Sleep pressure modulates single-neuron synapse number in zebrafish.

Sleep is a nearly universal behaviour with unclear functions1. The synaptic homeostasis hypothesis proposes that sleep is required to renormalize the increases in synaptic number and strength that occur during wakefulness2. Some studies examining either large neuronal populations3 or small patches of dendrites4 have found evidence consistent with the synaptic homeostasis hypothesis, but whether sleep merely functions as a permissive state or actively promotes synaptic downregulation at the scale of whole neurons is unclear. Here, by repeatedly imaging all excitatory synapses on single neurons across sleep-wake states of zebrafish larvae, we show that synapses are gained during periods of wake (either spontaneous or forced) and lost during sleep in a neuron-subtype-dependent manner. However, synapse loss is greatest during sleep associated with high sleep pressure after prolonged wakefulness, and lowest in the latter half of an undisrupted night. Conversely, sleep induced pharmacologically during periods of low sleep pressure is insufficient to trigger synapse loss unless adenosine levels are boosted while noradrenergic tone is inhibited. We conclude that sleep-dependent synapse loss is regulated by sleep pressure at the level of the single neuron and that not all sleep periods are equally capable of fulfilling the functions of synaptic homeostasis.

Genetic and chemical disruption of amyloid precursor protein processing impairs zebrafish sleep maintenance.

Amyloid precursor protein (APP) is a brain-rich, single pass transmembrane protein that is proteolytically processed into multiple products, including amyloid-beta (Aß), a major driver of Alzheimer disease (AD). Although both overexpression of APP and exogenously delivered Aß lead to changes in sleep, whether APP processing plays an endogenous role in regulating sleep is unknown. Here, we demonstrate that APP processing into Aß40 and Aß42 is conserved in zebrafish and then describe sleep/wake phenotypes in loss-of-function appa and appb mutants. Larvae with mutations in appa had reduced waking activity, whereas larvae that lacked appb had shortened sleep bout durations at night. Treatment with the γ-secretase inhibitor DAPT also shortened night sleep bouts, whereas the BACE-1 inhibitor lanabecestat lengthened sleep bouts. Intraventricular injection of P3 also shortened night sleep bouts, suggesting that the proper balance of Appb proteolytic processing is required for normal sleep maintenance in zebrafish.

The zebrafish mutant dreammist implicates sodium homeostasis in sleep regulation.

Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral insertion sleep screen in larval zebrafish, we identified a novel gene, dreammist (dmist), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed dmist gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na+,K+-ATPase regulator, FXYD1/Phospholemman. Disruption of either fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since atpa1a3a and dmist mutants have elevated intracellular Na+ levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na+ pump function modulates neuronal excitability to maintain normal sleep behaviour.

Noradrenergic tone is not required for neuronal activity-induced rebound sleep in zebrafish.

Sleep pressure builds during wakefulness, but the mechanisms underlying this homeostatic process are poorly understood. One zebrafish model suggests that sleep pressure increases as a function of global neuronal activity, such as during sleep deprivation or acute exposure to drugs that induce widespread brain activation. Given that the arousal-promoting noradrenergic system is important for maintaining heightened neuronal activity during wakefulness, we hypothesised that genetic and pharmacological reduction of noradrenergic tone during drug-induced neuronal activation would dampen subsequent rebound sleep in zebrafish larvae. During stimulant drug treatment, dampening noradrenergic tone with the α2-adrenoceptor agonist clonidine unexpectedly enhanced subsequent rebound sleep, whereas enhancing noradrenergic signalling with a cocktail of α1- and ß-adrenoceptor agonists did not enhance rebound sleep. Similarly, CRISPR/Cas9-mediated elimination of the dopamine ß-hydroxylase (dbh) gene, which encodes an enzyme required for noradrenalin synthesis, enhanced baseline sleep in larvae but did not prevent additional rebound sleep following acute induction of neuronal activity. Across all drug conditions, c-fos expression immediately after drug exposure correlated strongly with the amount of induced rebound sleep, but was inversely related to the strength of noradrenergic modulatory tone. These results are consistent with a model in which increases in neuronal activity, as reflected by brain-wide levels of c-fos induction, drive a sleep pressure signal that promotes rebound sleep independently of noradrenergic tone.

linc-mipep and linc-wrb encode micropeptides that regulate chromatin accessibility in vertebrate-specific neural cells.

