Jürgen Boeckh (1934-2023) and Vera Boeckh, née von Zwehl (1928-2022): pioneers of sensory physiology and neuroethology.

Jürgen Boeckh, a respected pioneer of insect olfaction died shortly after his beloved wife Vera Boeckh, née von Zwehl, who pioneered insect vision. Both met in 1958, at the Zoological Institute in Munich. There, Jürgen worked in the group of his PhD advisor Dietrich Schneider, while Vera finished her PhD with Werner Jacobs before she joined the group of Hansjochem Autrum. There, Vera characterized the spectral sensitivity of bee photoreceptors, laying the physiological foundation of Karl von Frisch´s behavioral experiments with bee color vision. Meanwhile, Jürgen focused on the physiological characterization of insect antennal olfactory sensilla. In 1962 Vera and Jürgen married in Munich. Sadly, but characteristic of German woman at these times, Vera´s career ended after her marriage, while Jürgen moved with his mentor Schneider to the Max Planck Institute of Behavioral Physiology in Seewiesen near Munich, which became a famous cradle of insect neuroethology. Vera accompanied and supported her husband Jürgen´s career during his scientific Wanderschaft which ended in 1969, when Jürgen received a full professorship at the University of Regensburg. There, Jürgen became an accomplished German professor, focusing on insect olfaction from peripheral sensory transduction to information processing in the brain´s antennal lobe. After Jürgens retirement in 2000 they moved to Hopfen, Enzensberg near Füssen, where they enjoyed happy years together, before especially Vera´s health deteriorated. Both died shortly after one another during the Corona pandemic. We lost a remarkable couple of insect scientists that will be remembered as pioneers of sensory physiology and neuroethology.

Mutagenesis of odorant coreceptor Orco fully disrupts foraging but not oviposition behaviors in the hawkmoth Manduca sexta.

The hawkmoth Manduca sexta and one of its preferred hosts in the North American Southwest, Datura wrightii, share a model insect-plant relationship based on mutualistic and antagonistic life-history traits. D. wrightii is the innately preferred nectar source and oviposition host for M. sexta Hence, the hawkmoth is an important pollinator while the M. sexta larvae are specialized herbivores of the plant. Olfactory detection of plant volatiles plays a crucial role in the behavior of the hawkmoth. In vivo, the odorant receptor coreceptor (Orco) is an obligatory component for the function of odorant receptors (ORs), a major receptor family involved in insect olfaction. We used CRISPR-Cas9 targeted mutagenesis to knock out (KO) the MsexOrco gene to test the consequences of a loss of OR-mediated olfaction in an insect-plant relationship. Neurophysiological characterization revealed severely reduced antennal and antennal lobe responses to representative odorants emitted by D. wrightii In a wind-tunnel setting with a flowering plant, Orco KO hawkmoths showed disrupted flight orientation and an ablated proboscis extension response to the natural stimulus. The Orco KO gravid female displayed reduced attraction toward a nonflowering plant. However, more than half of hawkmoths were able to use characteristic odor-directed flight orientation and oviposit on the host plant. Overall, OR-mediated olfaction is essential for foraging and pollination behaviors, but plant-seeking and oviposition behaviors are sustained through additional OR-independent sensory cues.

Cyclic nucleotide-dependent ionic currents in olfactory receptor neurons of the hawkmoth Manduca sexta suggest pull-push sensitivity modulation.

