Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Brain-wide neuronal circuit connectome of human glioblastoma.

Glioblastoma (GBM), a universally fatal brain cancer, infiltrates the brain and can be synaptically innervated by neurons, which drives tumor progression 1-6 . Synaptic inputs onto GBM cells identified so far are largely short-range and glutamatergic 7-9 . The extent of integration of GBM cells into brain-wide neuronal circuitry is not well understood. Here we applied a rabies virus-mediated retrograde monosynaptic tracing approach 10-12 to systematically investigate circuit integration of human GBM organoids transplanted into adult mice. We found that GBM cells from multiple patients rapidly integrated into brain-wide neuronal circuits and exhibited diverse local and long-range connectivity. Beyond glutamatergic inputs, we identified a variety of neuromodulatory inputs across the brain, including cholinergic inputs from the basal forebrain. Acute acetylcholine stimulation induced sustained calcium oscillations and long-lasting transcriptional reprogramming of GBM cells into a more invasive state via the metabotropic CHRM3 receptor. CHRM3 downregulation suppressed GBM cell invasion, proliferation, and survival in vitro and in vivo. Together, these results reveal the capacity of human GBM cells to rapidly and robustly integrate into anatomically and molecularly diverse neuronal circuitry in the adult brain and support a model wherein rapid synapse formation onto GBM cells and transient activation of upstream neurons may lead to a long-lasting increase in fitness to promote tumor infiltration and progression.

Inflammation-related pathology in the olfactory epithelium: its impact on the olfactory system in psychotic disorders.

Smell deficits and neurobiological changes in the olfactory bulb (OB) and olfactory epithelium (OE) have been observed in schizophrenia and related disorders. The OE is the most peripheral olfactory system located outside the cranium, and is connected with the brain via direct neuronal projections to the OB. Nevertheless, it is unknown whether and how a disturbance of the OE affects the OB in schizophrenia and related disorders. Addressing this gap would be the first step in studying the impact of OE pathology in the disease pathophysiology in the brain. In this cross-species study, we observed that chronic, local OE inflammation with a set of upregulated genes in an inducible olfactory inflammation (IOI) mouse model led to a volume reduction, layer structure changes, and alterations of neuron functionality in the OB. Furthermore, IOI model also displayed behavioral deficits relevant to negative symptoms (avolition) in parallel to smell deficits. In first episode psychosis (FEP) patients, we observed a significant alteration in immune/inflammation-related molecular signatures in olfactory neuronal cells (ONCs) enriched from biopsied OE and a significant reduction in the OB volume, compared with those of healthy controls (HC). The increased expression of immune/inflammation-related molecules in ONCs was significantly correlated to the OB volume reduction in FEP patients, but no correlation was found in HCs. Moreover, the increased expression of human orthologues of the IOI genes in ONCs was significantly correlated with the OB volume reduction in FEP, but not in HCs. Together, our study implies a potential mechanism of the OE-OB pathology in patients with psychotic disorders (schizophrenia and related disorders). We hope that this mechanism may have a cross-disease implication, including COVID-19-elicited mental conditions that include smell deficits.

Circuit mechanism underlying fragmented sleep and memory deficits in 16p11.2 deletion mouse model of autism.

Sleep disturbances are prevalent in children with autism spectrum disorder (ASD) and have a major impact on the quality of life. Strikingly, sleep problems are positively correlated with the severity of ASD symptoms, such as memory impairment. However, the neural mechanisms underlying sleep disturbances and cognitive deficits in ASD are largely unexplored. Here, we show that non-rapid eye movement sleep (NREMs) is highly fragmented in the 16p11.2 deletion mouse model of ASD. The degree of sleep fragmentation is reflected in an increased number of calcium transients in the activity of locus coeruleus noradrenergic (LC-NE) neurons during NREMs. Exposure to a novel environment further exacerbates sleep disturbances in 16p11.2 deletion mice by fragmenting NREMs and decreasing rapid eye movement sleep (REMs). In contrast, optogenetic inhibition of LC-NE neurons and pharmacological blockade of noradrenergic transmission using clonidine reverse sleep fragmentation. Furthermore, inhibiting LC-NE neurons restores memory. Rabies-mediated unbiased screening of presynaptic neurons reveals altered connectivity of LC-NE neurons with sleep- and memory regulatory brain regions in 16p11.2 deletion mice. Our findings demonstrate that heightened activity of LC-NE neurons and altered brain-wide connectivity underlies sleep fragmentation in 16p11.2 deletion mice and identify a crucial role of the LC-NE system in regulating sleep stability and memory in ASD.

