Optogenetic and pharmacological interventions link hypocretin neurons to impulsivity in mice.

Neurons in the lateral hypothalamus expressing the neuropeptide Hypocretin, also known as orexin, are known critical modulators of arousal stability. However, their role in the different components of the arousal construct such as attention and decision making is poorly understood. Here we study Hypocretin neuronal circuit dynamics during stop action impulsivity in a Go/NoGo task in mice. We show that Hypocretin neuronal activity correlates with anticipation of reward. We then assessed the causal role of Hypocretin neuronal activity using optogenetics in a Go/NoGo task. We show that stimulation of Hypocretin neurons during the cue period dramatically increases the number of premature responses. These effects are mimicked by amphetamine, reduced by atomoxetine, a norepinephrine uptake inhibitor, and blocked by a Hypocretin receptor 1 selective antagonist. We conclude that Hypocretin neurons have a key role in the integration of salient stimuli during wakefulness to produce appropriate and timely responses to rewarding and aversive cues.

Lateral hypothalamic galanin neurons are activated by stress and blunt anxiety-like behavior in mice.

Despite the prevalence of anxiety disorders, the molecular identity of neural circuits underlying anxiety remains unclear. The lateral hypothalamus (LH) is one brain region implicated in the regulation of anxiety, and our recent data found that chemogenetic activation of LH galanin neurons attenuated the stress response to a novel environment as measured by the marble burying test. Thus, we hypothesize that LH galanin neurons may contribute to anxiety-related behavior. We used chemogenetics and fiber photometry to test the ability of LH galanin neurons to influence anxiety and stress-related behavior. Chemogenetic activation of LH galanin neurons significantly decreased anxiety-like behavior in the elevated plus maze, open field test, and light dark test. However, LH galanin activation did not alter restraint stress induced HPA activation or freezing behavior in the fear conditioning paradigm. In vivo calcium monitoring by fiber photometry indicated that LH galanin neurons were activated by anxiogenic and/or stressful stimuli including tail suspension, novel mouse interaction, and predator odor. Further, in a fear conditioning task, calcium transients strongly increased during foot shock, but were not affected by the unconditioned stimulus tone. These data indicate that LH galanin neurons both respond to and modulate anxiety, with no influence on stress induced HPA activation or fear behaviors. Further investigation of LH galanin circuitry and functional mediators of behavioral output may offer a more refined pharmacological target as an alternative to first-line broad pharmacotherapies such as benzodiazepines.

Hyperexcitable arousal circuits drive sleep instability during aging.

Sleep quality declines with age; however, the underlying mechanisms remain elusive. We found that hyperexcitable hypocretin/orexin (Hcrt/OX) neurons drive sleep fragmentation during aging. In aged mice, Hcrt neurons exhibited more frequent neuronal activity epochs driving wake bouts, and optogenetic activation of Hcrt neurons elicited more prolonged wakefulness. Aged Hcrt neurons showed hyperexcitability with lower KCNQ2 expression and impaired M-current, mediated by KCNQ2/3 channels. Single-nucleus RNA-sequencing revealed adaptive changes to Hcrt neuron loss in the aging brain. Disruption of Kcnq2/3 genes in Hcrt neurons of young mice destabilized sleep, mimicking aging-associated sleep fragmentation, whereas the KCNQ-selective activator flupirtine hyperpolarized Hcrt neurons and rejuvenated sleep architecture in aged mice. Our findings demonstrate a mechanism underlying sleep instability during aging and a strategy to improve sleep continuity.

Periodic B- and N-doped phenalenyl π-aggregates: unexpected nonlinear optical properties by tuning pancake π-π bonding.

Phenalenyl (PLY) and its derivatives could form one-dimensional π-aggregates through pancake π-π bonding, which would lead to exotic optoelectronic properties. We will highlight the key aspects of the PLY derivatives from the design strategies to exploration of the electronic properties. Here, we primarily construct alternating boron (B)- and nitrogen (N)-doped PLY π-aggregates: dimer[12], trimer[12-1], trimer[12-2], tetramer[12]2, pentamer[12]2-1, pentamer[12]2-2, and hexamer[12]3. The geometric and electronic structures show that the short intermolecular distances of the π-aggregates drive the formation of pancake π-π bonding. Significantly, the molecular structures show periodic changes in the π-aggregates, but the first hyperpolarizabilities (ßtot) present unexpected changes, which are found to increase sharply with increasing even layer thickness due to intermolecular charge transfer. The ßtot value of hexamer[12]3 (5.72 × 104 a.u.) is 6.4 times that of tetramer[12]2 (8.95 × 103 a.u.), and is 22.4 times that of dimer[12] (2.55 × 103 a.u.). Thus, constructing π-aggregates can significantly improve the second-order NLO response, which is mainly due to intermolecular charge transfer through pancake π-π bonding of the interlayers.