Olfactory receptor neurons (ORNs) of the hawkmoth Manduca sexta sensitize via cAMP- and adapt via cGMP-dependent mechanisms. Perforated patch clamp recordings distinguished 11 currents in these ORNs. Derivatives of cAMP and/or cGMP antagonistically affected three of five K+ currents and two non-specific cation currents. The Ca2+ -dependent K+ current IK(Ca2+) and the sensitive pheromone-dependent K+ current IK(cGMP-) , which both express fast kinetics, were inhibited by 8bcGMP, while a slow K+ current, IK(cGMP+) , was activated by 8bcGMP. Furthermore, application of 8bcAMP blocked slowly activating, zero mV-reversing, non-specific cation currents, ILL and Icat(PKC?) , which remained activated in the presence of 8bcGMP. Their activations pull the membrane potential towards their 0-mV reversal potentials, in addition to increasing intracellular Ca2+ levels voltage- and ILL -dependently. Twenty minutes after application, 8bcGMP blocked a TEA-independent K+ current, IK(noTEA) , and a fast cation current, Icat(nRP) , which both shift the membrane potential to negative values. We conclude that conditions of sensitization are maintained at high levels of cAMP, via specific opening/closure of ion channels that allow for fast kinetics, hyperpolarized membrane potentials, and low intracellular Ca2+ levels. In contrast, adaptation is supported via cGMP, which antagonizes cAMP, opening Ca2+ -permeable channels with slow kinetics that stabilize depolarized resting potentials. The antagonistic modulation of peripheral sensory neurons by cAMP or cGMP is reminiscent of pull-push mechanisms of neuromodulation at central synapses underlying metaplasticity.

Circadian pacemaker neurons of the Madeira cockroach are inhibited and activated by GABAA and GABAB receptors.

GABA is the most abundant neurotransmitter in the circadian pacemaker circuits of mammals and insects. In the Madeira cockroach the accessory medulla (AME) in the brain's optic lobes is the circadian clock that orchestrates rest-activity rhythms in synchrony with light dark cycles. Three prominent GABAergic tracts connect the AME to termination sites of compound eye photoreceptors in the lamina and medulla. Parallel GABAergic light entrainment pathways were suggested to either advance or delay the clock for adjustment to changing photoperiods. In agreement with this hypothesis GABA activated or inhibited AME clock neurons, allowing for the distinction of three different GABA response types. Here, we examined which GABA receptors are responsible for these response types. We found that both ionotropic GABAA receptors and metabotropic GABAB receptors were expressed in AME clock cells. Via different signalling pathways, either one of them could account for all three GABA response types. The muscimol-dependently activated GABAA receptor formed a chloride channel, while the SKF 97541-dependently activated GABAB receptor signalled via G-proteins, apparently targeting potassium channels. Expression of chloride exporters or importers determined whether GABAA receptor activation hyper- or depolarized AME neurons. For GABAB receptor responses second messenger gated channels present in the clock cells appeared to decide about the polarity of the GABA response. In summary, circadian clock neurons co-expressed inhibitory and/or excitatory GABAA and GABAB receptors in various combinations, while cotransporter expression and the set of second messenger gated ion channels present allowed for distinct signalling in different clock neurons.

GABA- and serotonin-expressing neurons take part in inhibitory as well as excitatory input pathways to the circadian clock of the Madeira cockroach Rhyparobia maderae.

In the Madeira cockroach, pigment-dispersing factor-immunoreactive (PDF-ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain's optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light-dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple-label immunocytochemistry the serotonin (5-HT)-ir anterior fibre fan and GABA-ir pathways connecting the AME- and optic lobe neuropils. Colocalization of 5-HT with PDF was confirmed in PDF-ir lamina neurons (PDFLAs). Double-labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA-ir medial layer fibre tract connected the AME to the medulla's medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca2+ imaging on primary cell cultures of the AME and with loose-patch-clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5-HT in any optic lobe neuron, GABA- and 5-HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA- and excitatory 5-HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA- and 5-HT-immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed.

Identification and characterization of the bombykal receptor in the hawkmoth Manduca sexta.

Manduca sexta females attract their mates with the release of a species-specific sex-pheromone blend, with bombykal (E,Z)-10,12-hexadecadienal and (E,E,Z)-10,12,14-hexadecatrienal being the two major components. Here, we searched for the hawkmoth bombykal receptor in heterologous expression systems. The putative pheromone receptor MsexOr1 coexpressed with MsexOrco in Xenopus oocytes elicited dose-dependent inward currents upon bombykal application (10-300 µmol l-1), and coexpressed in HEK293 and CHO cells caused bombykal-dependent increases in the intracellular free Ca2+ concentration. In addition, the bombykal receptor of Bombyx mori BmOr3 coexpressed with MsexOrco responded to bombykal (30-100 µmol l-1) with inward currents. In contrast, MsexOr4 coexpressed with MsexOrco responded neither to bombykal (30-100 µmol l-1) nor to the (E,E,Z)-10,12,14-hexadecatrienal mimic. Thus, MsexOr1, but not MsexOrco and probably not MsexOr4, is the bombykal-binding pheromone receptor in the hawkmoth. Finally, we obtained evidence that phospholipase C and protein kinase C activity are involved in the hawkmoth's bombykal-receptor-mediated Ca2+ signals in HEK293 and CHO cells.