Inflammation-related pathology in the olfactory epithelium: its impact on the olfactory system in psychotic disorders.

Smell deficits and neurobiological changes in the olfactory bulb (OB) and olfactory epithelium (OE) have been observed in schizophrenia and related disorders. The OE is the most peripheral olfactory system located outside the cranium, and is connected with the brain via direct neuronal projections to the OB. Nevertheless, it is unknown whether and how a disturbance of the OE affects the OB in schizophrenia and related disorders. Addressing this gap would be the first step in studying the impact of OE pathology in the disease pathophysiology in the brain. In this cross-species study, we observed that chronic, local OE inflammation with a set of upregulated genes in an inducible olfactory inflammation (IOI) mouse model led to a volume reduction, layer structure changes, and alterations of neuron functionality in the OB. Furthermore, IOI model also displayed behavioral deficits relevant to negative symptoms (avolition) in parallel to smell deficits. In first episode psychosis (FEP) patients, we observed a significant alteration in immune/inflammation-related molecular signatures in olfactory neuronal cells (ONCs) enriched from biopsied OE and a significant reduction in the OB volume, compared with those of healthy controls (HC). The increased expression of immune/inflammation-related molecules in ONCs was significantly correlated to the OB volume reduction in FEP patients, but no correlation was found in HCs. Moreover, the increased expression of human orthologues of the IOI genes in ONCs was significantly correlated with the OB volume reduction in FEP, but not in HCs. Together, our study implies a potential mechanism of the OE-OB pathology in patients with psychotic disorders (schizophrenia and related disorders). We hope that this mechanism may have a cross-disease implication, including COVID-19-elicited mental conditions that include smell deficits.

Unraveling the Link between Olfactory Deficits and Neuropsychiatric Disorders.

Smell loss has caught public attention during the recent COVID-19 pandemic. Research on olfactory function in health and disease gains new momentum. Smell deficits have long been recognized as an early clinical sign associated with neuropsychiatric disorders. Here we review research on the associations between olfactory deficits and neuropathological conditions, focusing on recent progress in four areas: (1) human clinical studies of the correlations between smell deficits and neuropsychiatric disorders; (2) development of olfactory mucosa-derived tissue and cell models for studying the molecular pathologic mechanisms; (3) recent findings in brain imaging studies of structural and functional connectivity changes in olfactory pathways in neuropsychiatric disorders; and (4) application of preclinical animal models to validate and extend the findings from human subjects. Together, these studies have provided strong evidence of the link between the olfactory system and neuropsychiatric disorders, highlighting the relevance of deepening our understanding of the role of the olfactory system in pathophysiological processes. Following the lead of studies reviewed here, future research in this field may open the door to the early detection of neuropsychiatric disorders, personalized treatment approaches, and potential therapeutic interventions through nasal administration techniques, such as nasal brush or nasal spray.

Ventral striatal islands of Calleja neurons bidirectionally mediate depression-like behaviors in mice.

The ventral striatum is a reward center implicated in the pathophysiology of depression. It contains islands of Calleja, clusters of dopamine D3 receptor-expressing granule cells, predominantly in the olfactory tubercle (OT). These OT D3 neurons regulate self-grooming, a repetitive behavior manifested in affective disorders. Here we show that chronic restraint stress (CRS) induces robust depression-like behaviors in mice and decreases excitability of OT D3 neurons. Ablation or inhibition of these neurons leads to depression-like behaviors, whereas their activation ameliorates CRS-induced depression-like behaviors. Moreover, activation of OT D3 neurons has a rewarding effect, which diminishes when grooming is blocked. Finally, we propose a model that explains how OT D3 neurons may influence dopamine release via synaptic connections with OT spiny projection neurons (SPNs) that project to midbrain dopamine neurons. Our study reveals a crucial role of OT D3 neurons in bidirectionally mediating depression-like behaviors, suggesting a potential therapeutic target.

Intranasal Pressure Recording for Monitoring Mouse Respiration.