Voltage-independent GluN2A-type NMDA receptor Ca2+ signaling promotes audiogenic seizures, attentional and cognitive deficits in mice.

The NMDA receptor-mediated Ca2+ signaling during simultaneous pre- and postsynaptic activity is critically involved in synaptic plasticity and thus has a key role in the nervous system. In GRIN2-variant patients alterations of this coincidence detection provoked complex clinical phenotypes, ranging from reduced muscle strength to epileptic seizures and intellectual disability. By using our gene-targeted mouse line (Grin2aN615S), we show that voltage-independent glutamate-gated signaling of GluN2A-containing NMDA receptors is associated with NMDAR-dependent audiogenic seizures due to hyperexcitable midbrain circuits. In contrast, the NMDAR antagonist MK-801-induced c-Fos expression is reduced in the hippocampus. Likewise, the synchronization of theta- and gamma oscillatory activity is lowered during exploration, demonstrating reduced hippocampal activity. This is associated with exploratory hyperactivity and aberrantly increased and dysregulated levels of attention that can interfere with associative learning, in particular when relevant cues and reward outcomes are disconnected in space and time. Together, our findings provide (i) experimental evidence that the inherent voltage-dependent Ca2+ signaling of NMDA receptors is essential for maintaining appropriate responses to sensory stimuli and (ii) a mechanistic explanation for the neurological manifestations seen in the NMDAR-related human disorders with GRIN2 variant-meidiated intellectual disability and focal epilepsy.

Hypothalamic circuitry underlying stress-induced insomnia and peripheral immunosuppression.

The neural substrates of insomnia/hyperarousal induced by stress remain unknown. Here, we show that restraint stress leads to hyperarousal associated with strong activation of corticotropin-releasing hormone neurons in the paraventricular nucleus of hypothalamus (CRHPVN) and hypocretin neurons in the lateral hypothalamus (HcrtLH). CRHPVN neurons directly innervate HcrtLH neurons, and optogenetic stimulation of LH-projecting CRHPVN neurons elicits hyperarousal. CRISPR-Cas9-mediated knockdown of the crh gene in CRHPVN neurons abolishes hyperarousal induced by stimulating LH-projecting CRHPVN neurons. Genetic ablation of Hcrt neurons or crh gene knockdown significantly counteracts restraint stress-induced hyperarousal. Single-cell mass cytometry by time of flight (CyTOF) revealed extensive changes to immune cell distribution and functional responses in peripheral blood during hyperarousal upon optogenetic stimulation of CRHPVN neurons simulating stress-induced insomnia. Our findings suggest both central and peripheral systems are synergistically engaged in the response to stress via CRHPVN circuitry.

Brain Circuit of Claustrophobia-like Behavior in Mice Identified by Upstream Tracing of Sighing.

Emotions are distinct patterns of behavioral and physiological responses triggered by stimuli that induce different brain states. Elucidating the circuits is difficult because of challenges in interrogating emotional brain states and their complex outputs. Here, we leverage the recent discovery in mice of a neural circuit for sighing, a simple, quantifiable output of various emotions. We show that mouse confinement triggers sighing, and this "claustrophobic" sighing, but not accompanying tachypnea, requires the same medullary neuromedin B (Nmb)-expressing neurons as physiological sighing. Retrograde tracing from the Nmb neurons identified 12 forebrain centers providing presynaptic input, including hypocretin (Hcrt)-expressing lateral hypothalamic neurons. Confinement activates Hcrt neurons, and optogenetic activation induces sighing and tachypnea whereas pharmacologic inhibition suppresses both responses. The effect on sighing is mediated by HCRT directly on Nmbneurons. We propose that this HCRT-NMB neuropeptide relay circuit mediates claustrophobic sighing and that activated Hcrt neurons are a claustrophobia brain state that directly controls claustrophobic outputs.

The hypocretin (orexin) system: from a neural circuitry perspective.

Hypocretin/orexin neurons are distributed restrictively in the hypothalamus, a brain region known to orchestrate diverse functions including sleep, reward processing, food intake, thermogenesis, and mood. Since the hypocretins/orexins were discovered more than two decades ago, extensive studies have accumulated concrete evidence showing the pivotal role of hypocretin/orexin in diverse neural modulation. New method of viral-mediated tracing system offers the possibility to map the monosynaptic inputs and detailed anatomical connectivity of Hcrt neurons. With the development of powerful research techniques including optogenetics, fiber-photometry, cell-type/pathway specific manipulation and neuronal activity monitoring, as well as single-cell RNA sequencing, the details of how hypocretinergic system execute functional modulation of various behaviors are coming to light. In this review, we focus on the function of neural pathways from hypocretin neurons to target brain regions. Anatomical and functional inputs to hypocretin neurons are also discussed. We further briefly summarize the development of pharmaceutical compounds targeting hypocretin signaling. This article is part of the special issue on Neuropeptides.