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.

Neuropeptidergic input pathways to the circadian pacemaker center of the Madeira cockroach analysed with an improved injection technique.

Light entrainment pathways synchronize the circadian clock of almost all species of the animal and plant kingdom to the daily light dark cycle. In the Madeira cockroach Rhyparobia (Leucophaea) maderae, the circadian clock is located in the accessory medulla of the brain's optic lobes. The clock has abundant neuropeptides with unknown functions. Previous studies suggested that myoinhibitory peptides (MIPs), orcokinins (ORCs), and allatotropin (AT) take part in light input pathways to the circadian clock. As the sequences of AT and ORCs of R. maderae have not yet been determined, with matrix-assisted laser desorption/ionization-time of flight mass spectrometry, the respective Rhyparobia peptides were characterized. To search for light-like phase-shifting inputs to the circadian clock, Rhyparobia-MIP-1, Rhyparobia-AT, and Rhyparobia-ORC were injected at different circadian times, combined with locomotor activity assays. An improved, less invasive injection method was developed that allowed for the analysis of peptide effects within <2 weeks after injection. Rhyparobia-MIP-1 and Rhyparobia-AT injections resulted in dose-dependent monophasic phase response curves with maximum delays at the beginning of the subjective night, similar to light-dependent phase delays. In contrast to Manduca sexta-AT, Rhyparobia-AT did not phase advance locomotor activity rhythms. Only injections of Rhyparobia-ORCs resulted in a biphasic light-like phase response curve. Thus, it is hypothesized that Rhyparobia-MIP-1 and -AT are candidates for relaying light-dependent delays and/or non-photic inputs to the clock, whereas Rhyparobia-ORCs might be part of the light-entrainment pathways relaying phase delays and advances to the circadian clock of the Madeira cockroach.

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.

Calcium responses of circadian pacemaker neurons of the cockroach Rhyparobia maderae to acetylcholine and histamine.

The accessory medulla (aMe) is the pacemaker that controls circadian activity rhythms in the cockroach Rhyparobia maderae. Not much is known about the classical neurotransmitters of input pathways to the cockroach circadian system. The circadian pacemaker center receives photic input from the compound eye, via unknown excitatory and GABAergic inhibitory entrainment pathways. In addition, neuropeptidergic inputs couple both pacemaker centers. A histamine-immunoreactive centrifugal neuron connects the ventral aMe with projection areas in the lateral protocerebrum and may provide non-photic inputs. To identify neurotransmitters of input pathways to the circadian clock with Fura-2-dependent Ca(2+) imaging, primary cell cultures of the adult aMe were stimulated with acetylcholine (ACh), as the most prominent excitatory, and histamine, as common inhibitory neurotransmitter. In most of aMe neurons, ACh application caused dose-dependent increases in intracellular Ca(2+) levels via ionotropic nicotinic ACh receptors. These ACh-dependent rises in Ca(2+) were mediated by mibefradil-sensitive voltage-activated Ca(2+) channels. In contrast, histamine application decreased intracellular Ca(2+) levels in only a subpopulation of aMe cells via H2-type histamine receptor chloride channels. Thus, our data suggest that ACh is part of the light entrainment pathway while histamine is involved in a non-photic input pathway to the ventral circadian clock of the Madeira cockroach.

Contribution of membrane-associated oscillators to biological timing at different timescales.

