Dopamine's reach: Unlocked by sleep loss.

In this issue of Neuron, Wu et al.1 employ cutting-edge techniques to provide a mechanistic understanding of how sleep deprivation induces an altered affective state. They reveal a key function for dopaminergic signaling, and the formation of cortical spines, in this process.

DAMM for the detection and tracking of multiple animals within complex social and environmental settings.

Accurate detection and tracking of animals across diverse environments are crucial for behavioral studies in various disciplines, including neuroscience. Recently, machine learning and computer vision techniques have become integral to the neuroscientist's toolkit, enabling high-throughput behavioral studies. Despite advancements in localizing individual animals in simple environments, the task remains challenging in complex conditions due to intra-class visual variability and environmental diversity. These limitations hinder studies in ethologically-relevant conditions, such as when animals are concealed within nests or in obscured environments. Moreover, current tools are laborious and time-consuming to employ, requiring extensive, setup-specific annotation and model training/validation procedures. To address these challenges, we introduce the 'Detect Any Mouse Model' (DAMM), a pretrained object detector for localizing mice in complex environments, capable of robust performance with zero to minimal additional training on new experimental setups. Our approach involves collecting and annotating a diverse dataset that encompasses single and multi-housed mice in various lighting conditions, experimental setups, and occlusion levels. We utilize the Mask R-CNN architecture for instance segmentation and validate DAMM's performance with no additional training data (zero-shot inference) and with few examples for fine-tuning (few-shot inference). DAMM excels in zero-shot inference, detecting mice, and even rats, in entirely unseen scenarios and further improves with minimal additional training. By integrating DAMM with the SORT algorithm, we demonstrate robust tracking, competitively performing with keypoint-estimation-based methods. Finally, to advance and simplify behavioral studies, we made DAMM accessible to the scientific community with a user-friendly Python API, shared model weights, and a Google Colab implementation.

Neurophysiological and behavioral synchronization in group-living and sleeping mice.

Social interactions profoundly influence animal development, physiology, and behavior. Yet, how sleep-a central behavioral and neurophysiological process-is modulated by social interactions is poorly understood. Here, we characterized sleep behavior and neurophysiology in freely moving and co-living mice under different social conditions. We utilized wireless neurophysiological devices to simultaneously record multiple individuals within a group for 24 h, alongside video acquisition. We first demonstrated that mice seek physical contact before sleep initiation and sleep while in close proximity to each other (hereafter, "huddling"). To determine whether huddling during sleep is a motivated behavior, we devised a novel behavioral apparatus allowing mice to choose whether to sleep in close proximity to a conspecific or in solitude, under different environmental conditions. We also applied a deep-learning-based approach to classify huddling behavior. We demonstrate that mice are willing to forgo their preferred sleep location, even under thermoneutral conditions, to gain access to social contact during sleep. This strongly suggests that the motivation for prolonged physical contact-which we term somatolonging-drives huddling behavior. We then characterized sleep architecture under different social conditions and uncovered a social-dependent modulation of sleep. We also revealed coordination in multiple neurophysiological features among co-sleeping individuals, including in the timing of falling asleep and waking up and non-rapid eye movement sleep (NREMS) intensity. Notably, the timing of rapid eye movement sleep (REMS) was synchronized among co-sleeping male siblings but not co-sleeping female or unfamiliar mice. Our findings provide novel insights into the motivation for physical contact and the extent of social-dependent plasticity in sleep.

Neuro-orchestration of sleep and wakefulness.

Although considered an inactive state for centuries, sleep entails many active processes occurring at the cellular, circuit and organismal levels. Over the last decade, several key technological advances, including calcium imaging and optogenetic and chemogenetic manipulations, have facilitated a detailed understanding of the functions of different neuronal populations and circuits in sleep-wake regulation. Here, we present recent progress and summarize our current understanding of the circuitry underlying the initiation, maintenance and coordination of wakefulness, rapid eye movement sleep (REMS) and non-REMS (NREMS). We propose a de-arousal model for sleep initiation, in which the neuromodulatory milieu necessary for sleep initiation is achieved by engaging in repetitive pre-sleep behaviors that gradually reduce vigilance to the external environment and wake-promoting neuromodulatory tone. We also discuss how brain processes related to thermoregulation, hunger and fear intersect with sleep-wake circuits to control arousal. Lastly, we discuss controversies and lingering questions in the sleep field.

