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 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     

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.     

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.     

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.     

Ultrastructure of pigment-dispersing hormone-immunoreactive neurons in a three-dimensional model of the accessory medulla of the cockroach<Emphasis Type="Italic"> Leucophaea maderae</Emphasis>

Locomotor activity rhythms of the cockroach Leucophaea maderae are orchestrated by two bilaterally symmetric, mutually coupled, circadian pacemakers. They lie in the optic lobes of the brain and are confined to the accessory medulla (AMe), ventro-medially to the medulla. The AMe is innervated by approximately 12 pigment-dispersing hormone (PDH)-immunoreactive anterior medulla neurons (PDHMe), which are circadian pacemaker candidates in the fruitfly and the cockroach. We have developed a three-dimensional computer model of the AMe and associated structures as a framework for neuroanatomical studies. Our greatly improved understanding of this structure in space has allowed us further to subdivide the anterior PDHMe into three subgroups, i.e., large, medium-sized, and small anterior PDHMe. The synaptic connections of two of these subgroups have been examined within subcompartments of the AMe by light and electron microscopy. The large, intensely staining, anterior PDHMe contain medium-sized dense-core vesicles and form input and output synapses with profiles densely filled with clear vesicles primarily in the anterior and shell neuropil of the AMe. The medium-sized anterior PDHMe contain large dense-core vesicles and constitute input and output synapses either with profiles being densely filled with clear vesicles, or with profiles containing granular dense-core vesicles. The small, weakly staining anterior PDHMe belong to a morphological group different from the large and medium-sized PDHMe and cannot be further identified at the electron-microscopic level because of their weak PDH immunoreactivity.This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants STE 531/7-1, 2, 3, and Human Science Frontier     

Azadirachtin affects the circadian rhythm of locomotion in Leucophaea maderae

Abstract

Azadirachtin shortens the period length of the locomotor activity rhythm in the circadian rhythm of Leucophaea maderae and induces splitting of this rhythm in two components in about 40% of the animals. The phase relationship between the two components is 180°. Both shortening of period and splitting are more pronounced in animals possessing longer periods before the injection of azadirachtin.     

Immunocytochemical localization and immunochemical characterization of an insulin-related peptide in the insect Leucophaea maderae

Summary Immunocytochemical tests with eight monoclonal antibodies against either bovine or human insulin and seven polyclonal antibodies against bovine insulin were carried out to determine the presence of insulin-like neuropeptides in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Reaction products identified in the brain, subesophageal ganglion, and corpus cardiacum-corpus allatum complex indicate the presence of materials resembling mammalian insulins in its antigenic properties. The immunostaining observed with monoclonal antibodies appears to indicate the occurrence of an insulin-related peptide that shows sequential similarities with parts of both the A- and B-chains of mammalian insulin molecules. These suppositions are supported by the results of dot-blot and two-site time-resolved immunofluorometric assay (TR-IFMA) screenings of fractions of Leucophaea tissue extracts obtained by chromatography. The polyclonal antibodies yielded reaction products in some of the same areas and in additional parts of the neuroendocrine system not visualized by the monoclonal antibodies. Immunoreaction was observed in the following areas: the pars intercerebralis of the protocerebrum, the nervi corporis cardiaci I transporting insulin-like material to the corpus cardiacum, the dorsolateral protocerebral area and the optic lobes, the deutocerebrum, the tritocerebrum, and the subesophageal ganglion. In addition, smaller cell bodies with immunoreactive deposits occur at the border between proto- and deutocerebrum, and in the central area of the protocerebrum. The distribution of reactive material in the corpus cardiacum-corpus allatum complex after use of both groups of antibodies was the same. The fact that polyclonal and monoclonal antibodies yielded reaction products in different cells of the brain suggests either that the two groups of antibodies recognize different epitopes of the same molecule, or that they reveal two different types of immunoreactive molecules related to mammalian insulins. Together with the biochemical data reported by Nagasawa and coworkers (PNAS 83, 1986) the present immunocytochemical analysis has established a closer relationship between mammalian and insect insulins than was previously known.Supported in part by NIH grant NS 2344-02 (B.S.) and SNF grant 11-5082 and 11-7705 (G.N.H.)     