Environmental rhythms such as the daily light-dark cycle selected for endogenous clocks. These clocks predict regular environmental changes and provide the basis for well-timed adaptive homeostasis in physiology and behavior of organisms. Endogenous clocks are oscillators that are based on positive feedforward and negative feedback loops. They generate stable rhythms even under constant conditions. Since even weak interactions between oscillators allow for autonomous synchronization, coupling/synchronization of oscillators provides the basis of self-organized physiological timing. Amongst the most thoroughly researched clocks are the endogenous circadian clock neurons in mammals and insects. They comprise nuclear clockworks of transcriptional/translational feedback loops (TTFL) that generate ∼24 h rhythms in clock gene expression entrained to the environmental day-night cycle. It is generally assumed that this TTFL clockwork drives all circadian oscillations within and between clock cells, being the basis of any circadian rhythm in physiology and behavior of organisms. Instead of the current gene-based hierarchical clock model we provide here a systems view of timing. We suggest that a coupled system of autonomous TTFL and posttranslational feedback loop (PTFL) oscillators/clocks that run at multiple timescales governs adaptive, dynamic homeostasis of physiology and behavior. We focus on mammalian and insect neurons as endogenous oscillators at multiple timescales. We suggest that neuronal plasma membrane-associated signalosomes constitute specific autonomous PTFL clocks that generate localized but interlinked oscillations of membrane potential and intracellular messengers with specific endogenous frequencies. In each clock neuron multiscale interactions of TTFL and PTFL oscillators/clocks form a temporally structured oscillatory network with a common complex frequency-band comprising superimposed multiscale oscillations. Coupling between oscillator/clock neurons provides the next level of complexity of an oscillatory network. This systemic dynamic network of molecular and cellular oscillators/clocks is suggested to form the basis of any physiological homeostasis that cycles through dynamic homeostatic setpoints with a characteristic frequency-band as hallmark. We propose that mechanisms of homeostatic plasticity maintain the stability of these dynamic setpoints, whereas Hebbian plasticity enables switching between setpoints via coupling factors, like biogenic amines and/or neuropeptides. They reprogram the network to a new common frequency, a new dynamic setpoint. Our novel hypothesis is up for experimental challenge.

Ca²âº-dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons.

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20-70  Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca(2+) imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca(2+) transients in circadian pacemakers accompany tetrodotoxin (TTX)-blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX-sensitive sustained Na(+) channels. Instead, they are predominantly driven by mibefradil-sensitive, low-voltage-activated Ca(2+) channels and DK-AH269-sensitive hyperpolarisation-activated, cyclic nucleotide-gated cation channels. Rhythmic depolarisations activate voltage-gated Na(+) channels and nifedipine-sensitive high-voltage-activated Ca(2+) channels. Together with Ca(2+) rises, the depolarisations open repolarising small-conductance but not large-conductance Ca(2+) -dependent K(+) channels. In contrast, we hypothesise that P/Q-type Ca(2+) channels coupled to large-conductance Ca(2+) -dependent K(+) channels are involved in input-dependent activity.

Distribution and daily oscillation of GABA in the circadian system of the cockroach Rhyparobia maderae.

Gamma-aminobutyric acid (GABA) is the prevalent inhibitory neurotransmitter in nervous systems promoting sleep in both mammals and insects. In the Madeira cockroach, sleep-wake cycles are controlled by a circadian clock network in the brain's optic lobes, centered in the accessory medulla (AME) with its innervating pigment-dispersing factor (PDF) expressing clock neurons at the anterior-ventral rim of the medulla. GABA is present in cell clusters that innervate different circuits of the cockroach's AME clock, without colocalizing in PDF clock neurons. Physiological, immunohistochemical, and behavioral assays provided evidence for a role of GABA in light entrainment, possibly via the distal tract that connects the AME's glomeruli to the medulla. Furthermore, GABA was implemented in clock outputs to multiple effector systems in optic lobe and midbrain. Here, GABAergic brain circuits were analyzed further, focusing on the circadian system in search for sleep/wake controlling brain circuits. All GABA-immunoreactive neurons of the cockroach brain were also stained with an antiserum against the GABA-synthesizing enzyme glutamic acid decarboxylase. We found strong overlap of the distribution of GABA-immunoreactive networks with PDF clock networks in optic lobes and midbrain. Neurons in five of the six soma groups that innervate the clock exhibited GABA immunoreactivity. The intensity of GABA immunoreactivity in the distal tract showed daily fluctuations with maximum staining intensity in the middle of the day and weakest staining at the end of the day. Quantification via enzyme-linked immunosorbent assay and quantitative liquid chromatography coupled to electrospray ionization tandem mass spectrometry, likewise, showed higher GABA levels in the optic lobe during the inactivity phase of the cockroach during the day and lower levels during its activity phase at dusk. Our data further support the hypothesis that light- and PDF-dependently the circadian clock network of the cockroach controls GABA levels and thereby promotes sleep during the day.

