Different neuroendocrine cell types in the pars intercerebralis of Periplaneta americana produce their own specific IGF-related peptides.

Of the nine genes of the American cockroach, Periplaneta americana, coding for peptides related to insulin and insulin-like growth factor, seven show significant expression in the central nervous system as demonstrated by the polymerase chain reaction on reverse transcribed RNA. In situ hybridisation shows that five of those are expressed by cells in the pars intercerebralis. Antisera raised to the predicted peptides show that these cells are neuroendocrine in nature and project to the corpora cardiaca. Interestingly, there are at least three cell types that each express different genes. This contrasts with Drosophila where a single cell type expresses a number of genes expressing several such peptides. Whereas in Drosophila the neuroendocrine cells producing insulin-like peptides also express sulfakinins, the arthropod orthologs of gastrin and cholecystokinin, in Periplaneta the sulfakinins are produced by different cells. Other neuropeptides known to be produced by the pars intercerebralis in Periplaneta and other insect species, such as the CRF-like diuretic hormone, neuroparsin, leucokinin or myosuppressin, neither colocalize with an insulin-related peptide. The separate cellular localization of these peptides and the existence of multiple insulin receptors in this species implies a more complex regulation by insulin and IGF-related peptides in cockroaches than in the fruit fly.

Photoperiodic Response in the Pars Intercerebralis Neurons, Including Plast-MIP Neurons, in the Brown-Winged Green Bug, Plautia stali.

Many insects in temperate regions avoid environmental adversity for reproduction, and thus enter reproductive diapause according to photoperiod. This reproductive diapause is induced by inhibition of juvenile hormone biosynthesis in the corpus allatum. Some neuropeptides that have an effect on juvenile hormone biosynthesis have been detected in insect brains. Thus, the reproductive diapause may be photoperiodically regulated by these juvenile hormones-controlling neuropeptides. However, there is limited understanding of how the neurons expressing these neuropeptides respond to the photoperiod and control the peptide release accordingly. Here, we performed electrophysiological analyses in the pars intercerebralis (PI) of Plautia stali, where juvenile hormone inhibitory neuropeptides, Plautia stali myoinhibitory peptides (Plast-MIPs) are expressed. We found that the large neurons in the PI showed very high firing activity under diapause-inducing short day conditions. Neurotracer staining revealed that all recorded neurons projected to the nervus corporis cardiaci 1, which is known to be connected to the corpus cardiacum-corpus allatum complex. Finally, we determined how many of the large PI cells expressed Plast-MIP by single cell reverse transcription PCR. About half of large PI neurons coexpressed Plast-Mip and other neuropeptides, Diuretic hormone 44 and insulin-like peptide 1. The remaining cells only expressed Diuretic hormone 44 and insulin-like peptide 1. The present results suggested that large PI neurons, including Plast-MIP neurons, have enhanced activity under short day conditions, which may increase Plast-MIP release to the corpus cardiacum-corpus allatum complex and thus contribute to reproductive diapause.

The adverse effects of injected functionalized multi-walled carbon nanotube (f-MWCNT) on in vivo neurosecretory brain cells of Jamaican field cricket, Gryllus assimilis.

Carbon nanotubes (CNTs) have been increasingly more prevalent due to their use in product technology owing to their exceptional electrical and thermal conductivity and tensile strength because of their nanostructure and strength of the bonds among carbon atoms. The potential increase of CNTs in the environment is a concern, and studies to assess the toxic effects of these nanomaterials (NMs) are needed. However, so far, most of the studies are focused on aquatic species and much less is understood about the effects of NM in terrestrial organisms. This investigation used a functionalized multi-walled carbon nanotube (f-MWCNT) and the Jamaican cricket Gryllus assimilis to assess the effects of this NM. Cricket nymphs were injected with f-MWCNT suspension-at three different concentrations. The insecticide Fipronil was used as a positive control. Survival was monitored, and histological analysis was made in the brains. Pyknotic cells were quantified in two brain regions, a neurosecretory called Pars intercerebralis (PI), and an associative region called mushroom body (MB). No mortality was observed in any f-MWCNT concentration tested. A significant increase in pyknotic cells was observed as sub-lethal effect for the intermediate concentration of f-MWCNT, at PI, while any significant change was observed at the Kenyon cells of the MB. These results are discussed in the context of agglomeration and dispersion of the f-MWCNT at different concentrations, and availability of the f-MWCNT on the circulatory system, as well as the natural decay of pyknotic cells with time and different patterns of adult cricket neurogenesis. Our results showed that f-MWCNT had negative effects in the neurosecretory region of the brain.