Lateral hypothalamic neuronal ensembles regulate pre-sleep nest-building behavior.

The transition from wakefulness to sleep requires striking alterations in brain activity, physiology, and behavior, yet the precise neuronal circuit elements facilitating this transition remain unclear. Prior to sleep onset, many animal species display characteristic behaviors, including finding a safe location, performing hygiene-related behaviors, and preparing a space for sleep. It has been proposed that the pre-sleep period is a transitional phase in which engaging in a specific behavioral repertoire de-arouses the brain and facilitates the wake-to-sleep transition, yet both causal evidence for this premise and an understanding of the neuronal circuit elements involved are lacking. Here, we combine detailed behavioral observations, EEG-EMG recordings, selective targeting, and activity modulation of pre-sleep-active neurons to reveal the behaviors preceding sleep initiation and their underlying neurobiological mechanisms. We show that mice engage in temporally structured behaviors with stereotypic EEG signatures prior to sleep and that nest-building and grooming become significantly more prevalent with sleep proximity. We next demonstrate that the ability to build a nest promotes the initiation and consolidation of sleep and that the lack of nesting material chronically fragments sleep. Lastly, we identify broadly projecting and predominantly glutamatergic neuronal ensembles in the lateral hypothalamus that regulate the motivation to engage in pre-sleep nest-building behavior and gate sleep initiation and intensity. Our study provides causal evidence for the facilitatory role of pre-sleep behaviors in sleep initiation and consolidation and a functional characterization of the neuronal underpinnings regulating a sleep-related and goal-directed complex behavior.

Arousal State-Dependent Alterations in VTA-GABAergic Neuronal Activity.

Decades of research have implicated the ventral tegmental area (VTA) in motivation, learning and reward processing. We and others recently demonstrated that it also serves as an important node in sleep/wake regulation. Specifically, VTA-dopaminergic neuron activation is sufficient to drive wakefulness and necessary for the maintenance of wakefulness. However, the role of VTA-GABAergic neurons in arousal regulation is not fully understood. It is still unclear whether VTA-GABAergic neurons predictably alter their activity across arousal states, what is the nature of interactions between VTA-GABAergic activity and cortical oscillations, and how activity in VTA-GABAergic neurons relates to VTA-dopaminergic neurons in the context of sleep/wake regulation. To address these, we simultaneously recorded population activity from VTA subpopulations and electroencephalography/electromyography (EEG/EMG) signals during spontaneous sleep/wake states and in the presence of salient stimuli in freely-behaving mice. We found that VTA-GABAergic neurons exhibit robust arousal-state-dependent alterations in population activity, with high activity and transients during wakefulness and REM sleep. During wakefulness, population activity of VTA-GABAergic neurons, but not VTA-dopaminergic neurons, was positively correlated with EEG γ power and negatively correlated with θ power. During NREM sleep, population activity in both VTA-GABAergic and VTA-dopaminergic neurons negatively correlated with δ, θ, and σ power bands. Salient stimuli, with both positive and negative valence, activated VTA-GABAergic neurons. Together, our data indicate that VTA-GABAergic neurons, like their dopaminergic counterparts, drastically alter their activity across sleep-wake states. Changes in their activity predicts cortical oscillatory patterns reflected in the EEG, which are distinct from EEG spectra associated with dopaminergic neural activity.

Newly identified sleep-wake and circadian circuits as potential therapeutic targets.

