Single-cell transcriptomics reveals that glial cells integrate homeostatic and circadian processes to drive sleep-wake cycles.

The sleep-wake cycle is determined by circadian and sleep homeostatic processes. However, the molecular impact of these processes and their interaction in different brain cell populations are unknown. To fill this gap, we profiled the single-cell transcriptome of adult Drosophila brains across the sleep-wake cycle and four circadian times. We show cell type-specific transcriptomic changes, with glia displaying the largest variation. Glia are also among the few cell types whose gene expression correlates with both sleep homeostat and circadian clock. The sleep-wake cycle and sleep drive level affect the expression of clock gene regulators in glia, and disrupting clock genes specifically in glia impairs homeostatic sleep rebound after sleep deprivation. These findings provide a comprehensive view of the effects of sleep homeostatic and circadian processes on distinct cell types in an entire animal brain and reveal glia as an interaction site of these two processes to determine sleep-wake dynamics.

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.

Consequences of artificial light at night on behavior, reproduction, and development of Lymnaea stagnalis.

Light is an important zeitgeber that regulates many behavioral and physiological processes in animals. These processes may become disturbed due to the changes in natural patterns of light and dark via the introduction of artificial light at night (ALAN). The present study was designed to determine the effect of possible consequences of ALAN on reproduction, hatching success, developmental success, growth rate, feeding rate, mortality rate, and locomotor activity of the simultaneous hermaphrodite pond snail Lymnaea stagnalis. Snails were exposed to different light intensities at night that simulate actual ALAN measurements from the snail's night environment. The data revealed that exposure to ALAN at a low level significantly affected the cumulative number of laid eggs. At the same time, snails exposed to ALAN laid smaller eggs than those laid under normal light-dark cycles. Additionally, high light-intensity of ALAN delayed development and hatching of eggs of L. stagnalis while it showed no effect on hatching percentage. Furthermore, ALAN increased both the feeding and growth rates but did not lead to mortality. The results also show that snails exposed to dark conditions at night travel longer distances and do so faster than those exposed to ALAN. In light of these findings, it is clear that ALAN may have an influence on snails and their abundance in an environment, possibly disturbing ecological stability.

Slowly seeing the light: an integrative review on ecological light pollution as a potential threat for mollusks.

Seasonal changes in the natural light condition play a pivotal role in the regulation of many biological processes in organisms. Disruption of this natural condition via the growing loss of darkness as a result of anthropogenic light pollution has been linked to species-wide shifts in behavioral and physiological traits. This review starts with a brief overview of the definition of light pollution and the most recent insights into the perception of light. We then go on to review the evidence for some adverse effects of ecological light pollution on different groups of animals and will focus on mollusks. Taken together, the available evidence suggests a critical role for light pollution as a recent, growing threat to the regulation of various biological processes in these animals, with the potential to disrupt ecosystem stability. The latter indicates that ecological light pollution is an environmental threat that needs to be taken seriously and requires further research attention.

Astroglial Calcium Signaling Encodes Sleep Need in Drosophila.

Sleep is under homeostatic control, whereby increasing wakefulness generates sleep need and triggers sleep drive. However, the molecular and cellular pathways by which sleep need is encoded are poorly understood. In addition, the mechanisms underlying both how and when sleep need is transformed to sleep drive are unknown. Here, using ex vivo and in vivo imaging, we show in Drosophila that astroglial Ca2+ signaling increases with sleep need. We demonstrate that this signaling is dependent on a specific L-type Ca2+ channel and is necessary for homeostatic sleep rebound. Thermogenetically increasing Ca2+ in astrocytes induces persistent sleep behavior, and we exploit this phenotype to conduct a genetic screen for genes required for the homeostatic regulation of sleep. From this large-scale screen, we identify TyrRII, a monoaminergic receptor required in astrocytes for sleep homeostasis. TyrRII levels rise following sleep deprivation in a Ca2+-dependent manner, promoting further increases in astrocytic Ca2+ and resulting in a positive-feedback loop. Moreover, our findings suggest that astrocytes then transmit this sleep need to a sleep drive circuit by upregulating and releasing the interleukin-1 analog Spätzle, which then acts on Toll receptors on R5 neurons. These findings define astroglial Ca2+ signaling mechanisms encoding sleep need and reveal dynamic properties of the sleep homeostatic control system.

Effect of photoperiod and light intensity on learning ability and memory formation of the pond snail Lymnaea stagnalis.

