Significance of the clock gene period in photoperiodism in larval development and production of diapause eggs in the silkworm Bombyx mori.

Many insects living in seasonal environments sense seasonal changes from photoperiod and appropriately regulate their development and physiological activities. Genetic researches have indicated the importance of a circadian clock system in photoperiodic time-measurement for photoperiodic regulations. However, most previous studies have focused on the effects on a single photoperiodic phenotype, without elucidating whether the circadian clock is involved in the core photoperiodic mechanism or only in the production of one target phenotype, such as diapause. Here, we focused on two different phenotypes in a bivoltine Kosetsu strain of the silkworm Bombyx mori, namely, embryonic diapause and larval development, and examined their photoperiodic responses and relationship to the circadian clock gene period. Photoperiod during the larval stage clearly influenced the induction of embryonic diapause and duration of larval development in the Kosetsu strain; short-day exposure leaded to the production of diapause eggs and shortened the larval duration. Genetic knockout of period inhibited the short-day-induced embryonic diapause. Conversely, in the period-knockout silkworms, the larval duration was shortened, but the photoperiodic difference was maintained. In conclusion, our results indicate that the period gene is not causally involved in the photoperiodic response of larval development, while that is essential for the short-day-induced embryonic diapause.

Neural mechanism of circadian clock-based photoperiodism in insects and snails.

The photoperiodic mechanism distinguishes between long and short days, and the circadian clock system is involved in this process. Although the necessity of circadian clock genes for photoperiodic responses has been demonstrated in many species, how the clock system contributes to photoperiodic mechanisms remains unclear. A comprehensive study, including the functional analysis of relevant genes and physiology of their expressing cells, is necessary to understand the molecular and cellular mechanisms. Since Drosophila melanogaster exhibits a shallow photoperiodism, photoperiodic mechanisms have been studied in non-model species, starting with brain microsurgery and neuroanatomy, followed by genetic manipulation in some insects. Here, we review and discuss the involvement of the circadian clock in photoperiodic mechanisms in terms of neural networks in insects. We also review recent advances in the neural mechanisms underlying photoperiodic responses in insects and snails, and additionally circadian clock systems in snails, whose involvement in photoperiodism has hardly been addressed yet. Brain neurosecretory cells, insulin-like peptide/diuretic hormone44-expressing pars intercerebralis neurones in the bean bug Riptortus pedestris and caudo-dorsal cell hormone-expressing caudo-dorsal cells in the snail Lymnaea stagnalis, both promote egg laying under long days, and their electrical excitability is attenuated under short and medium days, which reduces oviposition. The photoperiodic responses of the pars intercerebralis neurones are mediated by glutamate under the control of the clock gene period. Thus, we are now able to assess the photoperiodic response by neurosecretory cell activity to investigate the upstream mechanisms, that is, the photoperiodic clock and counter.

Clock gene-dependent glutamate dynamics in the bean bug brain regulate photoperiodic reproduction.

Animals adequately modulate their physiological status and behavior according to the season. Many animals sense photoperiod for seasonal adaptation, and the circadian clock is suggested to play an essential role in photoperiodic time measurement. However, circadian clock-driven neural signals in the brain that convey photoperiodic information remain unclear. Here, we focused on brain extracellular dynamics of a classical neurotransmitter glutamate, which is widely used for brain neurotransmission, and analyzed its involvement in photoperiodic responses using the bean bug Riptortus pedestris that shows clear photoperiodism in reproduction. Extracellular glutamate levels in the whole brain were significantly higher under short-day conditions, which cause a reproductive diapause, than those under long-day conditions. The photoperiodic change in glutamate levels was clearly abolished by knockdown of the clock gene period. We also demonstrated that genetic modulation of glutamate dynamics by knockdown of glutamate-metabolizing enzyme genes, glutamate oxaloacetate transaminase (got) and glutamine synthetase (gs), attenuated photoperiodic responses in reproduction. Further, we investigated glutamate-mediated photoperiodic modulations at a cellular level, focusing on the pars intercerebralis (PI) neurons that photoperiodically change their neural activity and promote oviposition. Electrophysiological analyses showed that L-Glutamate acts as an inhibitory signal to PI neurons via glutamate-gated chloride channel (GluCl). Additionally, combination of electrophysiology and genetics revealed that knockdown of got, gs, and glucl disrupted cellular photoperiodic responses of the PI neurons, in addition to reproductive phenotypes. Our results reveal that the extracellular glutamate dynamics are photoperiodically regulated depending on the clock gene and play an essential role in the photoperiodic control of reproduction via inhibitory pathways.

Pigment-dispersing factor is involved in photoperiodic control of reproduction in the brown-winged green bug, Plautia stali.