A subset of DN1p neurons integrates thermosensory inputs to promote wakefulness via CNMa signaling.

Sleep is an essential and evolutionarily conserved behavior that is modulated by many environmental factors. Ambient temperature shifting usually occurs during climatic or seasonal change or travel from high-latitude area to low-latitude area that affects animal physiology. Increasing ambient temperature modulates sleep in both humans and Drosophila. Although several thermosensory molecules and neurons have been identified, the neural mechanisms that integrate temperature sensation into the sleep neural circuit remain poorly understood. Here, we reveal that prolonged increasing of ambient temperature induces a reversible sleep reduction and impaired sleep consolidation in Drosophila via activating the internal thermosensory anterior cells (ACs). ACs form synaptic contacts with a subset of posterior dorsal neuron 1 (DN1p) neurons and release acetylcholine to promote wakefulness. Furthermore, we identify that this subset of DN1ps promotes wakefulness by releasing CNMamide (CNMa) neuropeptides to inhibit the Dh44-positive pars intercerebralis (PI) neurons through CNMa receptors. Our study demonstrates that the AC-DN1p-PI neural circuit is responsible for integrating thermosensory inputs into the sleep neural circuit. Moreover, we identify the CNMa signaling pathway as a newly recognized wakefulness-promoting DN1 pathway.

Oviposition-promoting pars intercerebralis neurons show period-dependent photoperiodic changes in their firing activity in the bean bug.

Animals show photoperiodic responses in physiology and behavior to adapt to seasonal changes. Recent genetic analyses have demonstrated the significance of circadian clock genes in these responses. However, the importance of clock genes in photoperiodic responses at the cellular level and the physiological roles of the cellular responses are poorly understood. The bean bug Riptortus pedestris shows a clear photoperiodic response in its reproduction. In the bug, the pars intercerebralis (PI) is an important brain region for promoting oviposition. Here, we analyzed the role of the photoperiodic neuronal response and its relationship with clock genes, focusing on PI neurons. Large PI neurons exhibited photoperiodic firing changes, and high firing activities were primarily found under photoperiodic conditions suitable for oviposition. RNA interference-mediated knockdown of the clock gene period abolished the photoperiodic response in PI neurons, as well as the response in ovarian development. To clarify whether the photoperiodic response in the PI was dependent on ovarian development, we performed an ovariectomy experiment. Ovariectomy did not have significant effects on the firing activity of PI neurons. Finally, we identified the output molecules of the PI neurons and analyzed the relevance of the output signals in oviposition. PI neurons express multiple neuropeptides-insulin-like peptides and diuretic hormone 44-and RNA interference of these neuropeptides reduced oviposition. Our results suggest that oviposition-promoting peptidergic neurons in the PI exhibit a circadian clock-dependent photoperiodic firing response, which contributes to the photoperiodic promotion of oviposition.

Monitoring Electrical Activity in Drosophila Circadian Output Neurons.

Drosophila melanogaster is a powerful model organism used to study circadian rhythms, historically for elucidating the molecular basis of the clock and, more recently, for allowing for dissection of neural circuits underlying rhythmic behavior. The fly can be used to investigate the neuronal basis of complex behaviors at single-neuron resolution. Patch clamp electrophysiology permits single-neuron recording of resting membrane potential and action potential firing in response to genetic or environmental manipulations or application of drugs and neurotransmitters. Here we describe a protocol for dissecting Drosophila brains for electrophysiology, setting up and using a patch clamp system, and analyzing firing data around the circadian day and in stimulation-response experiments to test for functional neuronal connectivity in circadian circuits.

Immunohistochemical and Direct Mass Spectral Analyses of Plautia stali Myoinhibitory Peptides in the Cephalic Ganglia of the Brown-Winged Green Bug Plautia stali.

