Circadian modulation of microglial physiological processes and immune responses.

Microglia is considered the central nervous system (CNS) resident macrophages that establish an innate immune response against pathogens and toxins. However, the recent studies have shown that microglial gene and protein expression follows a circadian pattern; several immune activation markers and clock genes are expressed rhythmically without the need for an immune stimulus. Furthermore, microglia responds to an immune challenge with different magnitudes depending on the time of the day. This review examines the circadian control of microglia function and the possible physiological implications. For example, we discuss that synaptic prune is performed in the cortex at a certain moment of the day. We also consider the implications of daily microglial function for maintaining biological rhythms like general activity, body temperature, and food intake. We conclude that the developmental stage, brain region, and pathological state are not the only factors to consider for the evaluation of microglial functions; instead, emerging evidence indicates that circadian time as an essential aspect for a better understanding of the role of microglia in CNS physiology.

The Active Role of Pericytes During Neuroinflammation in the Adult Brain.

Microvessels in the central nervous system (CNS) have one of the highest populations of pericytes, indicating their crucial role in maintaining homeostasis. Pericytes are heterogeneous cells located around brain microvessels; they present three different morphologies along the CNS vascular tree: ensheathing, mesh, and thin-strand pericytes. At the arteriole-capillary transition ensheathing pericytes are found, while mesh and thin-strand pericytes are located at capillary beds. Brain pericytes are essential for the establishment and maintenance of the blood-brain barrier, which restricts the passage of soluble and potentially toxic molecules from the circulatory system to the brain parenchyma. Pericytes play a key role in regulating local inflammation at the CNS. Pericytes can respond differentially, depending on the degree of inflammation, by secreting a set of neurotrophic factors to promote cell survival and regeneration, or by potentiating inflammation through the release of inflammatory mediators (e.g., cytokines and chemokines), and the overexpression of cell adhesion molecules. Under inflammatory conditions, pericytes may regulate immune cell trafficking to the CNS and play a role in perpetuating local inflammation. In this review, we describe pericyte responses during acute and chronic neuroinflammation.

Sleep loss disrupts pericyte-brain endothelial cell interactions impairing blood-brain barrier function.

Sleep loss in the rat increases blood-brain barrier permeability to circulating molecules by disrupting interendothelial tight junctions. Despite the description of the ultrastructure of cerebral microvessels and the evidence of an apparent pericyte detachment from capillary wall in sleep restricted rats the effect of sleep loss on pericytes is unknown. Here we characterized the interactions between pericytes and brain endothelial cells after sleep loss using male Wistar rats. Animals were sleep-restricted 20 h daily with 4 h sleep recovery for 10 days. At the end of the sleep restriction, brain microvessels (MVs) were isolated from cerebral cortex and hippocampus and processed for Western blot and immunocytochemistry to evaluate markers of pericyte-endothelial cell interaction (connexin 43, PDGFR-ß), tight junction proteins, and proinflammatory mediator proteins (MMP9, A2A adenosine receptor, CD73, NFκB). Sleep restriction reduced PDGFR-ß and connexin 43 expression in MVs; in addition, scanning electron microscopy micrographs showed that pericytes were detached from capillary walls, but did not undergo apoptosis (as depicted by a reduced active caspase-3 expression). Sleep restriction also decreased tight junction protein expression in MVs and increased BBB permeability to low- and high-molecular weight tracers in in vivo permeability assays. Those alterations seemed to depend on a low-grade inflammatory status as reflected by the increased expression of phosphorylated NFκB and A2A adenosine receptor in brain endothelial cells from the sleep-restricted rats. Our data show that pericyte-brain endothelial cell interaction is altered by sleep restriction; this evidence is essential to understand the role of sleep in regulating blood-brain barrier function.

Chronic sleep loss disrupts blood-testis and blood-epididymis barriers, and reduces male fertility.

