Monika Stengl

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 flys 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.

Pigment dispersing hormone-immunoreactive neurons in the cockroach Leucophaea maderae share properties with circadian pacemaker neurons

Neurons immunoreactive with antisera against the crustacean peptide β-pigment dispersing hormone fullfill several anatomical criteria proposed for circadian pacemakers in the brain of the cockroach Leucophaea maderae. These include position of somata, projections to the lamina and midbrain and possible coupling pathways between the two pacemakers through commissural fibers. In behavioral experiments combined with lesion studies and immunocytochemical investigations we examined whether the presence of pigment-dispersing hormone-immunoreactive arborizations in the midbrain of the cockroach correlates with the presence of circadian locomotor activity. No rhythm was detected after severing both optic stalks in any animal for at least 12 days. Within the same time pigment-dispersing hormone-immunoreactive fibers in the midbrain disappeared. Two to seven weeks after the operation some of the cockroaches regained circadian locomotor activity, while others remained arrhythmic. In all cockroaches which regained rhythmic behavior pigment-dispersing hormone-immunoreactive fibers had regenerated and had largely found their original targets within the brain. In all arrhythmic cockroaches either none or very little regeneration had occurred. The period of the regained circadian activity inversely correlated with the number of regenerated immunoreactive commissural fibers. These data provide further evidence for the involvement of pigment-dispersing hormone-immunoreactive neurons in circadian clocks of orthopteroid insects.

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.

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.

Ectopic transplantation of the accessory medulla restores circadian locomotor rhythms in arrhythmic cockroaches (Leucophaea maderae).

SUMMARY The presence of an endogenous circadian clock in the brain of an animal was first demonstrated in the cockroach Leucophaea maderae. However, the clocks cellular basis remained elusive until pigment-dispersing hormone-immunoreactive neurons, which express the clock genes period and timeless in Drosophila, were proposed as pacemaker candidates. In several insect species, pigment-dispersing hormone-immunoreactive neurons are closely associated with the accessory medulla, a small neuropil in the optic lobe, which was suggested to be a circadian clock neuropil. Here, we demonstrate that ectopic transplantation of adult accessory medulla into optic lobe-less cockroaches restores circadian locomotor activity rhythms in L. maderae. All histologically examined cockroaches that regained circadian activity regenerated pigment-dispersing hormone-immunoreactive fibres from the grafts to original targets in the protocerebrum. The data show that the accessory medulla is the circadian pacemaker controlling locomotor activity rhythms in the cockroach. Whether pigment-dispersing hormone-immunoreactive neurons are the only circadian pacemaker cells controlling locomotor activity rhythms remains to be examined.

Pigment-Dispersing Factor and GABA Synchronize Cells of the Isolated Circadian Clock of the Cockroach Leucophaea maderae

Pigment-dispersing factor-immunoreactive circadian pacemaker cells, which arborize in the accessory medulla, control circadian locomotor activity rhythms in Drosophila as well as in the cockroach Leucophaea maderae via unknown mechanisms. Here, we show that circadian pacemaker candidates of the accessory medulla of the cockroach produce regular interspike intervals. Therefore, the membrane potential of the cells oscillates with ultradian periods. Most or all oscillating cells within the accessory medulla are coupled via synaptic and nonsynaptic mechanisms, forming different assemblies. The cells within an assembly share the same ultradian period (interspike interval) and the same phase (timing of spikes), whereas cells between assemblies differ in phase. Apparently, the majority of these assemblies are formed by inhibitory GABAergic synaptic interactions. Application of pigment-dispersing factor phase locked and thereby synchronized different assemblies. The data suggest that pigment-dispersing factor inhibits GABAergic interneurons, resulting in disinhibition and phase locking of their postsynaptic cells, which previously belonged to different assemblies. Our data suggest that phase control of action potential oscillations in the ultradian range is a main task of the circadian pacemaker network. We hypothesize that neuropeptide-dependent phase control is used to gate circadian outputs to locomotor control centers.

The role of the coreceptor Orco in insect olfactory transduction.

Insects sense odorants with specialized odorant receptors (ORs). Each antennal olfactory receptor neuron expresses one OR with an odorant binding site together with a conserved coreceptor called Orco which does not bind odorants. Orco is necessary for localization of ORs to dendritic membranes and, thus, is essential for odorant detection. It forms a spontaneously opening cation channel, activated via phosphorylation by protein kinase C. Thereafter, Orco is also activated via cyclic adenosine monophosphate (cAMP). Orco forms homo—as well as heteromers with ORs with unknown stoichiometry. Contradictory publications suggest different mechanisms of olfactory transduction. On the one hand, evidence accumulates for the employment of more than one G protein-coupled olfactory transduction cascade in different insects. On the other hand, results from other studies suggest that the OR–Orco complex functions as an odorant-gated cation channel mediating ionotropic signal transduction. This review analyzes conflicting hypotheses concerning the role of Orco in insect olfactory transduction. In conclusion, in situ studies in hawkmoths falsify the hypothesis that Orco underlies odorant-induced ionotropic signal transduction in all insect species. Instead, Orco forms a metabotropically gated, slow cation channel which controls odorant response threshold and kinetics of the sensory neuron.

