Regulation of stress-induced sleep fragmentation by preoptic glutamatergic neurons.

Sleep disturbances are detrimental to our behavioral and emotional well-being. Stressful events disrupt sleep, in particular by inducing brief awakenings (microarousals, MAs), resulting in sleep fragmentation. The preoptic area of the hypothalamus (POA) is crucial for sleep control. However, how POA neurons contribute to the regulation of MAs and thereby impact sleep quality is unknown. Using fiber photometry in mice, we examine the activity of genetically defined POA subpopulations during sleep. We find that POA glutamatergic neurons are rhythmically activated in synchrony with an infraslow rhythm in the spindle band of the electroencephalogram during non-rapid eye movement sleep (NREMs) and are transiently activated during MAs. Optogenetic stimulation of these neurons promotes MAs and wakefulness. Exposure to acute social defeat stress fragments NREMs and significantly increases the number of transients in the calcium activity of POA glutamatergic neurons during NREMs. By reducing MAs, optogenetic inhibition during spontaneous sleep and after stress consolidates NREMs. Monosynaptically restricted rabies tracing reveals that POA glutamatergic neurons are innervated by brain regions regulating stress and sleep. In particular, presynaptic glutamatergic neurons in the lateral hypothalamus become activated after stress, and stimulating their projections to the POA promotes MAs and wakefulness. Our findings uncover a novel circuit mechanism by which POA excitatory neurons regulate sleep quality after stress.

Prefrontal cortical regulation of REM sleep.

Rapid eye movement (REM) sleep is accompanied by intense cortical activity, underlying its wake-like electroencephalogram. The neural activity inducing REM sleep is thought to originate from subcortical circuits in brainstem and hypothalamus. However, whether cortical neurons can also trigger REM sleep has remained unknown. Here we show in mice that the medial prefrontal cortex (mPFC) strongly promotes REM sleep. Bidirectional optogenetic manipulations demonstrate that excitatory mPFC neurons promote REM sleep through their projections to the lateral hypothalamus and regulate phasic events, reflected in accelerated electroencephalogram theta oscillations and increased eye movement density during REM sleep. Calcium imaging reveals that the majority of lateral hypothalamus-projecting mPFC neurons are maximally activated during REM sleep and a subpopulation is recruited during phasic theta accelerations. Our results delineate a cortico-hypothalamic circuit for the top-down control of REM sleep and identify a critical role of the mPFC in regulating phasic events during REM sleep.

Nanocrystalline Apatites: Post-Immersion Acidification and How to Avoid It-Application to Antibacterial Bone Substitutes.

Biomimetic nanocrystalline apatites analogous to bone mineral can be prepared using soft chemistry. Due to their high similarity to bone apatite, as opposed to stoichiometric hydroxyapatite for example, they now represent an appealing class of compounds to produce bioactive ceramics for which drug delivery and ion exchange abilities have been described extensively. However, immersion in aqueous media of dried non-carbonated biomimetic apatite crystals may generate an acidification event, which is often disregarded and not been clarified to-date. Yet, this acidification process could limit their further development if it is not understood and overcome if necessary. This may, for example, alter biological test outcomes, during their evaluation as bone repair materials, due to potentially deleterious effects of the acidic environment on cells, especially in in vitro static conditions. In this study, we explore the origins of this acidification phenomenon based on complementary experimental data and we point out the central role of the hydrated ionic layer present on apatite nanocrystals. We then propose a practical strategy to circumvent this acidification effect using an adequate post-precipitation equilibration step that was optimized. Using this enutralization protocol, we then showed the possibility of performing (micro)biological assessments on such compounds and provide an illustration with the examples of post-equilibrated Cu2+- and Ag+-doped nanocrystalline apatites. We demonstrate their non-cytotoxicity to osteoblast cells and their antibacterial features as tested versus five major pathogens involved in bone infections, therefore pointing to their relevance in the field of antibacterial bone substitutes. The preliminary in vivo implantation of a relevant sample in a rat's calvarial defect confirmed its biocompatibility and the absence of adverse reaction. Understanding and eliminating this technical barrier should help promoting biomimetic apatites as a genuine new class of biomaterial-producing compounds for bone regeneration applications, e.g., with antibacterial features, far from being solely considered as "laboratory curiosities".