Diversity of prolactin systems in the insect Leucophaea maderae: Use of antiserum polyclonality for immunocytochemical detection of neuropeptide heterogeneity

Summary The presence of prolactin-like neuropeptides was demonstrated immunocytochemically in the brain and affiliated neuroendocrine structures of the insect Leucophaea maderae. Use of the unlabelled peroxidase-antiperoxidase method of Sternberger revealed a rather widespread and differential distribution of reaction products resembling human (hPRL) and ovine (oPRL) prolactin. Tests with antirat PRL antibody were negative. The specificity of the antibodies used was established by liquid-phase absorptions and confirmed in tissue control systems. In L. maderae, anti-oPRL identifies part of an oPRL-like molecule different from human and rat PRL. Anti-hPRL reveals part of a human and ovine PRL-like molecule different from rat prolactin. These results indicate the occurrence, in the nervous tissue of one insect species, of at least two types of prolactin-like molecules.Supported in part by SNF grants 11-5082 and 11-6652 (G.N.H.) and NIH Grant NS 22344-02 (B.S.). The authors are indebted to Mrs. Bente Hershøj for skillful technical assistance     

Neurohormones as putative circadian clock output signals in the central nervous system of two cricket species

Antisera to the neuropeptides corazonin (Crz) and crustacean cardioactive peptide (CCAP) and to the diapause hormone (DH) react with small sets of neurones in the cephalic ganglia of the crickets Dianemobius nigrofasciatus and Allonemobius allardi. The distribution of their immunoreactivities is similar in the two species and overlaps with the locations of presumed circadian clock components in the optic lobes, protocerebrum, tritocerebrum, suboesophageal ganglion (SOG) and frontal ganglion. D. nigrofasciatus contains two Crz-immunoreactive (Crz-ir) cells in each optic lobe, six cell groups in the protocerebrum, four in the tritocerebrum, and one in SOG, whereas A. allardi harbours only five Crz-ir groups in the protocerebrum and four in the tritocerebrum. CCAP immunoreactivity occurs in both species in four protocerebrum cell clusters, four tritocerebrum cell clusters, four SOG cell clusters, one frontal ganglion cell cluster, and two optic lobe cell clusters; D. nigrofasciatus possesses two additional cells with unique links to the lamina in the optic lobe. DH-related antigens are present in four cell clusters in the optic lobe, six (D. nigrofasciatus) or eight (A. allardi) in the protocerebrum, four in the tritocerebrum, and three (A. allardi) or five (D. nigrofasciatus) in the SOG. Some of the detected cells also react with antibody to the clock protein Period (PER) or lie close to PER-ir cells. Crickets reared at two different photoperiods do not differ in the distribution and intensity of immunoreactivities. No changes have been detected during the course of diurnal light/dark cycles, possibly because the antisera react with persistent prohormones, whereas circadian fluctuations may occur at the level of their processing or of hormone release. The projection of immunoreactive fibres to several brain regions, the stomatogastric nervous system and the neurohaemal organs indicates multiple functions of the respective hormones. The work was supported by the “Research for Future” program of the Japan Society for the Promotion of Science (JSPS, 99L01205) and by the JSPS Postdoctoral Fellowship for Foreign Researchers (no. P 04197).     

Pigment-dispersing hormone-immunoreactive neurons and their relation to serotonergic neurons in the blowfly and cockroach visual system

Summary The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects, which can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups (Rao and Riehm 1989). In this investigation we have extended these immunocytochemical studies to include the blowfly Phormia terraenovae and the cockroach Leucophaea maderae. In the former species tissue extracts were also tested in a bioassay: extracts of blowfly brains exhibited PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. Using standard immunocytochemical techniques, we could demonstrate a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroach. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies which also send processes into the medulla. These neurons share many features in their laminamedulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier (see Nässel 1988). It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies could be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina was found to be very similar to the PDH-IR ones. It is hence tempting to speculate that in both species the PDH-and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system. In the flies photoreceptor properties may be regulated by action of the two set of neurons at sites peripheral to the lamina synaptic layer, possibly by paracrine release of messengers.     

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     

Neuropeptides in interneurons of the insect brain

A large number of neuropeptides has been identified in the brain of insects. At least 35 neuropeptide precursor genes have been characterized in Drosophila melanogaster, some of which encode multiple peptides. Additional neuropeptides have been found in other insect species. With a few notable exceptions, most of the neuropeptides have been demonstrated in brain interneurons of various types. The products of each neuropeptide precursor seem to be co-expressed, and each precursor displays a unique neuronal distribution pattern. Commonly, each type of neuropeptide is localized to a relatively small number of neurons. We describe the distribution of neuropeptides in brain interneurons of a few well-studied insect species. Emphasis has been placed upon interneurons innervating specific brain areas, such as the optic lobes, accessory medulla, antennal lobes, central body, and mushroom bodies. The functional roles of some neuropeptides and their receptors have been investigated in D. melanogaster by molecular genetics techniques. In addition, behavioral and electrophysiological assays have addressed neuropeptide functions in the cockroach Leucophaea maderae. Thus, the involvement of brain neuropeptides in circadian clock function, olfactory processing, various aspects of feeding behavior, and learning and memory are highlighted in this review. Studies so far indicate that neuropeptides can play a multitude of functional roles in the brain and that even single neuropeptides are likely to be multifunctional.The original research in the authors’ laboratories was supported by DFG grants HO 950/14 and 950/16 (U.H.) and Swedish Research Council grant VR 621-2004-3715 (D.R.N).     