For seasonal adaptation, the brown-winged green bug Plautia stali (Hemiptera: Pentatomidae) enters reproductive diapause by suppressing juvenile hormone biosynthesis. Plautia stali myoinhibitory peptides (Plast-MIPs) are known to have allatostatic effects and to suppress juvenile hormone biosynthesis. We examined Plast-MIP-producing neurons in the brain with immunohistochemistry and Fourier transform ion cyclotron resonance mass spectrometry. Rabbit polyclonal antiserum against Plast-MIP revealed immunoreactive cells in seven regions of the brain, including the posterior antennal lobe, basal optic lobe, dorsal anterior protocerebrum, ventrolateral protocerebrum, pars intercerebralis, posterior protocerebrum, and dorsal posterior region to the calyx of the mushroom body, aside from the gnathal ganglion. Anatomical locations of the immunoreactive cells in the pars intercerebralis and dorsal posterior region to the mushroom body calyx partly overlapped with the cell body location stained by retrograde dye fills from the corpus allatum and corpus cardiacum complex. Direct mass spectrometry revealed the molecular ion peaks corresponding to the predictive mass of Plast-MIPs in the pars intercerebralis and the corpus allatum-corpus cardiacum complex. Plast-MIP immunoreactivity in different cell types suggests that Plast-MIPs have different functions in the cephalic ganglia. Considering the anatomical location of neurons projecting to the corpus allatum-corpus cardiacum and results of mass spectrometry, Plast-MIP immunoreactive cells in the pars intercerebralis may play a role in suppressing juvenile hormone biosynthesis.

Three kinds of regulatory signals for production of juvenile hormone in females of the linden bug, Pyrrhocoris apterus.

Three types of regulation of the corpus allatum (CA) activity were defined in females of the linden bug Pyrrhocoris apterus. First, short-term inhibition of the CA activity was found in starved or fed long-day females, or in short-day females. Inhibitory factor(s) are transmitted to the CA via nerves, but in vitro they might reach the CA via the incubation medium. Origin of the inhibition is the pars intercerebralis (PI). The inhibitory effect is reversible during short-term incubation in vitro. This short-term inhibition can be quickly restored by the presence of the brain-suboesophageal ganglion (BR-SG) with the PI or removed, by the presence of the BR-SG without the PI or by the absence of the BR-SG. Short-term inhibition is sufficient to inhibit the CA of starved long-day females, but it is not strong enough to inhibit the CA of diapausing bugs. Second, developmental stimulation of the CA activity by feeding in long-day females is associated with growth in size of the CA. Stimulation proceeds slowly (days) in vivo and reaches the CA from the PI via nerves. Activity of the CA is irreversible in vitro; it is maintained without any further stimulation by the PI, i.e. in the presence of the BR-SG without PI or in the absence of the BR-SG. In the intact BR-SG-CC-CA the developmental stimulation of the CA is compensated by short-term inhibition of similar strength. Therefore, the activity of large CA within the intact BR-SG-CC-CA (stimulated + inhibited) is similar to the activity of the small denervated CA (no stimulation + no inhibition). Third, long-term inhibition of the CA activity in short-day females, produced by the diapause inducing photoperiod in the PI, reaches the CA via nerves. However, in contrast to the short-term inhibition of the CA, it is irreversible during short-term incubation in vitro. The long-term inhibition can only be removed several days after disconnection of the CA from the brain in vivo.

Effects of pars intercerebralis removal on circatidal rhythm in the mangrove cricket, Apteronemobius asahinai.

The circatidal rhythm is an endogenous rhythm corresponding to the tidal cycles, and its neural mechanism remains unknown. The mangrove cricket, Apteronemobius asahinai, possesses both circatidal and circadian clocks, and simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In a previous study, we showed that surgical removal of the optic lobes, the principal circadian clock locus in crickets, disrupted their circadian rhythm, but not their circatidal rhythm. In this study, we focused on the pars intercerebralis (PI) because surgical removal of the PI disrupts the circadian rhythm and causes arrhythmic activity in some cricket species. After surgical removal of the PI, the proportion of crickets displaying circatidal rhythm decreased, and more than half of the crickets exhibited arrhythmic activity. Surgical removal of the regions around the PI also caused a similar effect on locomotor activity. Our results indicate that the PI and/or its surrounding regions are important not only for circadian but also for circatidal rhythm. This suggests the presence of a neural or hormonal pathway in the PI and/or its surrounding regions that is common to the circatidal and circadian rhythms.

CORL Expression and Function in Insulin Producing Neurons Reversibly Influences Adult Longevity in Drosophila.

CORL proteins (known as SKOR in mice, Fussel in humans and fussel in Flybase) are a family of CNS specific proteins related to Sno/Ski oncogenes. Their developmental and adult roles are largely unknown. A Drosophila CORL (dCORL) reporter gene is expressed in all Drosophila insulin-like peptide 2 (dILP2) neurons of the pars intercerebralis (PI) of the larval and adult brain. The transcription factor Drifter is also expressed in the PI in a subset of dCORL and dILP2 expressing neurons and in several non-dILP2 neurons. dCORL mutant virgin adult brains are missing all dILP2 neurons that do not also express Drifter. This phenotype is also seen when expressing dCORL-RNAi in neurosecretory cells of the PI. dCORL mutant virgin adults of both sexes have a significantly shorter lifespan than their parental strain. This longevity defect is completely reversed by mating (lifespan increases over 50% for males and females). Analyses of dCORL mutant mated adult brains revealed a complete rescue of dILP2 neurons without Drifter. Taken together, the data suggest that dCORL participates in a neural network connecting the insulin signaling pathway, longevity and mating. The conserved sequence and CNS specificity of all CORL proteins imply that this network may be operating in mammals.

