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Prober, D. A.

Publications and source records attributed to Prober, D. A..

10 recordsLinked to original sources

A neuron-glia circuit anticipates hypoxia to regulate organismal oxygen use

Organisms must regulate metabolic resources such as oxygen (O2) and nutrients despite environmental variability and the energetic costs of their own actions1-3. Such regulation can occur reactively, through homeostatic corrections of recent imbalances, or predictively, through allostatic adjustments that anticipate future demand4,5. Predictive regulation is particularly important because metabolic resources often continue to be consumed for seconds to minutes after motor actions cease as tissues repay incurred costs, making it advantageous to prevent depletion before it occurs6. However, the cellular and circuit mechanisms for allostatic control remain largely unknown5,7,8. Using whole-brain neuronal and astroglial imaging and O2 measurements in behaving zebrafish, we identified a noradrenergic-astroglial circuit that detects, anticipates, and prevents internal O2 depletion. We found that swimming exacerbated internal hypoxia with a multi-second delay, but behavioral adaptations occurred before such self-generated hypoxia manifested, suggesting predictive control, confirmed using computational modeling. Noradrenergic neurons in the nucleus of the solitary tract directly detected brain hypoxia and received efference copies of swimming actions; these inputs summed at the level of membrane voltage to increase spiking and norepinephrine release when actions and resource scarcity co-occurred. Astroglia integrated noradrenergic input into prolonged Ca2+ elevation that tracked the O2 cost of recent actions and thereby predicted O2 debt relative to O2 availability, rising ~8 s before O2 fell. This astroglial prediction reorganized brain-wide activity to suppress locomotion and promote respiration, preempting O2 depletion. Silencing noradrenergic neurons or astroglial signaling abolished these hypoxia coping behaviors, whereas selective activation evoked them. This neuronal-astroglial mechanism constitutes a predictive control system that integrates physiological state with behavioral intent to avert metabolic crisis, revealing a cellular substrate for proactive energy management.

neuroscience↗

stk32a links sleep homeostasis to suppression of sensory and motor systems

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood. To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies. We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain. Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

neuroscience↗

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

Pth4 neurons define a novel hypothalamic circuit that promotes sleep via brainstem monoaminergic neurons

Classical studies identified a critical role for the hypothalamus in regulating sleep and wake states, but few such hypothalamic neuronal populations have been identified. Here we describe a sleep-promoting population of hypothalamic neurons that expresses the neuropeptides QRFP and parathyroid hormone 4 (Pth4) in zebrafish. Optogenetic stimulation of these neurons results in a large increase in sleep that requires pth4 but not qrfp. Noradrenergic locus coeruleus (LC) neurons and serotonergic raphe neurons (RN) in the hindbrain express distinct pth receptors, and genetic epistasis and cell ablation experiments revealed that Pth4 neuron-induced sleep is suppressed in mutants that lack noradrenaline in the LC or lack the serotonergic RN. Pth4 neuron-induced sleep is also suppressed in serine/threonine kinase 32a (stk32a) mutants, possibly via stk32a-expressing neurons in the prethalamus that express pth receptors. These results identify QRFP/Pth4 neurons as a novel hypothalamic sleep-promoting population and support a model in which distinct sleep- and wake-promoting hypothalamic populations act via monoaminergic neurons in the hindbrain to control vigilance state.

neuroscience↗

Zebrafish sleep displays distinct sub-states

Sleep is an essential and evolutionarily-conserved behavior. While mammals and several other species have been shown to exhibit well-defined sleep sub-states, some of which have been ascribed specific functions, it remains unclear to what extent such differentiation exists across the animal kingdom. Here we show, using long-term behavioral data combined with Hidden Markov Modeling, that larval zebrafish display distinct deep and light sleep sub-states. Although both states occur primarily at night, fish respond differently to sleep deprivation and arousing stimuli depending on which sleep sub-state they are in. Moreover, the proportions of deep and light sleep are selectively altered by genetic and pharmacological manipulations of melatonin, serotonin, and norepinephrine signaling, offering new insights into how these neuromodulators shape sleep architecture. These results support zebrafish as a tractable model for dissecting the regulation and function of sleep sub-states. More broadly, they demonstrate that structured, multi-state sleep is a conserved feature of vertebrate behavior.

neuroscience↗

Norepinephrine changes behavioral state via astroglial purinergic signaling

Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.

neuroscience↗

Open-Source Thermometer, Temperature Controller, and Light Meter for Use in Animal Facilities and During Experiments