Optogenetics and chemogenetics are powerful tools, allowing the specific activation or inhibition of targeted neuronal subpopulations. Application of these techniques to sleep and circadian research has resulted in the unveiling of several neuronal populations that are involved in sleep-wake control, and allowed a comprehensive interrogation of the circuitry through which these nodes are coordinated to orchestrate the sleep-wake cycle. In this review, we discuss six recently described sleep-wake and circadian circuits that show promise as therapeutic targets for sleep medicine. The parafacial zone (PZ) and the ventral tegmental area (VTA) are potential druggable targets for the treatment of insomnia. The brainstem circuit underlying rapid eye movement sleep behavior disorder (RBD) offers new possibilities for treating RBD and neurodegenerative synucleinopathies, whereas the parabrachial nucleus, as a nexus linking arousal state control and breathing, is a promising target for developing treatments for sleep apnea. Therapies that act upon the hypothalamic circuitry underlying the circadian regulation of aggression or the photic regulation of arousal and mood pathway carry enormous potential for helping to reduce the socioeconomic burden of neuropsychiatric and neurodegenerative disorders on society. Intriguingly, the development of chemogenetics as a therapeutic strategy is now well underway and such an approach has the capacity to lead to more focused and less invasive therapies for treating sleep-wake disorders and related comorbidities.

Parallel circuits from the bed nuclei of stria terminalis to the lateral hypothalamus drive opposing emotional states.

Lateral hypothalamus (LH) neurons containing the neuropeptide hypocretin (HCRT; orexin) modulate affective components of arousal, but their relevant synaptic inputs remain poorly defined. Here we identified inputs onto LH neurons that originate from neuronal populations in the bed nuclei of stria terminalis (BNST; a heterogeneous region of extended amygdala). We characterized two non-overlapping LH-projecting GABAergic BNST subpopulations that express distinct neuropeptides (corticotropin-releasing factor, CRF, and cholecystokinin, CCK). To functionally interrogate BNST→LH circuitry, we used tools for monitoring and manipulating neural activity with cell-type-specific resolution in freely behaving mice. We found that Crf-BNST and Cck-BNST neurons respectively provide abundant and sparse inputs onto Hcrt-LH neurons, display discrete physiological responses to salient stimuli, drive opposite emotionally valenced behaviors, and receive different proportions of inputs from upstream networks. Together, our data provide an advanced model for how parallel BNST→LH pathways promote divergent emotional states via connectivity patterns of genetically defined, circuit-specific neuronal subpopulations.

Neuronal Mechanisms for Sleep/Wake Regulation and Modulatory Drive.

Humans have been fascinated by sleep for millennia. After almost a century of scientific interrogation, significant progress has been made in understanding the neuronal regulation and functions of sleep. The application of new methods in neuroscience that enable the analysis of genetically defined neuronal circuits with unprecedented specificity and precision has been paramount in this endeavor. In this review, we first discuss electrophysiological and behavioral features of sleep/wake states and the principal neuronal populations involved in their regulation. Next, we describe the main modulatory drives of sleep and wakefulness, including homeostatic, circadian, and motivational processes. Finally, we describe a revised integrative model for sleep/wake regulation.

To sleep or not to sleep: neuronal and ecological insights.

Daily, animals need to decide when to stop engaging in cognitive processes and behavioral responses to the environment, and go to sleep. The main processes regulating the daily organization of sleep and wakefulness are circadian rhythms and homeostatic sleep pressure. In addition, motivational processes such as food seeking and predator evasion can modulate sleep/wake behaviors. Here, we discuss the principal processes regulating the propensity to stay awake or go to sleep-focusing on neuronal and behavioral aspects. We first introduce the neuronal populations involved in sleep/wake regulation. Next, we describe the circadian and homeostatic drives for sleep. Then, we highlight studies demonstrating various effects of motivational processes on sleep/wake behaviors, and discuss possible neuronal mechanisms underlying their control.

Neuronal substrates for initiation, maintenance, and structural organization of sleep/wake states.

Animals continuously alternate between sleep and wake states throughout their life. The daily organization of sleep and wakefulness is orchestrated by circadian, homeostatic, and motivational processes. Over the last decades, much progress has been made toward determining the neuronal populations involved in sleep/wake regulation. Here, we will discuss how the application of advanced in vivo tools for cell type-specific manipulations now permits the functional interrogation of different features of sleep/wake state regulation: initiation, maintenance, and structural organization. We will specifically focus on recent studies examining the roles of wake-promoting neuronal populations.