Thousands of long intergenic non-coding RNAs (lincRNAs) are transcribed throughout the vertebrate genome. A subset of lincRNAs enriched in developing brains have recently been found to contain cryptic open-reading frames and are speculated to encode micropeptides. However, systematic identification and functional assessment of these transcripts have been hindered by technical challenges caused by their small size. Here, we show that two putative lincRNAs (linc-mipep, also called lnc-rps25, and linc-wrb) encode micropeptides with homology to the vertebrate-specific chromatin architectural protein, Hmgn1, and demonstrate that they are required for development of vertebrate-specific brain cell types. Specifically, we show that NMDA receptor-mediated pathways are dysregulated in zebrafish lacking these micropeptides and that their loss preferentially alters the gene regulatory networks that establish cerebellar cells and oligodendrocytes - evolutionarily newer cell types that develop postnatally in humans. These findings reveal a key missing link in the evolution of vertebrate brain cell development and illustrate a genetic basis for how some neural cell types are more susceptible to chromatin disruptions, with implications for neurodevelopmental disorders and disease.

High-throughput functional analysis of autism genes in zebrafish identifies convergence in dopaminergic and neuroimmune pathways.

Advancing from gene discovery in autism spectrum disorders (ASDs) to the identification of biologically relevant mechanisms remains a central challenge. Here, we perform parallel in vivo functional analysis of 10 ASD genes at the behavioral, structural, and circuit levels in zebrafish mutants, revealing both unique and overlapping effects of gene loss of function. Whole-brain mapping identifies the forebrain and cerebellum as the most significant contributors to brain size differences, while regions involved in sensory-motor control, particularly dopaminergic regions, are associated with altered baseline brain activity. Finally, we show a global increase in microglia resulting from ASD gene loss of function in select mutants, implicating neuroimmune dysfunction as a key pathway relevant to ASD biology.

Loss of slc39a14 causes simultaneous manganese hypersensitivity and deficiency in zebrafish.

Manganese neurotoxicity is a hallmark of hypermanganesemia with dystonia 2, an inherited manganese transporter defect caused by mutations in SLC39A14. To identify novel potential targets of manganese neurotoxicity, we performed transcriptome analysis of slc39a14-/- mutant zebrafish that were exposed to MnCl2. Differentially expressed genes mapped to the central nervous system and eye, and pathway analysis suggested that Ca2+ dyshomeostasis and activation of the unfolded protein response are key features of manganese neurotoxicity. Consistent with this interpretation, MnCl2 exposure led to decreased whole-animal Ca2+ levels, locomotor defects and changes in neuronal activity within the telencephalon and optic tectum. In accordance with reduced tectal activity, slc39a14-/- zebrafish showed changes in visual phototransduction gene expression, absence of visual background adaptation and a diminished optokinetic reflex. Finally, numerous differentially expressed genes in mutant larvae normalised upon MnCl2 treatment indicating that, in addition to neurotoxicity, manganese deficiency is present either subcellularly or in specific cells or tissues. Overall, we assembled a comprehensive set of genes that mediate manganese-systemic responses and found a highly correlated and modulated network associated with Ca2+ dyshomeostasis and cellular stress. This article has an associated First Person interview with the first author of the paper.

A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes.

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week.

Diverse species-specific phenotypic consequences of loss of function sorting nexin 14 mutations.

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

Sleep is bi-directionally modified by amyloid beta oligomers.