In vivo cell type-specific CRISPR knockdown of dopamine beta hydroxylase reduces locus coeruleus evoked wakefulness.

Locus coeruleus (LC) neurons in the brainstem have long been associated with attention and arousal. Optogenetic stimulation of LC-NE neurons induces immediate sleep-to-wake transitions. However, LC neurons also secrete other neurotransmitters in addition to NE. To interrogate the role of NE derived from the LC in regulating wakefulness, we applied in vivo cell type-specific CRISPR/Cas9 technology to disrupt the dopamine beta hydroxylase (dbh) gene selectively in adult LC-NE neurons. Unilateral dbh gene disruption abolished immediate arousal following optogenetic stimulation of LC. Bilateral LC-specific dbh disruption significantly reduced NE concentration in LC projection areas and reduced wake length even in the presence of salient stimuli. These results suggest that NE may be crucial for the awakening effect of LC stimulation and serve as proof-of-principle that CRISPR gene editing in adult neurons can be used to interrogate gene function within genetically-defined neuronal circuitry associated with complex behaviors.

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.

Optical probing of orexin/hypocretin receptor antagonists.

Study Objectives: The present study investigated the function of Hypocretin (Hcrt or Orexin/OX) receptor antagonists in sleep modulation and memory function with optical methods in transgenic mice. Methods: We used Hcrt-IRES-Cre knock-in mice and AAV vectors expressing channelrhodopsin-2 (ChR2) to render Hcrt neurons sensitive to blue light stimulation. We optogenetically stimulated Hcrt neurons and measured latencies to wakefulness in the presence or absence of OX1/2R antagonists and Zolpidem. We also examined endogenous Hcrt neuronal activity with fiber photometry. Changes in memory after optogenetic sleep disruption were evaluated by the novel object recognition test (NOR) and compared for groups treated with vehicle, OX1/2R antagonists, or Zolpidem. We also analyzed electroencephalogram (EEG) power spectra of wakefulness, rapid eye movement (REM) sleep, and non-REM (NREM) sleep following the injections of vehicle, OX1/2R antagonists, and Zolpidem in young adult mice. Results: Acute optogenetic stimulation of Hcrt neurons at different frequencies resulted in wakefulness. Treatment with dual OX1/2R antagonists (DORAs) DORA12 and MK6096, as well as selective OX2R antagonist MK1064 and Zolpidem, but not selective OX1R antagonist 1SORA1, significantly reduced the bout length of optogenetic stimulation-evoked wakefulness episode. Fiber photometry recordings of GCaMP6f signals showed that Hcrt neurons are active during wakefulness, even in the presence of OXR antagonists. Treatment with dual OX1/2R antagonists improved memory function despite optogenetic sleep fragmentation caused impaired memory function in a NOR test. Conclusions: Our results show DORAs and selective OX2R antagonists stabilize sleep and improve sleep-dependent cognitive processes even when challenged by optogenetic stimulation mimicking highly arousing stimuli.

Hypocretins and Arousal.

How the brain controls vigilance state transitions remains to be fully understood. The discovery of hypocretins, also known as orexins, and their link to narcolepsy has undoubtedly allowed us to advance our knowledge on key mechanisms controlling the boundaries and transitions between sleep and wakefulness. Lack of function of hypocretin neurons (a relatively simple and non-redundant neuronal system) results in inappropriate control of sleep states without affecting the total amount of sleep or homeostatic mechanisms. Anatomical and functional evidence shows that the hypothalamic neurons that produce hypocretins/orexins project widely throughout the entire brain and interact with major neuromodulator systems in order to regulate physiological processes underlying wakefulness, attention, and emotions. Here, we review the role of hypocretins/orexins in arousal state transitions, and discuss possible mechanisms by which such a relatively small population of neurons controls fundamental brain state dynamics.

In vivo assessment of behavioral recovery and circulatory exchange in the peritoneal parabiosis model.