3D-atlas of the brain of the cockroach Rhyparobia maderae.

The Madeira cockroach Rhyparobia maderae is a nocturnal insect and a prominent model organism for the study of circadian rhythms. Its master circadian clock, controlling circadian locomotor activity and sleep-wake cycles, is located in the accessory medulla of the optic lobe. For a better understanding of brain regions controlled by the circadian clock and brain organization of this insect in general, we created a three-dimensional (3D) reconstruction of all neuropils of the cerebral ganglia based on anti-synapsin and anti-γ-aminobutyric acid immunolabeling of whole mount brains. Forty-nine major neuropils were identified and three-dimensionally reconstructed. Single-cell dye fills complement the data and provide evidence for distinct subdivisions of certain brain areas. Most neuropils defined in the fruit fly Drosophila melanogaster could be distinguished in the cockroach as well. However, some neuropils identified in the fruit fly do not exist as distinct entities in the cockroach while others are lacking in the fruit fly. In addition to neuropils, major fiber systems, tracts, and commissures were reconstructed and served as important landmarks separating brain areas. Being a nocturnal insect, R. maderae is an important new species to the growing collection of 3D insect brain atlases and only the second hemimetabolous insect, for which a detailed 3D brain atlas is available. This atlas will be highly valuable for an evolutionary comparison of insect brain organization and will greatly facilitate addressing brain areas that are supervised by the circadian clock.

Circadian pacemaker coupling by multi-peptidergic neurons in the cockroach Leucophaea maderae.

Lesion and transplantation studies in the cockroach, Leucophaea maderae, have located its bilaterally symmetric circadian pacemakers necessary for driving circadian locomotor activity rhythms to the accessory medulla of the optic lobes. The accessory medulla comprises a network of peptidergic neurons, including pigment-dispersing factor (PDF)-expressing presumptive circadian pacemaker cells. At least three of the PDF-expressing neurons directly connect the two accessory medullae, apparently as a circadian coupling pathway. Here, the PDF-expressing circadian coupling pathways were examined for peptide colocalization by tracer experiments and double-label immunohistochemistry with antisera against PDF, FMRFamide, and Asn(13)-orcokinin. A fourth group of contralaterally projecting medulla neurons was identified, additional to the three known groups. Group one of the contralaterally projecting medulla neurons contained up to four PDF-expressing cells. Of these, three medium-sized PDF-immunoreactive neurons coexpressed FMRFamide and Asn(13)-orcokinin immunoreactivity. However, the contralaterally projecting largest PDF neuron showed no further peptide colocalization, as was also the case for the other large PDF-expressing medulla cells, allowing the easy identification of this cell group. Although two-thirds of all PDF-expressing medulla neurons coexpressed FMRFamide and orcokinin immunoreactivity in their somata, colocalization of PDF and FMRFamide immunoreactivity was observed in only a few termination sites. Colocalization of PDF and orcokinin immunoreactivity was never observed in any of the terminals or optic commissures. We suggest that circadian pacemaker cells employ axonal peptide sorting to phase-control physiological processes at specific times of the day.

Perfusion with cAMP analogue affects pheromone-sensitive trichoid sensilla of the hawkmoth Manduca sexta in a time-dependent manner.