CORL Expression in the Drosophila Central Nervous System Is Regulated by Stage Specific Interactions of Intertwined Activators and Repressors.

CORL proteins (SKOR in mice and Fussel in humans) are a subfamily of central nervous system (CNS) specific proteins related to Sno/Ski oncogenes. Their developmental and homeostatic roles are largely unknown. We previously showed that Drosophila CORL (dCORL; fussel in Flybase) functions between the Activin receptor Baboon and Ecdysone Receptor-B1 (EcR-B1) activation in mushroom body neurons of third instar larval brains. To better understand dCORL regulation and function we generated a series of reporter genes. We examined the embryonic and larval CNS and found that dCORL is regulated by stage specific interactions between intertwined activators and repressors spanning numerous reporters. The reporter AH.lacZ, which contains sequences 7-11kb upstream of dCORL exon1, reflects dCORL brain expression at all stages. Surprisingly, AH.lacZ was not detected in EcR-B1 expressing mushroom body neurons. In larvae AH.lacZ is coexpressed with Elav and the transcription factor Drifter in dILP2 insulin producing cells of the pars intercerebralis. The presence of dCORL in insulin producing cells suggests that dCORL functions non-autonomously in the regulation of EcR-B1 mushroom body activation via the modulation of insulin signaling. Overall, the high level of sequence conservation seen in all CORL/SKOR/Fussel family members and their common CNS specificity suggest that similarly complex regulation and a potential function in insulin signaling are associated with SKOR/Fussel proteins in mammals.

Sleep in Insects.

Sleep is essential for proper brain function in mammals and insects. During sleep, animals are disconnected from the external world; they show high arousal thresholds and changed brain activity. Sleep deprivation results in a sleep rebound. Research using the fruit fly, Drosophila melanogaster, has helped us understand the genetic and neuronal control of sleep. Genes involved in sleep control code for ion channels, factors influencing neurotransmission and neuromodulation, and proteins involved in the circadian clock. The neurotransmitters/neuromodulators involved in sleep control are GABA, dopamine, acetylcholine, serotonin, and several neuropeptides. Sleep is controlled by the interplay between sleep homeostasis and the circadian clock. Putative sleep-wake centers are located in higher-order brain centers that are indirectly connected to the circadian clock network. The primary function of sleep appears to be the downscaling of synapses that have been built up during wakefulness. Thus, brain homeostasis is maintained and learning and memory are assured.

Diet and endocrine effects on behavioral maturation-related gene expression in the pars intercerebralis of the honey bee brain.

Nervous and neuroendocrine systems mediate environmental conditions to control a variety of life history traits. Our goal was to provide mechanistic insights as to how neurosecretory signals mediate division of labor in the honey bee (Apis mellifera). Worker division of labor is based on a process of behavioral maturation by individual bees, which involves performing in-hive tasks early in adulthood, then transitioning to foraging for food outside the hive. Social and nutritional cues converge on endocrine factors to regulate behavioral maturation, but whether neurosecretory systems are central to this process is not known. To explore this, we performed transcriptomic profiling of a neurosecretory region of the brain, the pars intercerebralis (PI). We first compared PI transcriptional profiles for bees performing in-hive tasks and bees engaged in foraging. Using these results as a baseline, we then performed manipulative experiments to test whether the PI is responsive to dietary changes and/or changes in juvenile hormone (JH) levels. Results reveal a robust molecular signature of behavioral maturation in the PI, with a subset of gene expression changes consistent with changes elicited by JH treatment. In contrast, dietary changes did not induce transcriptomic changes in the PI consistent with behavioral maturation or JH treatment. Based on these results, we propose a new verbal model of the regulation of division of labor in honey bees in which the relationship between diet and nutritional physiology is attenuated, and in its place is a relationship between social signals and nutritional physiology that is mediated by JH.

Neuroanatomy of pars intercerebralis neurons with special reference to their connections with neurons immunoreactive for pigment-dispersing factor in the blow fly Protophormia terraenovae.

