Microglial homeostasis disruption modulates non-rapid eye movement sleep duration and neuronal activity in adult female mice.

Sleep is a natural physiological state, tightly regulated through several neuroanatomical and neurochemical systems, which is essential to maintain physical and mental health. Recent studies revealed that the functions of microglia, the resident immune cells of the brain, differ along the sleep-wake cycle. Inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, mainly produced by microglia in the brain, are also well-known to promote sleep. However, the contributing role of microglia on sleep regulation remains largely elusive, even more so in females. Given the higher prevalence of various sleep disorders in women, we aimed to determine the role of microglia in regulating the sleep-wake cycle specifically in female mice. Microglia were depleted in adult female mice with inhibitors of the colony-stimulating factor 1 receptor (CSF1R) (PLX3397 or PLX5622), which is required for microglial population maintenance. This led to a 65-73% reduction of the microglial population, as confirmed by immunofluorescence staining against IBA1 (marker of microglia/macrophages) and TMEM119 (microglia-specific marker) in the reticular nucleus of the thalamus and primary motor cortex. The spontaneous sleep-wake cycle was evaluated at steady-state, during microglial homeostasis disruption and after complete microglial repopulation, upon cessation of treatment with the inhibitors of CSF1R, using electroencephalography (EEG) and electromyography (EMG). We found that microglia-depleted female mice spent more time in non-rapid eye movement (NREM) sleep and had an increased number of NREM sleep episodes, which was partially restored after microglial total repopulation. To determine whether microglia could regulate sleep locally by modulating synaptic transmission, we used patch clamp to record spontaneous activity of pyramidal neurons in the primary motor cortex, which showed an increase of excitatory synaptic transmission during the dark phase. These changes in neuronal activity were modulated by microglial depletion in a phase-dependent manner. Altogether, our results indicate that microglia are involved in the sleep regulation of female mice, further strengthening their potential implication in the development and/or progression of sleep disorders. Furthermore, our findings indicate that microglial repopulation can contribute to normalizing sleep alterations caused by their partial depletion.

Role of Glia in the Regulation of Sleep in Health and Disease.

Sleep is a naturally occurring physiological state that is required to sustain physical and mental health. Traditionally viewed as strictly regulated by top-down control mechanisms, sleep is now known to also originate locally. Glial cells are emerging as important contributors to the regulation of sleep-wake cycles, locally and among dedicated neural circuits. A few pioneering studies revealed that astrocytes and microglia may influence sleep pressure, duration as well as intensity, but the precise involvement of these two glial cells in the regulation of sleep remains to be fully addressed, across contexts of health and disease. In this overview article, we will first summarize the literature pertaining to the role of astrocytes and microglia in the regulation of sleep under normal physiological conditions. Afterward, we will discuss the beneficial and deleterious consequences of glia-mediated neuroinflammation, whether it is acute, or chronic and associated with brain diseases, on the regulation of sleep. Sleep disturbances are a main comorbidity in neurodegenerative diseases, and in several brain diseases that include pain, epilepsy, and cancer. Identifying the relationships between glia-mediated neuroinflammation, sleep-wake rhythm disruption and brain diseases may have important implications for the treatment of several disorders. © 2020 American Physiological Society. Compr Physiol 10:687-712, 2020.

Inhibiting Microglia Expansion Prevents Diet-Induced Hypothalamic and Peripheral Inflammation.

Cell proliferation and neuroinflammation in the adult hypothalamus may contribute to the pathogenesis of obesity. We tested whether the intertwining of these two processes plays a role in the metabolic changes caused by 3 weeks of a high-saturated fat diet (HFD) consumption. Compared with chow-fed mice, HFD-fed mice had a rapid increase in body weight and fat mass and specifically showed an increased number of microglia in the arcuate nucleus (ARC) of the hypothalamus. Microglia expansion required the adequate presence of fats and carbohydrates in the diet because feeding mice a very high-fat, very low-carbohydrate diet did not affect cell proliferation. Blocking HFD-induced cell proliferation by central delivery of the antimitotic drug arabinofuranosyl cytidine (AraC) blunted food intake, body weight gain, and adiposity. AraC treatment completely prevented the increase in number of activated microglia in the ARC, the expression of the proinflammatory cytokine tumor necrosis factor-α in microglia, and the recruitment of the nuclear factor-κB pathway while restoring hypothalamic leptin sensitivity. Central blockade of cell proliferation also normalized circulating levels of the cytokines leptin and interleukin 1ß and decreased peritoneal proinflammatory CD86 immunoreactive macrophage number. These findings suggest that inhibition of diet-dependent microglia expansion hinders body weight gain while preventing central and peripheral inflammatory responses due to caloric overload.