VTA dopaminergic neurons regulate ethologically relevant sleep-wake behaviors.

Dopaminergic ventral tegmental area (VTA) neurons are critically involved in a variety of behaviors that rely on heightened arousal, but whether they directly and causally control the generation and maintenance of wakefulness is unknown. We recorded calcium activity using fiber photometry in freely behaving mice and found arousal-state-dependent alterations in VTA dopaminergic neurons. We used chemogenetic and optogenetic manipulations together with polysomnographic recordings to demonstrate that VTA dopaminergic neurons are necessary for arousal and that their inhibition suppresses wakefulness, even in the face of ethologically relevant salient stimuli. Nevertheless, before inducing sleep, inhibition of VTA dopaminergic neurons promoted goal-directed and sleep-related nesting behavior. Optogenetic stimulation, in contrast, initiated and maintained wakefulness and suppressed sleep and sleep-related nesting behavior. We further found that different projections of VTA dopaminergic neurons differentially modulate arousal. Collectively, our findings uncover a fundamental role for VTA dopaminergic circuitry in the maintenance of the awake state and ethologically relevant sleep-related behaviors.

Potent social synchronization can override photic entrainment of circadian rhythms.

Circadian rhythms in behaviour and physiology are important for animal health and survival. Studies with individually isolated animals in the laboratory have consistently emphasized the dominant role of light for the entrainment of circadian rhythms to relevant environmental cycles. Although in nature interactions with conspecifics are functionally significant, social signals are typically not considered important time-givers for the animal circadian clock. Our results challenge this view. By studying honeybees in an ecologically relevant context and using a massive data set, we demonstrate that social entrainment can be potent, may act without direct contact with other individuals and does not rely on gating the exposure to light. We show for the first time that social time cues stably entrain the clock, even in animals experiencing conflicting photic and social environmental cycles. These findings add to the growing appreciation for the importance of studying circadian rhythms in ecologically relevant contexts.

The colony environment modulates sleep in honey bee workers.

One of the most important and evolutionarily conserved roles of sleep is the processing and consolidation of information acquired during wakefulness. In both insects and mammals, environmental and social stimuli can modify sleep physiology and behavior, yet relatively little is known about the specifics of the wake experiences and their relative contribution to experience-dependent modulation of sleep. Honey bees provide an excellent model system in this regard because their behavioral repertoire is well characterized and the environment they experience during the day can be manipulated while keeping an ecologically and sociobiologically relevant context. We examined whether social experience modulates sleep in honey bees, and evaluated the relative contribution of different social signals. We exposed newly emerged bees to different components of their natural social environment and then monitored their sleep behavior in individual cages in a constant lab environment. We found that rich waking experience modulates subsequent sleep. Bees that experienced the colony environment for 1 or 2 days slept more than same-age sister bees that were caged individually or in small groups in the lab. Furthermore, bees placed in mesh-enclosures in the colony, that prevented direct contact with nestmates, slept similarly to bees freely moving in the colony. These results suggest that social signals that do not require direct or close distance interactions between bees are sufficiently rich to encompass almost the entire effect of the colony on sleep. Our findings provide a remarkable example of social experience-dependent modulation of an essential biological process.

Optogenetics in psychiatric diseases.

Optogenetic tools have revolutionized the field of neuroscience, and brought the study of neural circuits to a higher level. Optogenetics has significantly improved our understanding not only of the neuronal connections and function of the healthy brain, but also of the neuronal changes that lead to psychiatric disorders. In this review, we summarize recent optogenetic studies that explored different brain circuits involved in natural behaviors, such as sleep and arousal, reward, fear, and social and aggressive behavior. In addition, we describe how alterations in these circuits may lead to psychiatric disorders such as addiction, anxiety, depression, or schizophrenia.

Social influences on circadian rhythms and sleep in insects.