Respiration is a highly dynamic signal that influences voluntary behaviors including odor sampling and entrains rhythmic activity in the brain. Many techniques exist to record respiration with each exhibiting strengths and drawbacks given the ultimate goals of the respiration recording. Intranasal cannula implantation, coupled with pressure sensor recording, allows for temporal precision and detailed feature extraction of the respiratory waveform. Here we describe the implantation process and necessary recording equipment to effectively conduct intranasal pressure recording of respiration. This is an ideal method for understanding the dynamics of odor sampling in conjunction with olfactory sensory transmission.

Machine learning-based clustering and classification of mouse behaviors via respiratory patterns.

Breathing is dynamically modulated by metabolic needs as well as by emotional states. Even though rodents are invaluable models for investigating the neural control of respiration, current literature lacks systematic characterization of breathing dynamics across a broad spectrum of rodent behaviors. Here we uncover a wide diversity in breathing patterns across spontaneous, attractive odor-, stress-, and fear-induced behaviors in mice. Direct recordings of intranasal pressure afford more detailed respiratory information than more traditional whole-body plethysmography. K-means clustering groups 11 well-defined behavioral states into four clusters with distinct key respiratory features. Furthermore, we implement RUSBoost (random undersampling boost) classification, a supervised machine learning model, and find that breathing patterns can separate these behaviors with an accuracy of 80%. Taken together, our findings highlight the tight relationship between breathing and behavior and the potential use of breathing patterns to aid in distinguishing similar behaviors and inform about their internal states.

Self-directed orofacial grooming promotes social attraction in mice via chemosensory communication.

Self-grooming is a stereotyped behavior displayed by nearly all animals. Among other established functions, self-grooming is implicated in social communication. However, whether self-grooming specifically influences behaviors of nearby individuals has not been directly tested, partly because of the technical challenge of inducing self-grooming in a reliable and temporally controllable manner. We recently found that optogenetic activation of dopamine D3 receptor expressing neurons in the ventral striatal islands of Calleja robustly induces orofacial grooming in mice. Using this optogenetic manipulation, here we demonstrate that observer mice exhibit social preference for mice that groom more regardless of biological sex. Moreover, grooming-induced social attraction depends on volatile chemosensory cues broadcasted from grooming mice. Collectively, our study establishes self-grooming as a means of promoting social attraction among mice via volatile cues, suggesting an additional benefit for animals to allocate a significant amount of time to this behavior.

State-dependent olfactory processing in freely behaving mice.

Decreased responsiveness to sensory stimuli during sleep is presumably mediated via thalamic gating. Without an obligatory thalamic relay in the olfactory system, the anterior piriform cortex (APC) is suggested to be a gate in anesthetized states. However, olfactory processing in natural sleep states remains undetermined. Here, we simultaneously record local field potentials (LFPs) in hierarchical olfactory regions (olfactory bulb [OB], APC, and orbitofrontal cortex) while optogenetically activating olfactory sensory neurons, ensuring consistent peripheral inputs across states in behaving mice. Surprisingly, evoked LFPs in sleep states (both non-rapid eye movement [NREM] and rapid eye movement [REM]) are larger and contain greater gamma-band power and cross-region coherence (compared to wakefulness) throughout the olfactory pathway, suggesting the lack of a central gate. Single-unit recordings from the OB and APC reveal a higher percentage of responsive neurons during sleep with a higher incidence of suppressed firing. Additionally, nasal breathing is slower and shallower during sleep, suggesting a partial peripheral gating mechanism.

Olfactory modulation of the medial prefrontal cortex circuitry: Implications for social cognition.

Olfactory dysfunction is manifested in a wide range of neurological and psychiatric diseases, and often emerges prior to the onset of more classical symptoms and signs. From a behavioral perspective, olfactory deficits typically arise in conjunction with impairments of cognition, motivation, memory, and emotion. However, a conceptual framework for explaining the impact of olfactory processing on higher brain functions in health and disease remains lacking. Here we aim to provide circuit-level insights into this question by synthesizing recent advances in olfactory network connectivity with other cortical brain regions such as the prefrontal cortex. We will focus on social cognition as a representative model for exploring and critically evaluating the relationship between olfactory cortices and higher-order cortical regions in rodent models. Although rodents do not recapitulate all dimensions of human social cognition, they have experimentally accessible neural circuits and well-established behavioral tests for social motivation, memory/recognition, and hierarchy, which can be extrapolated to other species including humans. In particular, the medial prefrontal cortex (mPFC) has been recognized as a key brain region in mediating social cognition in both rodents and humans. This review will highlight the underappreciated connectivity, both anatomical and functional, between the olfactory system and mPFC circuitry, which together provide a neural substrate for olfactory modulation of social cognition and social behaviors. We will provide future perspectives on the functional investigation of the olfactory-mPFC circuit in rodent models and discuss how to translate such animal research to human studies.