Neuronal Hyperactivity Disturbs ATP Microgradients, Impairs Microglial Motility, and Reduces Phagocytic Receptor Expression Triggering Apoptosis/Microglial Phagocytosis Uncoupling.

Phagocytosis is essential to maintain tissue homeostasis in a large number of inflammatory and autoimmune diseases, but its role in the diseased brain is poorly explored. Recent findings suggest that in the adult hippocampal neurogenic niche, where the excess of newborn cells undergo apoptosis in physiological conditions, phagocytosis is efficiently executed by surveillant, ramified microglia. To test whether microglia are efficient phagocytes in the diseased brain as well, we confronted them with a series of apoptotic challenges and discovered a generalized response. When challenged with excitotoxicity in vitro (via the glutamate agonist NMDA) or inflammation in vivo (via systemic administration of bacterial lipopolysaccharides or by omega 3 fatty acid deficient diets), microglia resorted to different strategies to boost their phagocytic efficiency and compensate for the increased number of apoptotic cells, thus maintaining phagocytosis and apoptosis tightly coupled. Unexpectedly, this coupling was chronically lost in a mouse model of mesial temporal lobe epilepsy (MTLE) as well as in hippocampal tissue resected from individuals with MTLE, a major neurological disorder characterized by seizures, excitotoxicity, and inflammation. Importantly, the loss of phagocytosis/apoptosis coupling correlated with the expression of microglial proinflammatory, epileptogenic cytokines, suggesting its contribution to the pathophysiology of epilepsy. The phagocytic blockade resulted from reduced microglial surveillance and apoptotic cell recognition receptor expression and was not directly mediated by signaling through microglial glutamate receptors. Instead, it was related to the disruption of local ATP microgradients caused by the hyperactivity of the hippocampal network, at least in the acute phase of epilepsy. Finally, the uncoupling led to an accumulation of apoptotic newborn cells in the neurogenic niche that was due not to decreased survival but to delayed cell clearance after seizures. These results demonstrate that the efficiency of microglial phagocytosis critically affects the dynamics of apoptosis and urge to routinely assess the microglial phagocytic efficiency in neurodegenerative disorders.

Microglial activation enhances associative taste memory through purinergic modulation of glutamatergic neurotransmission.

The cerebral innate immune system is able to modulate brain functioning and cognitive processes. During activation of the cerebral innate immune system, inflammatory factors produced by microglia, such as cytokines and adenosine triphosphate (ATP), have been directly linked to modulation of glutamatergic system on one hand and learning and memory functions on the other hand. However, the cellular mechanisms by which microglial activation modulates cognitive processes are still unclear. Here, we used taste memory tasks, highly dependent on glutamatergic transmission in the insular cortex, to investigate the behavioral and cellular impacts of an inflammation restricted to this cortical area in rats. We first show that intrainsular infusion of the endotoxin lipopolysaccharide induces a local inflammation and increases glutamatergic AMPA, but not NMDA, receptor expression at the synaptic level. This cortical inflammation also enhances associative, but not incidental, taste memory through increase of glutamatergic AMPA receptor trafficking. Moreover, we demonstrate that ATP, but not proinflammatory cytokines, is responsible for inflammation-induced enhancement of both associative taste memory and AMPA receptor expression in insular cortex. In conclusion, we propose that inflammation restricted to the insular cortex enhances associative taste memory through a purinergic-dependent increase of glutamatergic AMPA receptor expression at the synapse.

Mechanisms involved in dual vasopressin/apelin neuron dysfunction during aging.

Normal aging is associated with vasopressin neuron adaptation, but little is known about its effects on the release of apelin, an aquaretic peptide colocalized with vasopressin. We found that plasma vasopressin concentrations were higher and plasma apelin concentrations lower in aged rats than in younger adults. The response of AVP/apelin neurons to osmotic challenge was impaired in aged rats. The overactivity of vasopressin neurons was sustained partly by the increased expression of Transient receptor potential vanilloid2 (Trpv2), because central Trpv blocker injection reversed the age-induced increase in plasma vasopressin concentration without modifying plasma apelin concentration. The morphofunctional plasticity of the supraoptic nucleus neuron-astrocyte network normally observed during chronic dehydration in adults appeared to be impaired in aged rats as well. IL-6 overproduction by astrocytes and low-grade microglial neuroinflammation may contribute to the modification of neuronal functioning during aging. Indeed, central treatment with antibodies against IL-6 decreased plasma vasopressin levels and increased plasma apelin concentration toward the values observed in younger adults. Conversely, minocycline treatment (inhibiting microglial metabolism) did not affect plasma vasopressin concentration, but increased plasma apelin concentration toward control values for younger adults. This study is the first to demonstrate dual vasopressin/apelin adaptation mediated by inflammatory molecules and neuronal Trpv2, during aging.