Sleep loss increases blood-brain barrier permeability. As the blood-brain barrier and the blood-tissue barriers in the reproductive tract (blood-testis and blood-epididymis barriers) share common characteristics, we hypothesized that sleep restriction may also modify their barrier function. Previous reports showed that sleep loss decreased sperm viability and progressive fast mobility, which may be a consequence of altered blood-testis and blood-epididymis barrier. Therefore, we quantified changes in blood-testis and blood-epididymis barrier after sleep loss and related them to male fertility. Adult male Wistar rats were sleep restricted using the multiple-platform technique in a protocol of 20 hr daily sleep deprivation plus 4 hr of sleep recovery in the home-cage. At the 10th day, barrier permeability assays were performed with Na-fluorescein, 10 kDa Cascade blue-dextrans and Evans blue, and the expression of tight junction proteins, actin and androgen receptor was quantified. At the 10th day of sleep restriction and after sleep recovery days 1-7, males were placed with sexually receptive females, sexual behaviour was tested, and the percentage of pregnancies was calculated. Sleep restriction increased the barrier permeability to low- and high-molecular-weight tracers, and decreased the expression of tight junction proteins, actin and androgen receptor. Concomitantly, sleep restriction reduced the percentage of ejaculating males and the number of pregnancies. Sleep recovery for 2-3 days progressively re-established fertility, as indicated by a higher percentage of ejaculating males and impregnated females. In conclusion, chronic sleep loss alters fertility concomitantly with the disruption of the blood-tissue barriers at the reproductive tract, the mechanism involves androgen signalling.

Concomitant Treatment with Doxycycline and Rifampicin in Balb/c Mice Infected with Brucella abortus 2308 Fails to Reduce Inflammation and Motor Disability.

Brucellosis is an infection widely distributed around the world, and in some countries it is considered a public health problem. Brucellosis causes insidious symptoms that make it difficult to diagnose. Infection can also trigger chronic pain and neuropsychiatric complications. Antibiotics are not always effective to eradicate infection, contributing to chronicity. We aimed to investigate the effects of antibiotic treatment on proinflammatory cytokines, neurotransmitters, corticosterone, and behavior in a murine model of infecrion of B. abortus strain 2308. Four study groups were created: (a) control; (b) antibiotic control; (c) infected with B. abortus 2308; and (d) infected and treated with rifampicin and doxycycline. We determined B. abortus 2308 colony-forming units (CFUs), the count of dendritic cells, and macrophages in the spleen; serum levels of cytokines and corticosterone; levels of serotonin, dopamine, epinephrine, and norepinephrine in the brain; and equilibrium, physical strength, anxiety, and hopelessness tests. The infected and treated mice group was compared with the control and infected mice to assess whether treatment is sufficient to recover neuroimmunoendocrine parameters. Our results showed that despite the treatment of brucellosis with rifampicin and doxycycline, antibiotic-treated mice showed a persistence of B. abortus 2308 CFUs, an increased count in macrophage number, and higher circulating levels of corticosterone. Furthermore, the levels of IL-12, IL-6, and TNF-α remained higher. We found a decrease in muscular strength and equilibrium concomitant to changes in neurotransmitters in the hippocampus, cerebellum, and frontal cortex. Our data suggest that the remaining bacterial load after antibiotic administration favors inflammatory, neurochemical, and behavioral alterations, partly explaining the widespread and paradoxical symptomatology experienced by patients with chronic brucellosis.

Sleep loss as a factor to induce cellular and molecular inflammatory variations.

A reduction in the amount of time spent sleeping occurs chronically in modern society. Clinical and experimental studies in humans and animal models have shown that immune function is impaired when sleep loss is experienced. Sleep loss exerts a strong regulatory influence on peripheral levels of inflammatory mediators of the immune response. An increasing number of research projects support the existence of reciprocal regulation between sleep and low-intensity inflammatory response. Recent studies show that sleep deficient humans and rodents exhibit a proinflammatory component; therefore, sleep loss is considered as a risk factor for developing cardiovascular, metabolic, and neurodegenerative diseases (e.g., diabetes, Alzheimer's disease, and multiple sclerosis). Circulating levels of proinflammatory mediators depend on the intensity and duration of the method employed to induce sleep loss. Recognizing the fact that the concentration of proinflammatory mediators is different between acute and chronic sleep-loss may expand the understanding of the relationship between sleep and the immune response. The aim of this review is to integrate data from recent published reports (2002-2013) on the effects of sleep loss on the immune response. This review may allow readers to have an integrated view of the mechanisms involved in central and peripheral deficits induced by sleep loss.