Suppression of neurotransmission on gonadotropin-releasing hormone neurons in letrozole-induced polycystic ovary syndrome: A mouse model.

Objective: Polycystic ovarian syndrome (PCOS) is a heterogeneous endocrine disorder in reproductive-age women, characterized by the accretion of small cystic follicles in the ovary associated with chronic anovulation and overproduction of androgens. Ovarian function in all mammals is controlled by gonadotropin-releasing hormone (GnRH) neurons, which are the central regulator of the hypothalamic-pituitary-gonadal (HPG) axis. However, the impact on the neurotransmitter system regulating GnRH neuronal function in the letrozole-induced PCOS mouse model remains unclear. Methods: In this study, we compared the response of various neurotransmitters and neurosteroids regulating GnRH neuronal activities between letrozole-induced PCOS and normal mice via electrophysiological techniques. Results: Response to neurotransmitter systems like GABAergic, glutamatergic and kisspeptinergic were suppressed in letrozole-fed compared to normal mice. In addition, neurosteroids tetrahydrodeoxycorticosterone (THDOC) and 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) mediated response on GnRH neurons were significantly smaller on letrozole-fed mice compared to normal mice. Furthermore, we also found that letrozole-fed mice showed irregularity in the estrous cycle, increased body weight, and anovulation in female mice. Conclusion: These findings suggest that PCOS is an endocrine disorder that may directly affect the neurotransmitter system regulating GnRH neuronal activity at the hypothalamic level and impact reproductive physiology.

Ventral striatal islands of Calleja neurons control grooming in mice.

The striatum comprises multiple subdivisions and neural circuits that differentially control motor output. The islands of Calleja (IC) contain clusters of densely packed granule cells situated in the ventral striatum, predominantly in the olfactory tubercle (OT). Characterized by expression of the D3 dopamine receptor, the IC are evolutionally conserved, but have undefined functions. Here, we show that optogenetic activation of OT D3 neurons robustly initiates self-grooming in mice while suppressing other ongoing behaviors. Conversely, optogenetic inhibition of these neurons halts ongoing grooming, and genetic ablation reduces spontaneous grooming. Furthermore, OT D3 neurons show increased activity before and during grooming and influence local striatal output via synaptic connections with neighboring OT neurons (primarily spiny projection neurons), whose firing rates display grooming-related modulation. Our study uncovers a new role of the ventral striatum's IC in regulating motor output and has important implications for the neural control of grooming.

Targeted Inhibition of Anti-Inflammatory Regulator Nrf2 Results in Breast Cancer Retardation In Vitro and In Vivo.

Nuclear factor erythroid-2 related factor-2 (Nrf2) is an oxidative stress-response transcriptional activator that promotes carcinogenesis through metabolic reprogramming, tumor promoting inflammation, and therapeutic resistance. However, the extension of Nrf2 expression and its involvement in regulation of breast cancer (BC) responses to chemotherapy remain largely unclear. This study determined the expression of Nrf2 in BC tissues (n = 46) and cell lines (MDA-MB-453, MCF-7, MDA-MB-231, MDA-MB-468) with diverse phenotypes. Immunohistochemical (IHC)analysis indicated lower Nrf2 expression in normal breast tissues, compared to BC samples, although the difference was not found to be significant. However, pharmacological inhibition and siRNA-induced downregulation of Nrf2 were marked by decreased activity of NADPH quinone oxidoreductase 1 (NQO1), a direct target of Nrf2. Silenced or inhibited Nrf2 signaling resulted in reduced BC proliferation and migration, cell cycle arrest, activation of apoptosis, and sensitization of BC cells to cisplatin in vitro. Ehrlich Ascites Carcinoma (EAC) cells demonstrated elevated levels of Nrf2 and were further tested in experimental mouse models in vivo. Intraperitoneal administration of pharmacological Nrf2 inhibitor brusatol slowed tumor cell growth. Brusatol increased lymphocyte trafficking towards engrafted tumor tissue in vivo, suggesting activation of anti-cancer effects in tumor microenvironment. Further large-scale BC testing is needed to confirm Nrf2 marker and therapeutic capacities for chemo sensitization in drug resistant and advanced tumors.