Disrupted sleep is a major feature of Alzheimer's disease (AD), often arising years before symptoms of cognitive decline. Prolonged wakefulness exacerbates the production of amyloid-beta (Aß) species, a major driver of AD progression, suggesting that sleep loss further accelerates AD through a vicious cycle. However, the mechanisms by which Aß affects sleep are unknown. We demonstrate in zebrafish that Aß acutely and reversibly enhances or suppresses sleep as a function of oligomer length. Genetic disruptions revealed that short Aß oligomers induce acute wakefulness through Adrenergic receptor b2 (Adrb2) and Progesterone membrane receptor component 1 (Pgrmc1), while longer Aß forms induce sleep through a pharmacologically tractable Prion Protein (PrP) signaling cascade. Our data indicate that Aß can trigger a bi-directional sleep/wake switch. Alterations to the brain's Aß oligomeric milieu, such as during the progression of AD, may therefore disrupt sleep via changes in acute signaling events.

Hierarchical Compression Reveals Sub-Second to Day-Long Structure in Larval Zebrafish Behavior.

Animal behavior is dynamic, evolving over multiple timescales from milliseconds to days and even across a lifetime. To understand the mechanisms governing these dynamics, it is necessary to capture multi-timescale structure from behavioral data. Here, we develop computational tools and study the behavior of hundreds of larval zebrafish tracked continuously across multiple 24-h day/night cycles. We extracted millions of movements and pauses, termed bouts, and used unsupervised learning to reduce each larva's behavior to an alternating sequence of active and inactive bout types, termed modules. Through hierarchical compression, we identified recurrent behavioral patterns, termed motifs. Module and motif usage varied across the day/night cycle, revealing structure at sub-second to day-long timescales. We further demonstrate that module and motif analysis can uncover novel pharmacological and genetic mutant phenotypes. Overall, our work reveals the organization of larval zebrafish behavior at multiple timescales and provides tools to identify structure from large-scale behavioral datasets.

Sleep Circuits and Physiology in Non-Mammalian Systems.

Research over the last 20 years has firmly established the existence of sleep states across the animal kingdom. Work in non-mammalian animal models such as nematodes, fruit flies, and zebrafish has now uncovered many evolutionarily conserved aspects of sleep physiology and regulation, including shared circuit architecture, homeostatic and circadian control elements, and principles linking sleep physiology to function. Non-mammalian sleep research is now shedding light on fundamental aspects of the genetic and neuronal circuit regulation of sleep, with direct implications for the understanding of how sleep is regulated in mammals.

Structural and functional conservation of non-lumenized lymphatic endothelial cells in the mammalian leptomeninges.

The vertebrate CNS is surrounded by the meninges, a protective barrier comprised of the outer dura mater and the inner leptomeninges, which includes the arachnoid and pial layers. While the dura mater contains lymphatic vessels, no conventional lymphatics have been found within the brain or leptomeninges. However, non-lumenized cells called Brain/Mural Lymphatic Endothelial Cells or Fluorescent Granule Perithelial cells (muLECs/BLECs/FGPs) that share a developmental program and gene expression with peripheral lymphatic vessels have been described in the meninges of zebrafish. Here we identify a structurally and functionally similar cell type in the mammalian leptomeninges that we name Leptomeningeal Lymphatic Endothelial Cells (LLEC). As in zebrafish, LLECs express multiple lymphatic markers, containing very large, spherical inclusions, and develop independently from the meningeal macrophage lineage. Mouse LLECs also internalize macromolecules from the cerebrospinal fluid, including Amyloid-ß, the toxic driver of Alzheimer's disease progression. Finally, we identify morphologically similar cells co-expressing LLEC markers in human post-mortem leptomeninges. Given that LLECs share molecular, morphological, and functional characteristics with both lymphatics and macrophages, we propose they represent a novel, evolutionary conserved cell type with potential roles in homeostasis and immune organization of the meninges.

The Neuropeptide Galanin Is Required for Homeostatic Rebound Sleep following Increased Neuronal Activity.