Suprachiasmatic nucleus promotes hyperglycemia induced by sleep delay.

Short sleep is linked to disturbances in glucose metabolism and may induce a prediabetic condition. The biological clock in the suprachiasmatic nucleus (SCN) regulates the glucose rhythm in the circulation and the sleep-wake cycle. SCN vasopressin neurons (SCNVP) control daily glycemia by regulating the entrance of glucose into the arcuate nucleus (ARC). Thus, we hypothesized that sleep delay may influence SCN neuronal activity. We, therefore, investigated the role of SCNVP when sleep is disrupted by forced locomotor activity. After 2 h of sleep delay, rats exhibited decreased SCNVP neuronal activity, a decrease in the glucose transporter GLUT1 expression in tanycytes lining the third ventricle, lowered glucose entrance into the ARC, and developed hyperglycemia. The association between reduced SCNVP neuronal activity and hyperglycemia in sleep-delayed rats was evidenced by injecting intracerebroventricular vasopressin; this increased GLUT1 immunoreactivity in tanycytes, thus promoting normoglycemia. Following sleep recovery, glucose levels decreased, whereas SCNVP neuronal activity increased. These results imply that sleep-delay-induced changes in SCNVP activity lead to glycemic impairment, inferring that disruption of biological clock function might represent a critical step in developing type 2 diabetes.

Minor Changes in Daily Rhythms Induced by a Skeleton Photoperiod Are Associated with Increased Adiposity and Glucose Intolerance.

Eating during the rest phase is associated with metabolic syndrome, proposed to result from a conflict between food consumption and the energy-saving state imposed by the circadian system. However, in nocturnal rodents, eating during the rest phase (day-feeding, DF) also implies food intake during light exposure. To investigate whether light exposure contributes to DF-induced metabolic impairments, animals receive food during the subjective day without light. A skeleton photoperiod (SP) is used to entrain rats to a 12:12 cycle with two short light pulses framing the subjective day. DF-induced adiposity is prevented by SP, suggesting that the conflict between light and feeding stimulates fat accumulation. However, all animals under SP conditions develop glucose intolerance regardless of their feeding schedule. Moreover, animals under SP with ad libitum or night-feeding have increased adiposity. SP animals show a delayed onset of the daily rise in body temperature and energy expenditure and shorter duration of nighttime activity, which may contribute to the metabolic disturbances. These data emphasize that metabolic homeostasis can only be achieved when all daily cycling variables are synchronized. Even small shifts in the alignment of different metabolic rhythms, such as those induced by SP, may predispose individuals to metabolic disease.

Suprachiasmatic nucleus-mediated glucose entry into the arcuate nucleus determines the daily rhythm in blood glycemia.

Glycemia is maintained within very narrow boundaries with less than 5% variation at a given time of the day. However, over the circadian cycle, glycemia changes with almost 50% difference. How the suprachiasmatic nucleus, the biological clock, maintains these day-night variations with such tiny disparities remains obscure. We show that via vasopressin release at the beginning of the sleep phase, the suprachiasmatic nucleus increases the glucose transporter GLUT1 in tanycytes. Hereby GLUT1 promotes glucose entrance into the arcuate nucleus, thereby lowering peripheral glycemia. Conversely, blocking vasopressin activity or the GLUT1 transporter at the daily trough of glycemia increases circulating glucose levels usually seen at the peak of the rhythm. Thus, biological clock-controlled mechanisms promoting glucose entry into the arcuate nucleus explain why peripheral blood glucose is low before sleep onset.

Vasopressin: An output signal from the suprachiasmatic nucleus to prepare physiology and behaviour for the resting phase.