Experiments with biological samples require precise control of environmental conditions. In our work we use zebrafish (Danio rerio) to understand the neurobiology of sleep, which requires precise control of temperature and lighting. Like many labs, lighting and temperature in the animal facility are centrally controlled in the building. During behavioral experiments and microscopy sessions, we use custom-built heating systems and perform occasional manual checks of conditions. However, without a system to precisely record conditions, gradual changes in temperature can go unnoticed for a long time, and temporary failures may be missed entirely. Here we present the design and characterization of affordable open-source tools to record temperature and light conditions during animal experiments using an Arduino microcontroller or a Raspberry Pi compact computer. The waterproof temperature sensor has high stability over 50 days of recording and is precise to 0.1{degrees}C. The Arduino device can be used through a common serial port interface for which we present code in Python and MATLAB. The Raspberry Pi version can be accessed through a web interface, for which we provide an installation guide. We use the device to record and review temperature and lighting conditions in two zebrafish animal facilities. We use our platform to add a water heating system to maintain temperature at 28{degrees}C during in vivo light-sheet imaging of larval zebrafish. We show that a change in temperature from 28{degrees}C to 32{degrees}C affects resting heart rate of the animal, highlighting the importance of maintaining and recording conditions. The protocols presented here do not require advanced engineering, fabrication, or software skills, and provide an approach to accurately record and report experimental conditions.

bioengineering↗

A genetic screen identifies dreammist as a regulator of sleep

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. Significance statementSleep is an essential behavioral state, but the genes that regulate sleep and wake states are still being uncovered. A viral insertion screen in zebrafish identified a novel sleep mutant called dreammist, in which a small, highly-conserved transmembrane protein is disrupted. The discovery of dreammist highlights the importance of a class of small transmembrane-protein modulators of the sodium pump in setting appropriate sleep duration.

neuroscience↗

Neuropeptide VF neurons promote sleep via the serotonergic raphe

Although several sleep-regulating neurons have been identified, little is known about how they interact with each other for sleep/wake control. We previously identified neuropeptide VF (NPVF) and the hypothalamic neurons that produce it as a sleep-promoting system (Lee et al., 2017). Here we use zebrafish to describe a neural circuit in which neuropeptide VF (npvf)-expressing neurons control sleep via the serotonergic raphe nuclei (RN), a hindbrain structure that promotes sleep in both diurnal zebrafish and nocturnal mice. Using genetic labeling and calcium imaging, we show that npvf-expressing neurons innervate and activate serotonergic RN neurons. We additionally demonstrate that optogenetic stimulation of npvf-expressing neurons induces sleep in a manner that requires NPVF and is abolished when the RN are ablated or lack serotonin. Finally, genetic epistasis demonstrates that NPVF acts upstream of serotonin in the RN to maintain normal sleep levels. These findings reveal a novel hypothalamic-hindbrain circuit for sleep/wake control.

neuroscience↗

Gaze-stabilizing central vestibular neurons project asymmetrically to extraocular motoneuron pools

Within reflex circuits, specific anatomical projections allow central neurons to relay sensations to effectors that generate movements. A major challenge is to relate anatomical features of central neural populations -- such as asymmetric connectivity -- to the computations the populations perform. To address this problem, we mapped the anatomy, modeled the function, and discovered a new behavioral role for a genetically-defined population of central vestibular neurons in rhombomeres 5-7 of larval zebrafish. First, we found that neurons within this central population project preferentially to motoneurons that move the eyes downward. Concor-dantly, when the entire population of asymmetrically-projecting neurons was stimulated collectively, only downward eye rotations were observed, demonstrating a functional correlate of the anatomical bias. When these neurons are ablated, fish failed to rotate their eyes following either nose-up or nose-down body tilts. This asymmetrically-projecting central population thus participates in both up and downward gaze stabilization. In addition to projecting to motoneurons, central vestibular neurons also receive direct sensory input from peripheral afferents. To infer whether asymmetric projections can facilitate sensory encoding or motor output, we modeled differentially-projecting sets of central vestibular neurons. Whereas motor command strength was independent of projection allocation, asymmetric projections enabled more accurate representation of nose-up stimuli. The model shows how asymmetric connectivity could enhance the representation of imbalance during nose-up postures while preserving gaze-stabilization performance. Finally, we found that central vestibular neurons were necessary for a vital behavior requiring maintenance of a nose-up posture: swim bladder inflation. These observations suggest that asymmetric connectivity in the vestibular system facilitates representation of ethologically-relevant stimuli without compromising reflexive behavior.\n\nSignificance StatementInterneuron populations use specific anatomical projections to transform sensations into reflexive actions. Here we examined how the anatomical composition of a genetically-defined population of balance interneurons in the larval zebrafish relates to the computations it performs. First, we found that the population of interneurons that stabilize gaze preferentially project to motoneurons that move the eyes downward. Next, we discovered through modeling that such projection patterns can enhance the encoding of nose-up sensations without compromising gaze stabilization. Finally we found that loss of these interneurons impairs a vital behavior, swim bladder inflation, that relies on maintaining a nose-up posture. These observations suggest that anatomical specialization permits neural circuits to represent relevant features of the environment without compromising behavior.

neuroscience↗