The sharing of circulation between two animals using a surgical procedure known as parabiosis has created a wealth of information towards our understanding of physiology, most recently in the neuroscience arena. The systemic milieu is a complex reservoir of tissues, immune cells, and circulating molecules that is surprisingly not well understood in terms of its communication across organ systems. While the model has been used to probe complex physiological questions for many years, critical parameters of recovery and exchange kinetics remain incompletely characterized, limiting the ability to design experiments and interpret results for complex questions. Here we provide evidence that mice joined by parabiosis gradually recover much physiology relevant to the study of brain function. Specifically, we describe the timecourse for a variety of recovery parameters, including those for general health and metabolism, motor coordination, activity, and sleep behavior. Finally, we describe the kinetics of chimerism for several lymphocyte populations as well as the uptake of small molecules into the brains of mice following parabiosis. Our characterization provides an important resource to those attempting to understand the complex interplay between the immune system and the brain as well as other organ systems.

Hypocretins, Neural Systems, Physiology, and Psychiatric Disorders.

The hypocretins (Hcrts), also known as orexins, have been among the most intensely studied neuropeptide systems since their discovery about two decades ago. Anatomical evidence shows that the hypothalamic neurons that produce hypocretins/orexins project widely throughout the entire brain, innervating the noradrenergic locus coeruleus, the cholinergic basal forebrain, the dopaminergic ventral tegmental area, the serotonergic raphe nuclei, the histaminergic tuberomammillary nucleus, and many other brain regions. By interacting with other neural systems, the Hcrt system profoundly modulates versatile physiological processes including arousal, food intake, emotion, attention, and reward. Importantly, interruption of the interactions between these systems has the potential to cause neurological and psychiatric diseases. Here, we review the modulation of diverse neural systems by Hcrts and summarize potential therapeutic strategies based on our understanding of the Hcrt system's role in physiology and pathophysiological processes.

D4 Receptor Activation Differentially Modulates Hippocampal Basal and Apical Dendritic Synapses in Freely Moving Mice.

Activation of D4 receptors (D4Rs) has been shown to improve cognitive performance, potentially affecting synaptic strength. We investigated the D4R agonist PD 168077 (PD) in hippocampal CA1 of freely moving mice. We electrically stimulated in stratum oriens (OR) or radiatum (RAD) and evoked local field potentials (LFPs). Intraperitoneally injected PD dose-dependently and reversibly attenuated LFPs for longer time in basal (OR) than apical (RAD) dendrites. High-frequency stimulation induced LTP that was stronger and more stable in OR than RAD. LTP lasted at least 4 h during which the paired-pulse ratio remained reduced. A PD concentration not affecting synaptic transmission was sufficient to reduce LTP in OR but not in RAD. A PD concentration reducing synaptic transmission reduced the early phase LTP in OR additionally and the late phase LTP in RAD exclusively. Furthermore, cell type-specific expression of mCherry in DATCre mice generated fluorescence in dorsal CA1 that was highest in lacunosum moleculare and similar in OR/RAD, indicating that midbrain dopaminergic fibers distribute evenly in OR/RAD. Together, the D4R-mediated modulation of hippocampal synaptic transmission and plasticity is stronger in OR than RAD. This could affect information processing in CA1 neurons, since signals arriving via basal and apical afferents are distinct.

Controlled variations in stimulus similarity during learning determine visual discrimination capacity in freely moving mice.

The mouse is receiving growing interest as a model organism for studying visual perception. However, little is known about how discrimination and learning interact to produce visual conditioned responses. Here, we adapted a two-alternative forced-choice visual discrimination task for mice and examined how training with equiprobable stimuli of varying similarity influenced conditioned response and discrimination performance as a function of learning. Our results indicate that the slope of the gradients in similarity during training determined the learning rate, the maximum performance and the threshold for successful discrimination. Moreover, the learning process obeyed an inverse relationship between discrimination performance and discriminative resolution, implying that sensitivity within a similarity range cannot be improved without sacrificing performance in another. Our study demonstrates how the interplay between discrimination and learning controls visual discrimination capacity and introduces a new training protocol with quantitative measures to study perceptual learning and visually-guided behavior in freely moving mice.

Discrimination learning with variable stimulus 'salience'.

BACKGROUND: In nature, sensory stimuli are organized in heterogeneous combinations. Salient items from these combinations 'stand-out' from their surroundings and determine what and how we learn. Yet, the relationship between varying stimulus salience and discrimination learning remains unclear. PRESENTATION OF THE HYPOTHESIS: A rigorous formulation of the problem of discrimination learning should account for varying salience effects. We hypothesize that structural variations in the environment where the conditioned stimulus (CS) is embedded will be a significant determinant of learning rate and retention level. TESTING THE HYPOTHESIS: Using numerical simulations, we show how a modified version of the Rescorla-Wagner model, an influential theory of associative learning, predicts relevant interactions between varying salience and discrimination learning. IMPLICATIONS OF THE HYPOTHESIS: If supported by empirical data, our model will help to interpret critical experiments addressing the relations between attention, discrimination and learning.