Optic lobe commissures in a three-dimensional brain model of the cockroach Leucophaea maderae: A search for the circadian coupling pathways

The circadian rhythm of locomotor activity in the cockroach Leucophaea maderae is controlled by bilaterally symmetric, apparently directly coupled, circadian pacemakers in the optic lobes. Strong evidence predicts that ventromedial to the medulla, the accessory medulla with associated pigment‐dispersing hormone‐immunoreactive neurons is this circadian clock. In search for direct coupling pathways between both clocks, we performed horseradish peroxidase backfills from one optic stalk as well as dextran and horseradish peroxidase injections into one accessory medulla. Seven commissures with projections in the contralateral optic lobe were identified and reconstructed. Three of these commissures connected both accessory medullae. Two of these resembled the arborization pattern of the pigment‐dispersing hormone‐immunoreactive neurons, which are circadian pacemaker candidates in insects. This finding suggests that some of these pacemaker candidates form a direct circadian coupling pathway. For better visualization of reconstructed commissures, we implemented the reconstructions into a three‐dimensional model of the cockroach brain. J. Comp. Neurol. 443:388–400, 2002.

Pheromone Transduction in Moths

Calling female moths attract their mates late at night with intermittent release of a species-specific sex-pheromone blend. Mean frequency of pheromone filaments encodes distance to the calling female. In their zig-zagging upwind search male moths encounter turbulent pheromone blend filaments at highly variable concentrations and frequencies. The male moth antennae are delicately designed to detect and distinguish even traces of these sex pheromones amongst the abundance of other odors. Its olfactory receptor neurons sense even single pheromone molecules and track intermittent pheromone filaments of highly variable frequencies up to about 30 Hz over a wide concentration range. In the hawkmoth Manduca sexta brief, weak pheromone stimuli as encountered during flight are detected via a metabotropic PLCβ-dependent signal transduction cascade which leads to transient changes in intracellular Ca2+ concentrations. Strong or long pheromone stimuli, which are possibly perceived in direct contact with the female, activate receptor-guanylyl cyclases causing long-term adaptation. In addition, depending on endogenous rhythms of the moths physiological state, hormones such as the stress hormone octopamine modulate second messenger levels in sensory neurons. High octopamine levels during the activity phase maximize temporal resolution cAMP-dependently as a prerequisite to mate location. Thus, I suggest that sliding adjustment of odor response threshold and kinetics is based upon relative concentration ratios of intracellular Ca2+ and cyclic nucleotide levels which gate different ion channels synergistically. In addition, I propose a new hypothesis for the cyclic nucleotide-dependent ion channel formed by insect olfactory receptor/coreceptor complexes. Instead of being employed for an ionotropic mechanism of odor detection it is proposed to control subthreshold membrane potential oscillation of sensory neurons, as a basis for temporal encoding of odors.

Adaptation in pheromone-sensitive trichoid sensilla of the hawkmoth Manduca sexta

SUMMARY In extracellular tip recordings from long trichoid sensilla of male Manduca sexta moths, we studied dose–response relationships in response to bombykal stimuli of two different durations in the adapted and the non-adapted state. Bombykal-responsive cells could be distinguished from non-bombykal-sensitive cells in each trichoid sensillum because the bombykal-responsive cell always generated the action potentials of larger initial amplitude. The bombykal cell, which was recorded at a defined location within a distal flagellar annulus, can resolve at least four log10-units of pheromone concentrations but is apparently unable to encode all stimulus durations tested. Parameters of the amplitude-modulated sensillar potential and the frequency-modulated action potential responses were examined in different states of adaptation. Evidence is presented for the existence of several mechanisms of adaptation, which affect distinct steps of the transduction cascade. After adapting pheromone stimuli, the sensillar potential rises to a lower amplitude and declines faster compared with the non-adapted response. In addition, the frequency of the adapted action potential response is reduced. Only the time of rise of the sensillar potential is differentially affected by adapting pheromone stimuli of different duration. The time of rise does not increase after short, but only after long, adapting stimuli. Both short and long adapting stimuli shift the dose–response curves of the sensillar potential amplitude, as well as the initial slope of its rising phase, to higher stimulus concentrations by approximately one log10-unit. The shift in the dose–response curve of the action potential response is larger than for the sensillar potential response, suggesting that an additional adaptation mechanism acts at the level of action potential generation. Furthermore, a faster decline of the sensillar potential after short and long adapting stimuli suggests that the resting potential of the olfactory receptor neuron is stabilized.