Sleep pressure increases during wake and dissipates during sleep, but the molecules and neurons that measure homeostatic sleep pressure remain poorly understood. We present a pharmacological assay in larval zebrafish that generates short-term increases in wakefulness followed by sustained rebound sleep after washout. The intensity of global neuronal activity during drug-induced wakefulness predicted the amount of subsequent rebound sleep. Whole-brain mapping with the neuronal activity marker phosphorylated extracellular signal-regulated kinase (pERK) identified preoptic Galanin (Galn)-expressing neurons as selectively active during rebound sleep, and the relative induction of galn transcripts was predictive of total rebound sleep time. Galn is required for sleep homeostasis, as galn mutants almost completely lacked rebound sleep following both pharmacologically induced neuronal activity and physical sleep deprivation. These results suggest that Galn plays a key role in responding to sleep pressure signals derived from neuronal activity and functions as an output arm of the vertebrate sleep homeostat.

Pitpnc1a Regulates Zebrafish Sleep and Wake Behavior through Modulation of Insulin-like Growth Factor Signaling.

The lipid transporters of the phosphatidylinositol transfer protein (PITP) family dictate phosphoinositide compartmentalization, and specific phosphoinositides play crucial roles in signaling cascades, membrane traffic, ion channel regulation, and actin dynamics. Although PITPs are enriched in the brain, their physiological functions in neuronal signaling pathways in vivo remain ill defined. We describe a CRISPR/Cas9-generated zebrafish mutant in a brain-specific, conserved class II PITP member, pitpnc1a. Zebrafish pitpnc1a mutants are healthy but display widespread aberrant neuronal activity and increased wakefulness across the day-night cycle. The loss of Pitpnc1a increases insulin-like growth factor (IGF) signaling in the brain, and inhibition of IGF pathways is sufficient to rescue both neuronal and behavioral hyperactivity in pitpnc1a mutants. We propose that Pitpnc1a-expressing neurons alter behavior via modification of neuro-modulatory IGF that acts on downstream wake-promoting circuits.

The zebrafish as a promising tool for modeling human brain disorders: A review based upon an IBNS Symposium.

The zebrafish represents an excellent compromise between system complexity and practical simplicity, features that make it useful for modeling and mechanistic analysis of complex brain disorders. Also promising are screens for psychoactive drugs with effects on larval and adult zebrafish behavior. This review, based upon a recent symposium held at the 2016 IBNS Congress, provides different perspectives on how the zebrafish may be utilized to advance research into human central nervous system disorders. It starts with a discussion on an important bottleneck in zebrafish research, measuring the behavior of this species (specifically shoaling), and continues with examples on research on autism spectrum disorder in larval zebrafish, on screening natural products for compounds with psychoactive properties in adult zebrafish, and on the development of a zebrafish model of fetal alcohol spectrum disorders. By providing information on a broad spectrum of brain disorders, experimental methods, and scientific approaches using both larval and adult zebrafish, the review is intended to showcase this underutilized laboratory species for behavioral neuroscience and psychopharmacology research.

Neuropeptide Y Regulates Sleep by Modulating Noradrenergic Signaling.

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both necessary for normal daytime sleep duration and sufficient to promote sleep. We show that overexpression of NPY increases sleep, whereas mutation of npy or ablation of npy-expressing neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we tested for interactions with several systems known to regulate sleep, and provide anatomical, molecular, genetic, and pharmacological evidence that NPY promotes sleep by inhibiting noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator and link NPY signaling to an established arousal-promoting system.

Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish.

Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.

Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development.

The lymphatic system controls fluid homeostasis and the clearance of macromolecules from interstitial compartments. In mammals brain lymphatics were only recently discovered, with significant implications for physiology and disease. We examined zebrafish for the presence of brain lymphatics and found loosely connected endothelial cells with lymphatic molecular signature covering parts of the brain without forming endothelial tubular structures. These brain lymphatic endothelial cells (BLECs) derive from venous endothelium, are distinct from macrophages, and are sensitive to loss of Vegfc. BLECs endocytose macromolecules in a selective manner, which can be blocked by injection of mannose receptor ligands. This first report on brain lymphatic endothelial cells in a vertebrate embryo identifies cells with unique features, including the uptake of macromolecules at a single cell level. Future studies will address whether this represents an uptake mechanism that is conserved in mammals and how these cells affect functions of the embryonic and adult brain.