Octopamine causes time-dependent disadaptation of pheromone-sensitive olfactory receptor neurons (ORNs) of Manduca sexta. Because the majority of insect octopamine receptors are positively coupled to adenylyl cyclases we examined whether cyclic adenosine monophosphate (cAMP) mimics octopamine-dependent modulation of pheromone transduction in a time-dependent manner. Long-term tip recordings of single trichoid sensilla of Manduca sexta were performed during three zeitgeber times (ZTs, ZT 0=lights on), while stimulating the sensilla with two doses of the main pheromone component bombykal in a non-adapting protocol. The membrane-permeable cAMP analogue 8bcAMP increased the normalized sensillar potential amplitude in a time- and bombykal dose-dependent way. At the higher bombykal dose only, the applied 8bcAMP antagonized an endogenous decrease in the mean sensillar potential amplitude at ZT 1-4 and ZT 8-11 when ORNs were adapted but not at ZT 22-1, when ORNs were sensitized. In contrast to octopamine, 8bcAMP did not consistently affect the initial pheromone-dependent action potential frequency, the phasic/tonic response pattern, or the time-dependent shift to lower mean action potential frequencies at ZT 8-11. Furthermore, 8bcAMP increased the spontaneous action potential frequency time dependently, but differently from octopamine. In conclusion, our results show that cAMP only partly mimics the octopamine-dependent disadaptation of olfactory receptor neurons during photophase, apparently due to another missing octopamine-dependent synergistic factor such as defined intracellular calcium levels.

Candidates for photic entrainment pathways to the circadian clock via optic lobe neuropils in the Madeira cockroach.

The compound eye of cockroaches is obligatory for entrainment of the Madeira cockroach's circadian clock, but the cellular nature of its entrainment pathways is enigmatic. Employing multiple-label immunocytochemistry, histochemistry, and backfills, we searched for photic entrainment pathways to the accessory medulla (AME), the circadian clock of the Madeira cockroach. We wanted to know whether photoreceptor terminals could directly contact pigment-dispersing factor-immunoreactive (PDF-ir) circadian pacemaker neurons with somata in the lamina (PDFLAs) or somata next to the AME (PDFMEs). Short green-sensitive photoreceptor neurons of the compound eye terminated in lamina layers LA1 and LA2, adjacent to PDFLAs and PDFMEs that branched in LA3. Long UV-sensitive compound eye photoreceptor neurons terminated in medulla layer ME2 without direct contact to ipsilateral PDFMEs that arborized in ME4. Multiple neuropeptide-ir interneurons branched in ME4, connecting the AME to ME2. Before, extraocular photoreceptors of the lamina organ were suggested to send terminals to accessory laminae. There, they overlapped with PDFLAs that mostly colocalized PDF, FMRFamide, and 5-HT immunoreactivities, and with terminals of ipsi- and contralateral PDFMEs. We hypothesize that during the day cholinergic activation of the largest PDFME via lamina organ photoreceptors maintains PDF release orchestrating phases of sleep-wake cycles. As ipsilateral PDFMEs express excitatory and contralateral PDFMEs inhibitory PDF autoreceptors, diurnal PDF release keeps both PDF-dependent clock circuits in antiphase. Future experiments will test whether ipsilateral PDFMEs are sleep-promoting morning cells, while contralateral PDFMEs are activity-promoting evening cells, maintaining stable antiphase via the largest PDFME entrained by extraocular photoreceptors of the lamina organ.

Neither per, nor tim1, nor cry2 alone are essential components of the molecular circadian clockwork in the Madeira cockroach.

Circadian clocks control rhythms in physiology and behavior entrained to 24 h light-dark cycles. Despite of conserved general schemes, molecular circadian clockworks differ between insect species. With RNA interference (RNAi) we examined an ancient circadian clockwork in a basic insect, the hemimetabolous Madeira cockroach Rhyparobia maderae. With injections of double-stranded RNA (dsRNA) of cockroach period (Rm´per), timeless 1 (Rm´tim1), or cryptochrome 2 (Rm´cry2) we searched for essential components of the clock´s core negative feedback loop. Single injections of dsRNA of each clock gene into adult cockroaches successfully and permanently knocked down respective mRNA levels within ~two weeks deleting daytime-dependent mRNA rhythms for Rm´per and Rm´cry2. Rm´perRNAi or Rm´cry2RNAi affected total mRNA levels of both genes, while Rm´tim1 transcription was independent of both, also keeping rhythmic expression. Unexpectedly, circadian locomotor activity of most cockroaches remained rhythmic for each clock gene knockdown employed. It expressed weakened rhythms and unchanged periods for Rm´perRNAi and shorter periods for Rm´tim1RNAi and Rm´cry2RNAi.As a hypothesis of the cockroach´s molecular clockwork, a basic network of switched differential equations was developed to model the oscillatory behavior of clock cells expressing respective clock genes. Data were consistent with two synchronized main groups of coupled oscillator cells, a leading (morning) oscillator, or a lagging (evening) oscillator that couple via mutual inhibition. The morning oscillators express shorter, the evening oscillators longer endogenous periods based on core feedback loops with either PER, TIM1, or CRY2/PER complexes as dominant negative feedback of the clockwork. We hypothesize that dominant morning oscillator cells with shorter periods express PER, but not CRY2, or TIM1 as suppressor of clock gene expression, while two groups of evening oscillator cells with longer periods either comprise TIM1 or CRY2/PER suppressing complexes. Modelling suggests that there is an additional negative feedback next to Rm´PER in cockroach morning oscillator cells.