Astrocyte-derived adenosine modulates increased sleep pressure during inflammatory response.

Activation of the immune system elicits several behavioral changes collectively called sickness. Among the behavioral changes, systemic infections induce an increase in time spent in nonrapid-eye-movement (NREM) sleep and an increase of slow wave activity (or "sleep pressure"). Using an inducible, astrocyte-specific transgenic dominant negative SNARE (dnSNARE) mouse line we recently demonstrated that gliotransmission plays an important role in sleep homeostasis through an adenosine receptor 1 (A1R)-sensitive pathway. It has been hypothesized that systemic infection, mimicked by peripheral administration of lipopolysaccharide (LPS), increases sleeping behavior in part through upregulation of central adenosine levels. Moreover, as a source of immunologically relevant factors, astrocytes play a pivotal role in the central nervous system immune and inflammatory responses. However, little is known about the role of astrocytes in the CNS response to a peripheral immune challenge. We hypothesize that LPS impacts sleep homeostasis through the modulation of astrocyte-derived adenosine accumulation. We therefore used dnSNARE mice to determine whether astrocytes contribute to the increased sleep pressure under immune challenge and whether this is a result of changes in adenosine signaling. We demonstrate that dnSNARE-mediated gliotransmission is required for the ability of LPS to elevate sleep pressure as measured by the power of slow wave activity during NREM sleep. Moreover, in agreement with a role of astrocyte-derived adenosine in modulating sleep homeostasis, we find that intracerebroventricular infusion of the A1R antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT) mimics this dnSNARE phenotype. Taken together, our data demonstrate that astrocytic adenosine acting through A1 receptors contributes to the modulation of sleep pressure by LPS.

Brain cyclooxygenase-2 mediates interleukin-1-induced cellular activation in preoptic and arcuate hypothalamus, but not sickness symptoms.

Interleukin-1beta acts on the CNS to induce fever, neuroendocrine activation, and behavioral changes, but cannot passively cross the blood-brain barrier. According to a widely accepted hypothesis interleukin-1beta induces the synthesis of cyclooxygenase-2 at the blood-brain interface, which produces prostaglandins that diffuse into brain parenchyma to activate neurons. We studied the role of brain cyclooxygenase-2 in interleukin-1beta-induced fever, neuroendocrine and behavioral responses and cellular activation by intracerebroventricular infusion of the cyclooxygenase-2 inhibitor NS-398. Central cyclooxygenase-2 inhibition attenuated extracellular signal-regulated kinase-1/2 phosphorylation and c-Fos induction in the median preoptic area and arcuate hypothalamus, but not in other hypothalamic or brainstem structures, after intraperitoneal interleukin-1beta administration. However, the same treatment did not affect interleukin-1beta-induced fever, rises in corticosterone or anorexia. These findings moderate the prevailing view and indicate that brain cyclooxygenase-2-dependent prostaglandin production is important to activation of the median preoptic and arcuate hypothalamus, but not necessarily involved in fever, rises in plasma corticosterone and anorexia after peripheral interleukin-1beta administration.

High frequency stimulation of the entopeduncular nucleus sets the cortico-basal ganglia network to a new functional state in the dystonic hamster.

High frequency stimulation (HFS) of the internal pallidum is effective for the treatment of dystonia. Only few studies have investigated the effects of stimulation on the activity of the cortex-basal ganglia network. We here assess within this network the effect of entopeduncular nucleus (EP) HFS on the expression of c-Fos and cytochrome oxidase subunit I (COI) in the dt(sz)-hamster, a well-characterized model of paroxysmal dystonia. In dt(sz)-hamsters, we identified abnormal activity in motor cortex, basal ganglia and thalamus. These structures have already been linked to the pathophysiology of human dystonia. EP-HFS (i) increased striatal c-Fos expression in controls and dystonic hamsters and (ii) reduced thalamic c-Fos expression in dt(sz)-hamsters. EP-HFS had no effect on COI expression. The present results suggest that EP-HFS induces a new network activity state which may improve information processing and finally reduces the severity of dystonic attacks in dt(sz)-hamsters.