Chitinase-Like Protein Ym2 (Chil4) Regulates Regeneration of the Olfactory Epithelium via Interaction with Inflammation.

The adult olfactory epithelium (OE) regenerates sensory neurons and nonsensory supporting cells from resident stem cells after injury. How supporting cells contribute to OE regeneration remains largely unknown. In this study, we elucidated a novel role of Ym2 (also known as Chil4 or Chi3l4), a chitinase-like protein expressed in supporting cells, in regulating regeneration of the injured OE in vivo in both male and female mice and cell proliferation/differentiation in OE colonies in vitro We found that Ym2 expression was enhanced in supporting cells after OE injury. Genetic knockdown of Ym2 in supporting cells attenuated recovery of the injured OE, while Ym2 overexpression by lentiviral infection accelerated OE regeneration. Similarly, Ym2 bidirectionally regulated cell proliferation and differentiation in OE colonies. Furthermore, anti-inflammatory treatment reduced Ym2 expression and delayed OE regeneration in vivo and cell proliferation/differentiation in vitro, which were counteracted by Ym2 overexpression. Collectively, this study revealed a novel role of Ym2 in OE regeneration and cell proliferation/differentiation of OE colonies via interaction with inflammatory responses, providing new clues to the function of supporting cells in these processes.SIGNIFICANCE STATEMENT The mammalian olfactory epithelium (OE) is a unique neural tissue that regenerates sensory neurons and nonsensory supporting cells throughout life and postinjury. How supporting cells contribute to this process is not entirely understood. Here we report that OE injury causes upregulation of a chitinase-like protein, Ym2, in supporting cells, which facilitates OE regeneration. Moreover, anti-inflammatory treatment reduces Ym2 expression and delays OE regeneration, which are counteracted by Ym2 overexpression. This study reveals an important role of supporting cells in OE regeneration and provides a critical link between Ym2 and inflammation in this process.

Infrared attenuation due to phase change from amorphous to crystalline observed in astrochemical propargyl ether ices.

Astrochemical ices are known to undergo morphological changes, from amorphous to crystalline, upon warming the ice from lower (10 K) to higher temperatures. Phase changes are mostly identified by the observation of significant changes in the InfraRed (IR) spectrum, where the IR bands that are broad in the amorphous phase are narrower and split when the ice turns crystalline. To-date all the molecules that are studied under astrochemical conditions are observed to follow such a behaviour without significant attenuation in the IR wavelength. However, in this paper we report a new observation when propargyl ether (C3H3OC3H3) is warmed from the amorphous phase, at 10 K, through the phase transition temperature of 170 K, the crystalline ice being found to strongly attenuate IR photons at the mid-IR wavelengths.

Aversive Learning Increases Release Probability of Olfactory Sensory Neurons.

Predicting danger from previously associated sensory stimuli is essential for survival. Contributions from altered peripheral sensory inputs are implicated in this process, but the underlying mechanisms remain elusive. Here, we use the mammalian olfactory system to investigate such mechanisms. Primary olfactory sensory neurons (OSNs) project their axons directly to the olfactory bulb (OB) glomeruli, where their synaptic release is subject to local and cortical influence and neuromodulation. Pairing optogenetic activation of a single glomerulus with foot shock in mice induces freezing to light stimulation alone during fear retrieval. This is accompanied by an increase in OSN release probability and a reduction in GABAB receptor expression in the conditioned glomerulus. Furthermore, freezing time is positively correlated with the release probability of OSNs in fear-conditioned mice. These results suggest that aversive learning increases peripheral olfactory inputs at the first synapse, which may contribute to the behavioral outcome.

Residue from vacuum ultraviolet irradiation of benzene ices: Insights into the physical structure of astrophysical dust.

We have irradiated benzene ices deposited at 4 K on a cold, interstellar dust analog with vacuum ultraviolet (9 eV) irradiation for periods lasting from several hours to nearly a day, after which the irradiated ice was warmed to room temperature. Vacuum ultraviolet photoabsorption spectra of the aromatic residue left at room temperature were recorded and showed the synthesis of benzene derivatives. The residue was also imaged using an electron microscope and revealed crystals of various sizes and shapes. The result of our experiments suggests such geometrically shaped dust particles may be a key component of interstellar dust.