hormone nuclear receptor (EcR) exhibits circadian cycling in certain tissues, but not others, during development in Rhodnius prolixus (Hemiptera)

The insect moulting hormones, viz. the ecdysteroids, regulate gene expression during development by binding to an intracellular protein, the ecdysteroid receptor (EcR). In the insect Rhodnius prolixus, circulating levels of ecdysteroids exhibit a robust circadian rhythm. This paper demonstrates associated circadian rhythms in the abundance and distribution of EcR in several major target tissues of ecdysteroids, but not in others. Quantitative analysis of immunofluorescence images obtained by confocal laser-scanning microscopy following the use of anti-EcR has revealed a marked daily rhythm in the nuclear abundance of EcR in cells of the abdominal epidermis, brain, fat body, oenocytes and rectal epithelium of Rhodnius. This EcR rhythm is synchronous with the rhythm of circulating hormone levels. It free-runs in continuous darkness for several cycles, showing that EcR nuclear abundance is under circadian control. Circadian control of a nuclear receptor has not been shown previously in any animal. We infer that the above cell types detect and respond to the temporal signals in the rhythmic ecdysteroid titre. In several cell types, the rhythm in cytoplasmic EcR peaks several hours prior to the EcR peak in the nucleus each day, thereby implying a daily migration of EcR from the cytoplasm to the nucleus. This finding shows that EcR is not a constitutive nuclear receptor, as has previously been assumed. In the brain, rhythmic nuclear EcR has been found in peptidergic neurosecretory cells, indicating a potential pathway for feedback regulation of the neuroendocrine system by ecdysteroids, and also in regions containing circadian clock neurons, suggesting that the circadian timing system in the brain is also sensitive to rhythmic ecdysteroid signals. This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada.     

Phospholipid synthesis in the fat body endoplasmic reticulum during primary and secondary juvenile hormone stimulation of vitellogenesis inLeucophaea maderae

Summary Juvenile hormone (JH) treatment coordinately stimulated the dose-dependent synthesis of vitellogenin and endoplasmic reticulum (ER) membrane phospholipids in fat body cells from allatectomized adult females ofLeucophaea maderae. Animals were pulse-labeled in vivo with [³²P] to simultaneously measure the rates of synthesis of the phosphorylated subunits of vitellogenin and the structural phospholipids of the ER membranes. Phospholipid synthesis in ER membranes from nontarget tissues for JH such as thoracic muscle, midgut, and larval fat body was unresponsive to hormone treatment. The proliferation of ER in response to JH treatment was thus restricted to tissue that was competent to synthesize vitellogenin.Primary and secondary vitellogenin induction was measured in allatectomized adult females treated 12 days apart with JH-III. The time-course of the primary response for vitellogenin and ER phospholipid synthesis was characterized by a 24 h latent period, a rapid increase to a maximum at 72 h, and then a gradual decline. During secondary induction, vitellogenin accumulated in the hemolymph nearly twice as fast as before and peaked at a concentration of 38 g/l. This vitellogenin titer was approximately two-fold higher than that found at the height of the primary response. During both primary and secondary stimulation with JH, ER phospholipid synthesis, as measured by [¹⁴C]choline incorporation into microsomal phosphatidylcholine, was stimulated five-fold over the untreated control animals. The amplified production of vitellogenin during the secondary response was associated with a 24 h-earlier peak of ER phospholipid synthesis in the fat body.     

Development of the circadian clock in the German cockroach, Blattella germanica

The cell distribution and immunoreactivity (ir) against period (PER), pigment dispersing factor (PDF) and corazonin (CRZ), were compared between adults and nymphs in the central nervous system of the German cockroach. Although PER-ir cells in the optic lobes (OL) were expressed in the nymphs from the first instar, the links between major clock cells became more elaborated after second/third instar. A circadian rhythm of locomotion was initiated at the fourth/fifth instar. The results suggest that the clock was running from hatching, but the control network needed more time to develop. In addition, the putative downstream regulators, PDF-ir and CRZ-ir, are co-localized in various regions of the brain, indicating potential output routes of the circadian clock. CRZ-ir cells with typical morphology of neurosecretory cells in the dorsolateral protocerebrum send out three neural fibers to reach the ipsilateral corpora cardiaca (CC), the antennal lobe and two hemispheres of the protocerebrum. Based on co-localization with some PER-ir/PDF-ir cells, the CRZ-ir cells have the potential to serve as a bridge between circadian neural signals and endocrine regulation. Based on PDF's role in the regulation of locomotion, our results support the finding that the locomotor circadian rhythm is possibly controlled by a hormonal route.