Interleukin-6 activates arginine vasopressin neurons in the supraoptic nucleus during immune challenge in rats.

The increase of plasma arginin-vasopressin (AVP) release, which translates hypothalamic AVP neuron activation in response to immune challenge, appears to occur independently of plasma osmolality or blood pressure changes. Many studies have shown that major inflammatory mediators produced in response to peripheral inflammation, such as prostaglandin (PG)-E(2) and interleukin (IL)-1beta, excite AVP neurons. However, in vivo electrical activation of AVP neurons was still not assessed in relation to plasma AVP release, osmolality, or blood pressure or to the expression and role of inflammatory molecules like PG-E(2), IL-1beta, IL-6, and tumor necrosis factor-alpha (TNFalpha). This study aims at elucidating those factors that underlie the activation of AVP neurons in response to immune stimulation mimicked by an intraperitoneal injection of lipopolysaccharide (LPS) in male Wistar rats. LPS treatment concomittanlty decreased diuresis and increased plasma AVP as well as AVP neuron activity in vivo, and these effects occurred as early as 30 min. Activation was sustained for more than 6 h. Plasma osmolality did not change, whereas blood pressure only transiently increased during the first hour post-LPS. PG-E(2), IL-1beta, and TNFalpha mRNA expression were raised 3 h after LPS, whereas IL-6 mRNA level increased 30 min post-LPS. In vivo electrophysiological recordings showed that brain IL-6 injection increased AVP neuron activity similarly to peripheral LPS treatment. In contrast, brain injection of anti-IL-6 antibodies prevented the LPS induced-activation of AVP neurons. Taken together, these results suggest that the early activation of AVP neurons in response to LPS injection is induced by brain IL-6.

Inactivation of the cerebral NFkappaB pathway inhibits interleukin-1beta-induced sickness behavior and c-Fos expression in various brain nuclei.

The behavioral effects of peripherally administered interleukin-1beta (IL-1beta) are mediated by the production of cytokines and other proinflammatory mediators at the level of the blood-brain interface and by activation of neural pathway. To assess whether this action is mediated by NFkappaB activation, rats were injected into the lateral ventricle of the brain with a specific inhibitor of NFkappaB activation, the NEMO Binding Domain (NBD) peptide that has been shown previously to abolish completely IL-1beta-induced NFkappaB activation and Cox-2 synthesis in the brain microvasculature. NFkappaB pathway inactivation significantly blocked the behavioral effects of intraperitoneally administered IL-1beta in the form of social withdrawal and decreased food intake, and dramatically reduced IL-1beta-induced c-Fos expression in various brain regions as paraventricular nucleus, supraoptic nucleus, and lateral part of the central amygdala. These findings strongly support the hypothesis that IL-1beta-induced NFkappaB activation at the blood-brain interface is a crucial step in the transmission of immune signals from the periphery to the brain that underlies further events responsible of sickness behavior.

NFkappaB activates in vivo the synthesis of inducible Cox-2 in the brain.

Interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression in many of its cellular targets resulting in production and release of prostaglandins. Although IL-1beta-induced Cox-2 expression most likely requires activation of nuclear transcription factor kappa B (NFkappaB) pathway, this has never been formally demonstrated in vivo. We tested this using a specific inhibitor of NFkappaB activation, the NEMO binding domain (NBD) peptide, that has been shown previously to be effective in various in vivo models of acute inflammation. Incubation of rat glioma cells with the NBD peptide blocked IL-1beta-induced NFkappaB nuclear translocation. Furthermore, after injection of a biotinylated version of the NBD peptide into the lateral ventricle of the brain, we found that it readily diffused to its potential cellular targets in vivo. To test the effects of the peptide on NFkappaB activation and Cox-2 expression in the brain, we injected it intracerebroventricularly (36 microg/rat) into rats before intraperitoneal injection of IL-1beta (60 microg/kg). Treatment with NBD peptide completely abolished IL-1beta-induced NFkappaB activation and Cox-2 synthesis in microvasculature. In contrast, the peptide had no effect on constitutive neuronal Cox-2. These findings strongly support the hypothesis that IL-1beta-induced NFkappaB activation plays a major role in transmission of immune signals from the periphery to the brain.