Vasopressin (VP) is an important hormone produced in the supraoptic (SON) and paraventricular nucleus (PVN) with antidiuretic and vasoconstrictor functions in the periphery. As one of the first discovered peptide hormones, VP was also shown to act as a neurotransmitter, where VP is produced and released under the influence of various stimuli. VP is one of the core signals via which the biological clock, the suprachiasmatic nucleus (SCN), imposes its rhythm on its target structures and its production and release is influenced by the rhythm of clock genes and the light/dark cycle. This is contrasted with VP production and release from the bed nucleus of the stria terminalis and the medial amygdala, which is influenced by gonadal hormones, as well as with VP originating from the PVN and SON, which is released in the neural lobe and central targets. The release of VP from the SCN signals the near arrival of the resting phase in rodents and prepares their physiology accordingly by down-modulating corticosterone secretion, the reproductive cycle and locomotor activity. All these circadian variables are regulated within very narrow boundaries at a specific time of the day, where day-to-day variation is less than 5% at any particular hour. However, the circadian peak values can be at least ten times higher than the circadian trough values, indicating the need for an elaborate feedback system to inform the SCN and other participating nuclei about the actual levels reached during the circadian cycle. In short, the interplay between SCN circadian output and peripheral feedback to the SCN is essential for the adequate organisation of all circadian rhythms in physiology and behaviour.

Selective Regional Isolation of Brain Microvessels.

The study of the regionalized function of the blood-brain barrier at the level of brain endothelial cells and pericytes is essential to understand the biological properties and molecular mechanisms regulating this biological barrier. The isolation of blood vessels from specific brain regions will allow to understand regional differences in susceptibility to pathological phenomena such as ischemia, traumatic brain injury, and neurodegenerative diseases, such as Alzheimer disease. Here, we propose an efficient and fast method to isolate brain endothelial cells and pericytes from a specific cerebral region. The isolated brain endothelial cells and pericytes are viable to perform conventional molecular and histological techniques such as Western blots, immunocytofluorescence, and scanning electron microscopy.

Behavioral consequences of postnatal undernutrition and enriched environment during later life.

In rat models, large litter groups during suckling are used in the study of undernutrition. Large litter sizes are known to promote alterations in memory processes and anxiety-like behavior. Nevertheless, the effect of large litter size on sexual behavior and the reproductive system is still unknown. Environmental enrichment has been reported to (EE) enhance behavior and to correct some of the alterations produced by postnatal undernutrition. We used the Elevated Plus Maze (EPN), Morris Water Maze (MWM), Object Recognition test (OR) and several parameters of sexual behavior to determine the effect of large litter size on rats exposed to enriched and non-enriched environments. Newborn Wistar rats of both sexes were assigned to be suckled under lactation conditions, in litters of 8 pups or 16 pups. The large litter size (16 pups) caused a reduction in weight gain during the lactation period. On PND 45, four experimental groups were established for both sexes: Well-nourished Non-enriched (WN); Well-nourished Enriched (WE); undernourished Non-enriched (UN); Undernourished Enriched (UE). On PND 90, the UN males spent more time in the open arms on EPM. On PND 100, the UE females increased the latency to find the platform in training days (D1-4) in MWM. On probe day (D5) the UE males spent more time in the target quadrants in MWM. On PND 110, irrespective of EE the large litter size had increased the exploration time in both groups (UN) and (UE) in OR test. On PND 120, the performance of sexual behavior was more evident by effect of EE irrespective of the litter size. In conclusion, the large litter size showed no effects on sexual behavior, in contrast, EE has a sharp influence on sexual behavior. Conversely, memory processes and anxiety-like behavior are altered by large litter size.

Metabolic Disturbances Induced by Sleep Restriction as Potential Triggers for Alzheimer's Disease.

Sleep has a major role in learning, memory consolidation, and metabolic function. Although it is known that sleep restriction increases the accumulation of amyloid ß peptide (Aß) and the risk to develop Alzheimer's disease (AD), the mechanism behind these effects remains unknown. In this review, we discuss how chronic sleep restriction induces metabolic and cognitive impairments that could result in the development of AD in late life. Here, we integrate evidence regarding mechanisms whereby metabolic signaling becomes disturbed after short or chronic sleep restriction in the context of cognitive impairment, particularly in the accumulation of Aß in the brain. We also discuss the role of the blood-brain barrier in sleep restriction with an emphasis on the transport of metabolic signals into the brain and Aß clearance. This review presents the unexplored possibility that the alteration of peripheral metabolic signals induced by sleep restriction, especially insulin resistance, is responsible for cognitive deficit and, subsequently, implicated in AD development.

