (NPY)-like immunoreactivity and its colocalization with FMRFamide were investigated in the optic lobe and peduncle complex of the octopus ( Octopus vulgaris) by using immunohistochemical techniques. In the optic lobe cortex, NPY-immunoreactive (NPY-IR) fibers were observed in the plexiform layer, although no NPY-IR somata were observed in the outer or inner granular cell layers. In the optic lobe medulla, NPY-IR somata were seen in the cell islands, and abundant NPY-IR varicose fibers were observed in the neuropil. Most of the NPY-IR structures in the medulla showed FMRFamide-like immunoreactivity. In the peduncle lobe, abundant NPY-IR and FMRFamide-IR (NPY/FMRF-IR) varicose fibers were seen in the basal zone neuropil of the peduncle lobe. In the olfactory lobe, NPY/FMRF-IR varicose fibers were also abundant in the neuropil of the three lobules. NPY/FMRF-IR somata, with processes running to various neuropils, were scattered in the median and posterior lobules. In the optic gland, many NPY/FMRF-IR varicose fibers formed a honeycomb pattern. These observations suggest that NPY/FMRF-IR neurons in the optic lobes participate in the modulation of visual information and that those in the optic gland are involved in the regulation of endocrine function.