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.     

The cockroach Leucophaea maderae needs more than juvenile hormone, vitellogenin and reserves to make a yolky egg

For the cockroach Leucophaea maderae the developmental profile of lipophorin (Lp) concentrations in the hemolymph was determined through the entire vitellogenic period. At mid-vitellogenesis the concentrations of Lp had risen to 6 times the level at emergence and then declined to 2/3 of such high values at ovulation. The racemic 10R,10S-JH-III bound to Lp with an affinity of K(d) = 5.76 nM and the natural enantiomer 10R-JH-III with a K(d) = 1.60 nM. Injections of anti-Lp into mated females caused a significantly reduced rate of oocyte growth and a substantial degree of oosorption. Injections of gamma-globulin did not significantly reduce oocyte growth and caused only a small number of oocytes to resorb. Starvation after mating had similar effects as treatment with anti-Lp. Because of the high affinity of JH to Lp and since Lp occurs in micromolar concentrations during vitellogenesis one can assume that practically all JH is bound and not available for hydrolysis by the JH esterases. Lp appears to function as an inhibitor of JH metabolism by the JHEs through substrate depletion. One may conclude that a normal rate of egg growth is only achieved when titers of Lp exceed those of JH and remove major portions of this substrate from degradation by the JHEs.     

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.     

The polypeptide structure of Vitellogenin and Vitellin from the cockroach,Leucophaea maderae

Vitellogenin (Vg) synthesized by the fat body of Leucophaea maderaeis made up of four polypeptides with molecular weights of 160,000, 105,000, 98,000, and 57,000. Other polypeptides previously reported as part of Vg are associated with other proteins. Vitellin (Vt), the yolk protein (YP) isolated from mature oocytes and from newly formed oothecae, is a protein with a sedimentation coefficient of 28s and consists of three polypeptides with molecular weights of 105,000, 85,000, and 57,000. During vitellogenesis, the YP of developing oocytes contains both Vt and a 14s component. The 14s component is made up of four polypeptides with molecular weights of 105,000, 90,000, 85,000, and 57,000. The data suggest that 14s may not be a discrete protein but rather a form in transition between Vg and Vt in which the 98,000 dalton polypeptide is converted to the 85,000 dalton polypeptide of Vt through a 90,000 dalton intermediate. The 160,000 dalton peptide of Vg does not appear to be a part of Vt. Under alkaline conditions, both the 14s component and Vt are reduced to a polypeptide with a lower sedimentation rate in sucrose gradients. When acid conditions are restored, a protein resembling 14s is obtained. This suggests that the YP is a loosely held aggregate of similar or identical proteins with a molecular weight of about 250,000.     

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     

Innervation of the foregut of the cockroach Leucophaea maderae and inhibition of spontaneous contractile activity by callatostatin neuropeptides

Abstract. The innervation of the gut of the cockroach Leucophaea madera (F.) has been studied by means of wholemount immunocytochemistry with antisera raised against Leu-callatostatin, a cockroach allatostatin homologue identified from neuropeptide isolation and gene studies in the blowfly Calliphora vomitoria. Leu-callatostatin-imunoreactive neurones in the brain, with axon trajectories in the stomatogastric nervous system, innervate the foregut and midgut. Neurones in the last abdominal ganglion supply the hindgut and the midgut via the proctodeal nerve. In addition to a rich callatostatin-immunoreactive nerve supply, the midgut, including the midgut caeca, contain numerous callatostatin-immunoreactive endocrine cells. Physiological studies show that the spontaneous contractile activities of the foregut, but not the hindgut, are inhibited by callatostatin neuropeptides. Leu-callatostatin 3 was the most potent of the range of Leu-and Met-callatostatins tested, with a dose-dependent response between 10^-13 and 10^-7 M. This is similar to the results obtained with the previously identified myoinhibitory peptide of L. maderae , leucomyosuppressin. However, this peptide, with a different type of structure to the allatostatins, inhibits both foregut and hindgut motility equally. Experiments with a series of analogues of the Met-callatostatins showed that the free acid (as opposed to the carboxyamidated peptide) and N-terminally truncated peptides were inactive. These morphological and physiological results are thought to be representative of the, as yet unidentified, naturally occurring allatostatin homologues of L. maderae. This family of peptides should be added to the increasing list of insect gut myoinhibitory substances.     

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     

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).     

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.