The circadian system: From clocks to physiology.

The circadian system, composed of the central autonomous clock, the suprachiasmatic nucleus (SCN), and systems of the body that follow the signals of the SCN, continuously change the homeostatic set points of the body over the day-night cycle. Changes in the body's physiological state that do not agree with the time of the day feedback to the hypothalamus, and provide input to the SCN to adjust the condition, thus reaching another set point required by the changed conditions. This allows the adjustment of the set points to another level when environmental conditions change, which is thought to promote adaptation and survival. In fasting, the body temperature drops to a lower level only at the beginning of the sleep phase. Stressful conditions raise blood pressure relatively more during the active period than during the rest phase. Extensive, mostly reciprocal SCN interactions, with hypothalamic networks, induce these physiological adjustments by hormonal and autonomic control of the body's organs. More importantly, in addition to SCN's hormonal and autonomic influences, SCN induced behavior, such as rhythmic food intake, induces the oscillation of many genes in all tissues, including the so-called clock genes, which have an essential role as a transcriptional driving force for numerous cellular processes. Consequently, the light-dark cycle, the rhythm of the SCN, and the resulting rhythm in behavior need to be perfectly synchronized, especially where it involves synchronizing food intake with the activity phase. If these rhythms are not synchronous for extended periods of times, such as during shift work, light exposure at night, or frequent night eating, disease may develop. As such, our circadian system is a perfect illustration of how hypothalamic-driven processes depend on and interact with each other and need to be in seamless synchrony with the body's physiology.

Immunoendocrine Peripheral Effects Induced by Atypical Antipsychotics.

Atypical antipsychotics (AAP) or second-generation antipsychotics are the clinical option for schizophrenia treatment during acute psychoses, but they are also indicated for maintenance during lifetime, even though they are being used for other psychiatric conditions in clinical practice such as affective disorders and autism spectrum disorder, among others. These drugs are differentiated from typical antipsychotics based on their clinical profile and are a better choice because they cause fewer side effects regarding extrapyramidal symptoms (EPS). Even though they provide clear therapeutic benefits, AAP induce peripheral effects that trigger phenotypic, functional, and systemic changes outside the Central Nervous System (CNS). Metabolic disease is frequently associated with AAP and significantly impacts the patient's quality of life. However, other peripheral changes of clinical relevance are present during AAP treatment, such as alterations in the immune and endocrine systems as well as the intestinal microbiome. These less studied alterations also have a significant impact in the patient's health status. This manuscript aims to revise the peripheral immunological, endocrine, and intestinal microbiome changes induced by AAP consumption recommended in the clinical guidelines for schizophrenia and other psychiatric disorders.

Postnatal overnutrition alters the orexigenic effects of melanin-concentrating hormone (MCH) and reduces MCHR1 hypothalamic expression on spontaneous feeding and fasting.

One of the approaches to induce obesity in rodents consists in reducing litter size to 3 pups during the lactation period. Animals submitted to this manipulation are heavier, hyperphagic and develop several metabolic diseases for the rest of their lives. In the present study, under the premise that melanin-concentrating hormone (MCH), an orexigenic peptide synthesized by neurons of the lateral hypothalamus, is involved in food intake regulation, we aimed to measure the hypothalamic expression of its receptor, MCHR1, in adult early overfed obese animals and normoweight controls at both ad libitum and food deprived conditions. Additionally, we administered MCH, or an antiMCH antibody, into the third ventricle of ad libitum-fed rats, or fasted rats, respectively, and evaluated chow consumption. Typical nocturnal hyperphagia in rodents was elevated in obese animals compared to normoweight controls, accompanied by a lower expression of MCHR1 and leptin receptor (Ob-R). Following a 24 h fasting, MCHR1 remained lower in SL rats. After 4 h of re-feeding, obese animals ate more than normoweight controls. MCH failed to enhance appetite in early overfed obese animals and immunoneutralization of the peptide only reduced fasted induced-hyperphagia in normoweight controls. These results support the notion that both peptide and brain endogenous MCH exert a physiological relevant action in food intake regulation in normoweight rats, but that postnatal overnutrition disturbs this system, as reflected by MCHR1 downregulation at both ad libitum and fasted conditions and in the lack of response to MCH in both positive- and negative-energetic states in early overfed obese animals.