Analysis of Pigment-Dispersing Factor Neuropeptides and Their Receptor in a Velvet Worm.

Pigment-dispersing factor neuropeptides (PDFs) occur in a wide range of protostomes including ecdysozoans (= molting animals) and lophotrochozoans (mollusks, annelids, flatworms, and allies). Studies in insects revealed that PDFs play a role as coupling factors of circadian pacemaker cells, thereby controlling rest-activity rhythms. While the last common ancestor of protostomes most likely possessed only one pdf gene, two pdf homologs, pdf-I and pdf-II, might have been present in the last common ancestors of Ecdysozoa and Panarthropoda (Onychophora + Tardigrada + Arthropoda). One of these homologs, however, was subsequently lost in the tardigrade and arthropod lineages followed by independent duplications of pdf-I in tardigrades and decapod crustaceans. Due to the ancestral set of two pdf genes, the study of PDFs and their receptor (PDFR) in Onychophora might reveal the ancient organization and function of the PDF/PDFR system in panarthropods. Therefore, we deorphanized the PDF receptor and generated specific antibodies to localize the two PDF peptides and their receptor in the onychophoran Euperipatoides rowelli. We further conducted bioluminescence resonance energy transfer (BRET) experiments on cultured human cells (HEK293T) using an Epac-based sensor (Epac-L) to examine cAMP responses in transfected cells and to reveal potential differences in the interaction of PDF-I and PDF-II with PDFR from E. rowelli. These data show that PDF-II has a tenfold higher potency than PDF-I as an activating ligand. Double immunolabeling revealed that both peptides are co-expressed in E. rowelli but their respective levels of expression differ between specific cells: some neurons express the same amount of both peptides, while others exhibit higher levels of either PDF-I or PDF-II. The detection of the onychophoran PDF receptor in cells that additionally express the two PDF peptides suggests autoreception, whereas spatial separation of PDFR- and PDF-expressing cells supports hormonal release of PDF into the hemolymph. This suggests a dual role of PDF peptides-as hormones and as neurotransmitters/neuromodulators-in Onychophora.

Octopamine and tyramine modulate pheromone-sensitive olfactory sensilla of the hawkmoth Manduca sexta in a time-dependent manner.

In moths octopamine improved pheromone-dependent mate search time dependently. In the nocturnal hawkmoth Manduca sexta long-term tip recordings of trichoid sensilla were performed to investigate whether biogenic amines modulate pheromone transduction time dependently. At three Zeitgebertimes octopamine, tyramine and the octopamine antagonist epinastine were applied during non-adapting pheromone-stimulation. At ZT 8-11, during the photophase, when sensilla were adapted, octopamine and to a lesser extent tyramine increased the bombykal-dependent sensillar potential amplitude and initial action potential (AP) frequency. In addition, during the photophase, when sensilla are less able to resolve pheromone pulses, octopamine rendered pheromone responses more phasic and sensitive, and raised the spontaneous AP frequency. During the late scotophase, at ZT 22-1, when the antenna appeared maximally sensitized for pheromone pulse detection and endogenous octopamine levels are high, exogenously applied octopamine was ineffective. Epinastine blocked the pheromone-dependent AP response at ZT 8-11 and slightly affected it at ZT 22-1, while it had no effect on the sensillar potential amplitude. Epinastine decreased the spontaneous AP activity during photophase and scotophase and rendered pheromone responses more tonic in the scotophase. We hypothesize that the presence of octopamine in the antenna is obligatory for the detection of intermittent pheromone pulses at all Zeitgebertimes.