A noradrenergic-hypothalamic neural substrate for stress-induced sleep disturbances.

In our daily life, we are exposed to uncontrollable and stressful events that disrupt our sleep. However, the underlying neural mechanisms deteriorating the quality of non-rapid eye movement sleep (NREMs) and REM sleep are largely unknown. Here, we show in mice that acute psychosocial stress disrupts sleep by increasing brief arousals (microarousals [MAs]), reducing sleep spindles, and impairing infraslow oscillations in the spindle band of the electroencephalogram during NREMs, while reducing REMs. This poor sleep quality was reflected in an increased number of calcium transients in the activity of noradrenergic (NE) neurons in the locus coeruleus (LC) during NREMs. Opto- and chemogenetic LC-NE activation in naïve mice is sufficient to change the sleep microarchitecture similar to stress. Conversely, chemogenetically inhibiting LC-NE neurons reduced MAs during NREMs and normalized their number after stress. Specifically inhibiting LC-NE neurons projecting to the preoptic area of the hypothalamus (POA) decreased MAs and enhanced spindles and REMs after stress. Optrode recordings revealed that stimulating LC-NE fibers in the POA indeed suppressed the spiking activity of POA neurons that are activated during sleep spindles and REMs and inactivated during MAs. Our findings reveal that changes in the dynamics of the stress-regulatory LC-NE neurons during sleep negatively affect sleep quality, partially through their interaction with the POA.

An Excitatory Circuit in the Perioculomotor Midbrain for Non-REM Sleep Control.

The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.

Heterodimeric BMP-2/7 for nucleus pulposus regeneration-In vitro and ex vivo studies.

Intervertebral disc (IVD) degeneration is the leading trigger of low back pain, which causes disability and leads to enormous healthcare toll worldwide. Biological treatment with growth factors has evolved as potential therapy for IVD regeneration. Bone morphogenetic protein 2 (BMP-2) and BMP-7 have shown promise in this regard. In the current study, we evaluated the effect of BMP-2/7 heterodimer for disc regeneration both in vitro and in organ culture. Nucleus pulposus (NP) cells isolated from bovine caudal disc were cultured in a fibrin-hyaluronan (FBG-HA) hydrogel for up to 14 days. BMP-2/7 heterodimer covalently incorporated within the hydrogel up-regulated the aggrecan and type II collagen gene expression, and glycosaminoglycan synthesis of NP cells. The activity of the BMP-2/7 heterodimer was dose dependent. The higher dose of BMP-2/7 was further assessed in an IVD whole organ system. After 14 days of culture with cyclic dynamic load, the BMP-2/7 heterodimer delivered into the nucleotomized region showed potential to stimulate the gene expression and synthesis of proteoglycan in the remaining NP tissue after partial nucleotomy. The gene expression level of type I collagen and alkaline phosphatase in the native disc tissue were not affected by BMP-2/7 treatment, indicating no adverse fibroblastic or osteogenic effect on the disc tissue. Intradiscal delivery of BMP-2/7 heterodimer may be a promising therapeutic approach for NP regeneration. The current IVD whole organ partial nucleotomy model may be utilized for screening of other biomaterials or drugs to treat early degenerative disc disorders. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:51-60, 2017.

Circuit-based interrogation of sleep control.

Sleep is a fundamental biological process observed widely in the animal kingdom, but the neural circuits generating sleep remain poorly understood. Understanding the brain mechanisms controlling sleep requires the identification of key neurons in the control circuits and mapping of their synaptic connections. Technical innovations over the past decade have greatly facilitated dissection of the sleep circuits. This has set the stage for understanding how a variety of environmental and physiological factors influence sleep. The ability to initiate and terminate sleep on command will also help us to elucidate its functions within and beyond the brain.