Animals in temperate regions breed in the appropriate season by sensing seasonal changes through photoperiodism. Many studies suggest the involvement of a circadian clock system in the photoperiodic regulation of reproduction. Pigment-dispersing factor (PDF) is a known brain neuropeptide involved in the circadian control in various insects. Here, we investigated the localization and projection of PDF neurons in the brain and their involvement in the photoperiodic control of reproduction in the females of the brown-winged green bug, Plautia stali. Immunohistochemical analyses revealed a dense cluster of PDF-immunoreactive cells localized in the proximal medulla of the optic lobe, which corresponded to the cluster known as PDFMe cells. PDF-immunoreactive cells projected their fibers to the lamina through the medulla surface. PDF-immunoreactive fibers were also found in the protocerebrum and seemed to connect both PDF cell bodies in the optic lobes. RNA interference-mediated knockdown of pdf inhibited oviposition arrest induced by the transfer from long- to short-day conditions. Additionally, the knockdown of pdf delayed oviposition onset after the change from short- to long-day conditions. In conclusion, the study results indicate that PDF is locally expressed in a cell cluster at the proximal medulla and involved in the photoperiodic control of reproduction in P. stali females.

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.

Immunoreactive Response of Plast-MIPs to Fasting and Their Functional Role in the Reduction of Hemolymph Reducing Sugars in the Brown-Winged Green Bug, Plautia stali.

Animals survive nutrient deficiency by controlling their physiology, such as sugar metabolism and energy-consuming developmental events. Although research on the insect neural mechanisms of the starvation-induced modulation has progressed, the mechanisms have not been fully understood due to their complexity. Myoinhibitory peptides are known to be neuropeptides involved in various physiological activities, development, and behavior. Here, we analyzed the responsiveness of Plautia stali myoinhibitory peptides (Plast-MIPs) to starvation and their physiological role in the brown-winged green bug, P. stali. First, we performed immunohistochemical analyses to investigate the response of Plast-MIP neurons in the cephalic ganglion to fasting under long day conditions. Fasting significantly enhanced the immunoreactivity to Plast-MIPs in the pars intercerebralis (PI), which is known to be a brain region related to various endocrine regulations. Next, to analyze the physiological role of Plast-MIPs, we performed RNA interference-mediated knockdown of Plast-Mip and injection of synthetic Plast-MIP in normally fed and fasted females. The knockdown of Plast-Mip did not have significant effects on the body weight or proportions of ovarian development in each feeding condition. On the other hand, the knockdown of Plast-Mip increased the gonadosomatic index of normally fed females whereas it did not have a significant effect on food intake. Notably, the knockdown of Plast-Mip diminished the fasting-induced reduction of hemolymph reducing sugar levels. Additionally, injection of synthetic Plast-MIP acutely decreased the hemolymph reducing sugar level. Our results suggested responsiveness of Plast-MIPs in the PI to fasting and their functional role in reduction of the hemolymph reducing sugar level.

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.

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.

High-Frequency Firing Activity of GnRH1 Neurons in Female Medaka Induces the Release of GnRH1 Peptide From Their Nerve Terminals in the Pituitary.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons play an important role in promoting secretion of pituitary luteinizing hormone (LH) and ovulation by releasing GnRH peptide. The release of GnRH peptide is generally assumed to be mainly modulated according to the firing activity of GnRH neurons. However, the relationship between the firing activity and the release of GnRH peptide has been elusive. We analyzed the relationship using two lines of transgenic medaka (gnrh1:enhanced green fluorescent protein and lhb:inverse-pericam) for the combined electrophysiological and Ca2+ imaging analyses. We show that a high-frequency firing activity induced by an excitatory neurotransmitter, glutamate, strongly increases [Ca2+]i in the cell bodies of GnRH1 neurons, which should lead to stimulation of GnRH release. We examined whether this high-frequency firing actually leads to the release of endogenous GnRH1 peptide from the nerve terminals projecting to the pituitary LH cells using a whole brain-pituitary preparation of a fish generated by crossing the two types of transgenic fish. Ca2+ imaging analyses showed that local glutamate activation of GnRH1 cell bodies, but not their nerve terminals in the pituitary, induced a substantial Ca2+ response in LH cells that was abolished in the presence of a GnRH receptor antagonist, Analog M. These results suggest that such an evoked high-frequency firing activity of GnRH1 cell body stimulates the release of endogenous GnRH1 peptide from the axon terminals to the pituitary LH cells. Thus, the findings of the present study have clearly demonstrated the relationship between the firing activity of hypothalamic GnRH neurons and the release of GnRH peptide.

Female-Specific Glucose Sensitivity of GnRH1 Neurons Leads to Sexually Dimorphic Inhibition of Reproduction in Medaka.