The diverse social lifestyle and the small and accessible nervous system of insects make them valuable for research on the adaptive value and the organization principles of circadian rhythms and sleep. We focus on two complementary model insects, the fruit fly Drosophila melanogaster, which is amenable to extensive transgenic manipulations, and the honey bee Apis mellifera, which has rich and well-studied social behaviors. Social entrainment of activity rhythms (social synchronization) has been studied in many animals. Social time givers appear to be specifically important in dark cavity-dwelling social animals, but here there are no other clear relationships between the degree of sociality and the effectiveness of social entrainment. The olfactory system is important for social entrainment in insects. Little is known, however, about the molecular and neuronal pathways linking olfactory neurons to the central clock. In the honey bee, the expression, phase, and development of circadian rhythms are socially regulated, apparently by different signals. Peripheral clocks regulating pheromone synthesis and the olfactory system have been implicated in social influences on circadian rhythms in the fruit fly. An enriched social environment increases the total amount of sleep in both fruit flies and honey bees. In fruit flies, these changes have been linked to molecular and neuronal processes involved in learning, memory, and synaptic plasticity. The studies on insects suggest that social influences on the clock are richer than previously appreciated and have led to important breakthroughs in our understanding of the mechanisms underlying social influences on sleep and circadian rhythms.

The colony environment, but not direct contact with conspecifics, influences the development of circadian rhythms in honey bees.

Honey bee (Apis mellifera) workers emerge from the pupae with no circadian rhythms in behavior or brain clock gene expression but show strong rhythms later in life. This postembryonic development of circadian rhythms is reminiscent of that of infants of humans and other primates but contrasts with most insects, which typically emerge from the pupae with strong circadian rhythms. Very little is known about the internal and external factors regulating the ontogeny of circadian rhythms in bees or in other animals. We tested the hypothesis that the environment during early life influences the later expression of circadian rhythms in locomotor activity in young honey bees. We reared newly emerged bees in various social environments, transferred them to individual cages in constant laboratory conditions, and monitored their locomotor activity. We found that the percentage of rhythmic individuals among bees that experienced the colony environment for their first 48 h of adult life was similar to that of older sister foragers, but their rhythms were weaker. Sister bees isolated individually in the laboratory for the same period were significantly less likely to show circadian rhythms in locomotor activity. Bees experiencing the colony environment for only 24 h, or staying for 48 h with 30 same-age sister bees in the laboratory, were similar to bees individually isolated in the laboratory. By contrast, bees that were caged individually or in groups in single- or double-mesh enclosures inside a field colony were as likely to exhibit circadian rhythms as their sisters that were freely moving in the same colony. These findings suggest that the development of the circadian system in young adult honey bees is faster in the colony than in isolation. Direct contact with the queen, workers, or the brood, contact pheromones, and trophallaxis, which are all important means of communication in honey bees, cannot account for the influence of the colony environment, since they were all withheld from the bees in the double-mesh enclosures. Our results suggest that volatile pheromones, the colony microenvironment, or both influence the ontogeny of circadian rhythms in honey bees.

Maternity-related plasticity in circadian rhythms of bumble-bee queens.

Unlike most animals studied so far in which the activity with no circadian rhythms is pathological or linked to deteriorating performance, worker bees and ants naturally care for their sibling brood around the clock with no apparent ill effects. Here, we tested whether bumble-bee queens that care alone for their first batch of offspring are also capable of a similar chronobiological plasticity. We monitored locomotor activity of Bombus terrestris queens at various life cycle stages, and queens for which we manipulated the presence of brood or removed the ovaries. We found that gynes typically emerged from the pupae with no circadian rhythms, but after several days showed robust rhythms that were not affected by mating or diapauses. Colony-founding queens with brood showed attenuated circadian rhythms, irrespective of the presence of ovaries. By contrast, queens that lost their brood switched again to activity with strong circadian rhythms. The discovery that circadian rhythms in bumble-bee queens are regulated by the life cycle and the presence of brood suggests that plasticity in the circadian clock of bees is ancient and related to maternal behaviour or physiology, and is not a derived trait that evolved with the evolution of the worker caste.