Input regions of pars intercerebralis (PI) neurons are examined by confocal and electron microscopies with special reference to their connections with neurons immunoreactive for pigment-dispersing factor (PDF) in the blow fly, Protophormia terraenovae. PI neurons are a prerequisite for ovarian development under long-day conditions. Backfills from the cardiac recurrent nerve after severance of the posterior lateral tracts labeled thin fibers derived from the PI neurons in the superior medial protocerebrum. These PI fibers were mainly synapsin-negative and postsynaptic to unknown varicose profiles containing dense-core vesicles. Backfilled fibers in the periesophageal neuropils, derived from the PI neurons or neurons with somata in the subesophageal zone, were varicose and some were synapsin-positive. Electron microscopy revealed the presence of both presynaptic and postsynaptic sites in backfilled fibers in the periesophageal neuropils. Many PDF-immunoreactive varicosities were found in the superior medial and lateral protocerebrum and double-labeling showed that 60-88 % of PDF-immunoreactive varicosities were also synapsin-immunoreactive. Double-labeling with the backfills and PDF immunocytochemistry showed that the PI fibers and PDF-immunoreactive varicosities were located close to each other in the superior medial protocerebrum. Results of triple-labeling of PI neurons, PDF-immunoreactive neurons and synapsin-immunoreactive terminals demonstrated that the synapsin-positive PDF-immunoreactive varicosities contacted the PI fibers. These data suggest that PI neurons receive synaptic contacts from PDF-immunoreactive fibers, which are derived from circadian clock neurons, of small ventral lateral neurons (previously called OL2) or posterior dorsal (PD) neurons with somata in the pars lateralis.

An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima.

The apical plate of primary marine larvae is characterized by a common set of transcription factors comprising six3, rx, hbn, nk2.1 and FoxQ2. It harbours the apical organ, a neural and ciliary structure with neurosecretory properties. Recent studies in lophotrochozoans have found that apical organ cells form the anterior tip of the developing central nervous system. We identify an anterior medial tissue in the embryonic centipede head that shares the transcriptional profile of the apical plate of marine larvae, including nested domains of FoxQ2 and six3 expression. This domain gives rise to an anterior medial population of neural precursors distinct from those arising within the segmental neuroectoderm. These medial cells do not express achaete scute homologue in proneural clusters, but express collier, a marker for post mitotic cells committed to a neural fate, while they are still situated in the surface ectodermal layer. They then sink under the surface to form a compact cell cluster. Once internalized these cells extend axons that pioneer the primary axonal scaffold of the central nervous system. The same cells express phc2, a neural specific prohormone convertase, which suggests that they form an early active neurosecretory centre. Some also express markers of hypothalamic neurons, including otp, vtn and vax1. These medial neurosecretory cells of the centipede are distinct from those of the pars intercerebralis, the anterior neurosecretory part of the insect brain. The pars intercerebralis derives from vsx positive placodal-like invagination sites. In the centipede, vsx expressing invaginating ectoderm is situated bilaterally adjacent to the medial pioneer cell population. Hence the pars intercerebralis is present in both insect and centipede brains, whereas no prominent anterior medial cluster of pioneer neurons is present in insects. These observations suggest that the arthropod brain retained ancestrally an anterior medial population of neurosecretory cells homologous to those of the apical plate in other invertebrate phyla, but that this cell population has been lost or greatly reduced in insects.

Endocrine regulation of non-circadian behavior of circadian genes in insect gut.

The linden bug Pyrrhocoris apterus exhibits a robust diapause response to photoperiod. Photoperiod strongly affected basal levels of circadian gene transcripts in the gut, via the neuroendocrine system. Cryptochrome 2 (cry2) mRNA level was much higher in diapause promoting short days (SD) than in reproduction promoting long days (LD), while Par Domain Protein 1 (Pdp1) mRNA level was higher in LD than in SD. The effect of photoperiod on gene expression was mediated by the neurosecretory cells of the pars intercerebralis (PI) and the juvenile hormone (JH) producing corpus allatum (CA). In LD-females, CA ablation resulted in SD-like levels of gene transcripts, while PI ablation had little effect. Conversely, in SD-females, CA ablation had only a little effect, while PI ablation resulted in LD-like levels of gene transcripts. Thus, the CA is responsible for LD-like characteristics of gene expression in reproducing females and the PI is responsible for SD-like characteristics of gene expression in diapausing females. A simultaneous ablation of both PI and CA revealed two roles of PI in SD-females: (1) inhibition of CA, and (2) weak CA-independent stimulation of cry2 mRNA. Overall, our results indicate that peripheral circadian gene expression in the gut reflects the physiological state of females (with respect to diapause or reproduction) rather than the external light-dark cycle.