Close interaction exists between energy-consuming reproduction and nutritional status. However, there are differences in costs and priority for reproduction among species and even between sexes, which leads to diversification of interactions between reproduction and nutritional status. Despite such diversified interactions among species and sexes, most of the analysis of the nutritional status-dependent regulation of reproduction has been limited to an endothermic vertebrate, mammalian species of either sex. Therefore, the mechanisms underlying the diversified interactions remain elusive. In the present study, we demonstrated the effects of malnutritional status on reproduction at both organismal and cellular levels in an ectothermic vertebrate, a teleost medaka of both sexes. First, we analyzed the effects of malnutrition by fasting on gonadosomatic index, number of spawned/fertilized eggs, and courtship behavior. Fasting strongly suppressed reproduction in females but, surprisingly, not in males. Next, we analyzed the effects of fasting on firing activity of hypothalamic GnRH1 neurons, which form the final common pathway for the control of reproduction. An electrophysiological analysis showed that low glucose, which is induced by fasting, directly suppresses the firing activity of GnRH1 neurons specifically in females through intracellular ATP-sensitive potassium channels and AMP-activated protein kinase pathways. Based on the fact that such suppressions occurred only in females, we conclude that nutritional status-dependent, glucose-sensing in GnRH1 neurons may contribute to the most fitted reproductive regulation for each sex.

Nitric oxide/cGMP/PKG signaling pathway activated by M1-type muscarinic acetylcholine receptor cascade inhibits Na+-activated K+ currents in Kenyon cells.

The interneurons of the mushroom body, known as Kenyon cells, are essential for the long-term memory of olfactory associative learning in some insects. Some studies have reported that nitric oxide (NO) is strongly related to this long-term memory in Kenyon cells. However, the target molecules and upstream and downstream NO signaling cascades are not completely understood. Here we analyzed the effect of the NO signaling cascade on Na(+)-activated K(+) (KNa) channel activity in Kenyon cells of crickets (Gryllus bimaculatus). We found that two different NO donors, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetyl-dl-penicillamine (SNAP), strongly suppressed KNa channel currents. Additionally, this inhibitory effect of GSNO on KNa channel activity was diminished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase (sGC), and KT5823, an inhibitor of protein kinase G (PKG). Next, we analyzed the role of ACh in the NO signaling cascade. ACh strongly suppressed KNa channel currents, similar to NO donors. Furthermore, this inhibitory effect of ACh was blocked by pirenzepine, an M1 muscarinic ACh receptor antagonist, but not by 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) and mecamylamine, an M3 muscarinic ACh receptor antagonist and a nicotinic ACh receptor antagonist, respectively. The ACh-induced inhibition of KNa channel currents was also diminished by the PLC inhibitor U73122 and the calmodulin antagonist W-7. Finally, we found that ACh inhibition was blocked by the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME). These results suggested that the ACh signaling cascade promotes NO production by activating NOS and NO inhibits KNa channel currents via the sGC/cGMP/PKG signaling cascade in Kenyon cells.

Kiss1 neurons drastically change their firing activity in accordance with the reproductive state: insights from a seasonal breeder.

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.

Ionic channel mechanisms mediating the intrinsic excitability of Kenyon cells in the mushroom body of the cricket brain.

Intrinsic neurons within the mushroom body of the insect brain, called Kenyon cells, play an important role in olfactory associative learning. In this study, we examined the ionic mechanisms mediating the intrinsic excitability of Kenyon cells in the cricket Gryllus bimaculatus. A perforated whole-cell clamp study using ß-escin indicated the existence of several inward and outward currents. Three types of inward currents (INaf, INaP, and ICa) were identified. The transient sodium current (INaf) activated at -40 mV, peaked at -26 mV, and half-inactivated at -46.7 mV. The persistent sodium current (INaP) activated at -51 mV, peaked at -23 mV, and half-inactivated at -30.7 mV. Tetrodotoxin (TTX; 1 µM) completely blocked both INaf and INaP, but 10nM TTX blocked INaf more potently than INaP. Cd(2+) (50 µM) potently blocked INaP with little effect on INaf. Riluzole (>20 µM) nonselectively blocked both INaP and INaf. The voltage-dependent calcium current (ICa) activated at -30 mV, peaked at -11.3 mV, and half-inactivated at -34 mV. The Ca(2+) channel blocker verapamil (100 µM) blocked ICa in a use-dependent manner. Cell-attached patch-clamp recordings showed the presence of a large-conductance Ca(2+)-activated K(+) (BK) channel, and the activity of this channel was decreased by removing the extracellular Ca(2+) or adding verapamil or nifedipine, and increased by adding the Ca(2+) agonist Bay K8644, indicating that Ca(2+) entry via the L-type Ca(2+) channel regulates BK channel activity. Under the current-clamp condition, membrane depolarization generated membrane oscillations in the presence of 10nM TTX or 100 µM riluzole in the bath solution. These membrane oscillations disappeared with 1 µM TTX, 50 µM Cd(2+), replacement of external Na(+) with choline, and blockage of Na(+)-activated K(+) current (IKNa) with 50 µM quinidine, indicating that membrane oscillations are primarily mediated by INaP in cooperation with IKNa. The plateau potentials observed either in Ca(2+)-free medium or in the presence of verapamil were eliminated by blocking INaP with 50 µM Cd(2+). Taken together, these results indicate that INaP and IKNa participate in the generation of membrane oscillations and that INaP additionally participates in the generation of plateau potentials and initiation of spontaneous action potentials. ICa, through L-type Ca(2+) channels, was also found to play a role in the rapid membrane repolarization of action potentials by functional coupling with BK channels.