Natural light is regarded as a key regulator of biological systems and typically serves as a Zeitgeber for biological rhythms. As a natural abiotic factor, it is recognized to regulate multiple behavioral and physiological processes in animals. Disruption of the natural light regime due to light pollution may result in significant effects on animal learning and memory development. Here, we investigated whether sensitivity to various photoperiods or light intensities had an impact on intermediate-term memory (ITM) and long-term memory (LTM) formation in the pond snail Lymnaea stagnalis. We also investigated the change in the gene expression level of molluscan insulin-related peptide II (MIP II) is response to the given light treatments. The results show that the best light condition for proper LTM formation is exposure to a short day (8 h light) and low light intensity (1 and 10 lx). Moreover, the more extreme light conditions (16 h and 24 h light) prevent the formation of both ITM and LTM. We found no change in MIP II expression in any of the light treatments, which may indicate that MIP II is not directly involved in the operant conditioning used here, even though it is known to be involved in learning. The finding that snails did not learn in complete darkness indicates that light is a necessary factor for proper learning and memory formation. Furthermore, dim light enhances both ITM and LTM formation, which suggests that there is an optimum since both no light and too bright light prevented learning and memory. Our findings suggest that the upsurge of artificial day length and/or night light intensity may also negatively impact memory consolidation in the wild.

Functions of corazonin and histamine in light entrainment of the circadian pacemaker in the Madeira cockroach, Rhyparobia maderae.

The circadian pacemaker of the Madeira cockroach, Rhyparobia (Leucophaea) maderae, is located in the accessory medulla (AME). Ipsi- and contralateral histaminergic compound eyes are required for photic entrainment. Light pulses delay locomotor activity rhythm during the early night and advance it during the late night. Thus, different neuronal pathways might relay either light-dependent delays or advances to the clock. Injections of neuroactive substances combined with running-wheel assays suggested that GABA, pigment-dispersing factor, myoinhibitory peptides (MIPs), and orcokinins (ORCs) were part of both entrainment pathways, whereas allatotropin (AT) only delayed locomotor rhythms at the early night. To characterize photic entrainment further, histamine and corazonin were injected. Histamine injections resulted in light-like phase delays and advances, indicating that the neurotransmitter of the compound eyes participates in both entrainment pathways. Because injections of corazonin only advanced during the late subjective night, it was hypothesized that corazonin is only part of the advance pathway. Multiple-label immunocytochemistry in combination with neurobiotin backfills demonstrated that a single cell expressed corazonin in the optic lobes that belonged to the group of medial AME interneurons. It colocalized GABA and MIP but not AT or ORC immunoreactivity. Corazonin-immunoreactive (-ir) terminals overlapped with projections of putatively light-sensitive interneurons from the ipsi- and contralateral compound eye. Thus, we hypothesize that the corazonin-ir medial neuron integrates ipsi- and contralateral light information as part of the phase-advancing light entrainment pathway to the circadian clock. J. Comp. Neurol. 525:1250-1272, 2017. © 2016 Wiley Periodicals, Inc.

Signaling of pigment-dispersing factor (PDF) in the Madeira cockroach Rhyparobia maderae.

The insect neuropeptide pigment-dispersing factor (PDF) is a functional ortholog of vasoactive intestinal polypeptide, the coupling factor of the mammalian circadian pacemaker. Despite of PDF's importance for synchronized circadian locomotor activity rhythms its signaling is not well understood. We studied PDF signaling in primary cell cultures of the accessory medulla, the circadian pacemaker of the Madeira cockroach. In Ca²âº imaging studies four types of PDF-responses were distinguished. In regularly bursting type 1 pacemakers PDF application resulted in dose-dependent long-lasting increases in Ca²âº baseline concentration and frequency of oscillating Ca²âº transients. Adenylyl cyclase antagonists prevented PDF-responses in type 1 cells, indicating that PDF signaled via elevation of intracellular cAMP levels. In contrast, in type 2 pacemakers PDF transiently raised intracellular Ca²âº levels even after blocking adenylyl cyclase activity. In patch clamp experiments the previously characterized types 1-4 could not be identified. Instead, PDF-responses were categorized according to ion channels affected. Application of PDF inhibited outward potassium or inward sodium currents, sometimes in the same neuron. In a comparison of Ca²âº imaging and patch clamp experiments we hypothesized that in type 1 cells PDF-dependent rises in cAMP concentrations block primarily outward K⁺ currents. Possibly, this PDF-dependent depolarization underlies PDF-dependent phase advances of pacemakers. Finally, we propose that PDF-dependent concomitant modulation of K⁺ and Na⁺ channels in coupled pacemakers causes ultradian membrane potential oscillations as prerequisite to efficient synchronization via resonance.