Characterization of sleep-pattern and neuro-immune-endocrine markers at 24 hour post-injection of a single low dose of lipopolysaccharide in male Wistar rats.

The long-term effect of immune system activation by a low dose of lipopolysaccharide on neuro-immune-endocrine regulation is unclear. Sleep, neurotransmitter concentrations, TNF-α, and corticosterone levels were evaluated in male Wistar rats implanted with conventional sleep recordings. In this work, we found that REM sleep was reduced in the first 4 h post-injection, without affecting the total sleep time, while adrenaline concentration was reduced in the hippocampus at 24 h post-injection of LPS. Our results demonstrated that, although the acute immune response was not evident 24 h after the injection of LPS, it was able to promote the reduction of AD in the hippocampus, which may explain in part the depressive behavior reported in rodents following LPS administration.

A2A Adenosine Receptor Antagonism Reverts the Blood-Brain Barrier Dysfunction Induced by Sleep Restriction.

Chronic sleep restriction induces blood-brain barrier disruption and increases pro-inflammatory mediators in rodents. Those inflammatory mediators may modulate the blood-brain barrier and constitute a link between sleep loss and blood-brain barrier physiology. We propose that adenosine action on its A2A receptor may be modulating the blood-brain barrier dynamics in sleep-restricted rats. We administrated a selective A2A adenosine receptor antagonist (SCH58261) in sleep-restricted rats at the 10th day of sleep restriction and evaluated the blood-brain barrier permeability to dextrans coupled to fluorescein (FITC-dextrans) and Evans blue. In addition, we evaluated by western blot the expression of tight junction proteins (claudin-5, occludin, ZO-1), adherens junction protein (E-cadherin), A2A adenosine receptor, adenosine-synthesizing enzyme (CD73), and neuroinflammatory markers (Iba-1 and GFAP) in the cerebral cortex, hippocampus, basal nuclei and cerebellar vermis. Sleep restriction increased blood-brain barrier permeability to FITC-dextrans and Evans blue, and the effect was reverted by the administration of SCH58261 in almost all brain regions, excluding the cerebellum. Sleep restriction increased the expression of A2A adenosine receptor only in the hippocampus and basal nuclei without changing the expression of CD73 in all brain regions. Sleep restriction reduced the expression of tight junction proteins in all brain regions, except in the cerebellum; and SCH58261 restored the levels of tight junction proteins in the cortex, hippocampus and basal nuclei. Finally, sleep restriction induced GFAP and Iba-1 overexpression that was attenuated with the administration of SCH58261. These data suggest that the action of adenosine on its A2A receptor may have a crucial role in blood-brain barrier dysfunction during sleep loss probably by direct modulation of brain endothelial cell permeability or through a mechanism that involves gliosis with subsequent inflammation and increased blood-brain barrier permeability.

Immunomodulatory Effects Mediated by Dopamine.

Dopamine (DA), a neurotransmitter in the central nervous system (CNS), has modulatory functions at the systemic level. The peripheral and central nervous systems have independent dopaminergic system (DAS) that share mechanisms and molecular machinery. In the past century, experimental evidence has accumulated on the proteins knowledge that is involved in the synthesis, reuptake, and transportation of DA in leukocytes and the differential expression of the D1-like (D1R and D5R) and D2-like receptors (D2R, D3R, and D4R). The expression of these components depends on the state of cellular activation and the concentration and time of exposure to DA. Receptors that are expressed in leukocytes are linked to signaling pathways that are mediated by changes in cAMP concentration, which in turn triggers changes in phenotype and cellular function. According to the leukocyte lineage, the effects of DA are associated with such processes as respiratory burst, cytokine and antibody secretion, chemotaxis, apoptosis, and cytotoxicity. In clinical conditions such as schizophrenia, Parkinson disease, Tourette syndrome, and multiple sclerosis (MS), there are evident alterations during immune responses in leukocytes, in which changes in DA receptor density have been observed. Several groups have proposed that these findings are useful in establishing clinical status and clinical markers.