Luciana Pinato

Glia-Pinealocyte Network: The Paracrine Modulation of Melatonin Synthesis by Tumor Necrosis Factor (TNF)

The pineal gland, a circumventricular organ, plays an integrative role in defense responses. The injury-induced suppression of the pineal gland hormone, melatonin, which is triggered by darkness, allows the mounting of innate immune responses. We have previously shown that cultured pineal glands, which express toll-like receptor 4 (TLR4) and tumor necrosis factor receptor 1 (TNFR1), produce TNF when challenged with lipopolysaccharide (LPS). Here our aim was to evaluate which cells present in the pineal gland, astrocytes, microglia or pinealocytes produced TNF, in order to understand the interaction between pineal activity, melatonin production and immune function. Cultured pineal glands or pinealocytes were stimulated with LPS. TNF content was measured using an enzyme-linked immunosorbent assay. TLR4 and TNFR1 expression were analyzed by confocal microscopy. Microglial morphology was analyzed by immunohistochemistry. In the present study, we show that although the main cell types of the pineal gland (pinealocytes, astrocytes and microglia) express TLR4, the production of TNF induced by LPS is mediated by microglia. This effect is due to activation of the nuclear factor kappa B (NF-kB) pathway. In addition, we observed that LPS activates microglia and modulates the expression of TNFR1 in pinealocytes. As TNF has been shown to amplify and prolong inflammatory responses, its production by pineal microglia suggests a glia-pinealocyte network that regulates melatonin output. The current study demonstrates the molecular and cellular basis for understanding how melatonin synthesis is regulated during an innate immune response, thus our results reinforce the role of the pineal gland as sensor of immune status.

Molecular Basis for Defining the Pineal Gland and Pinealocytes as Targets for Tumor Necrosis Factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target of TNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response.

Influence of sleep disorders on the behavior of individuals with autism spectrum disorder

The aim of this study was to investigate the correlation between sleep disorders and the behavior of subjects with autism spectrum disorder (ASD) and control subjects using specific questionnaires. A small percentage (1.8%) of the control subjects had symptoms indicative of sleep-breathing disorders (SBD) and nocturnal sweating. Fifty-nine percent of the subjects with ASD had symptoms indicative of at least one sleep disorder, with SBD the most commonly reported (38%). In the control group, the symptoms of SBD were correlated with social, thought, attentional, aggression, externalizing and behavioral problems. In the ASD group, disorders of arousal (DA) were correlated with thinking problems, and disorders of excessive somnolence were correlated with thinking and behavioral problems. These results suggest that children and adolescents with ASD have a high frequency of sleep disorders, which in turn correlate with some of the behavioral traits that they already exhibit. Furthermore, sleep disturbances, when present in the typically developing children, also correlated with behavioral problems.

Intrinsic organization of the suprachiasmatic nucleus in the capuchin monkey

The suprachiasmatic nucleus (SCN), which is the main circadian biological clock in mammals, is composed of multiple cells that function individually as independent oscillators to express the self-sustained mRNA and protein rhythms of the so-called clock genes. Knowledge regarding the presence and localization of the proteins and neuroactive substances of the SCN are essential for understanding this nucleus and for its successful manipulation. Although there have been advances in the investigation of the intrinsic organization of the SCN in rodents, little information is available in diurnal species, especially in primates. This study, which explores the pattern of expression and localization of PER2 protein in the SCN of capuchin monkey, evaluates aspects of the circadian system that are common to both primates and rodents. Here, we showed that PER2 protein immunoreactivity is higher during the light phase. Additionally, the complex organization of cells that express vasopressin, vasoactive intestinal polypeptide, neuron-specific nuclear protein, calbindin and calretinin in the SCN, as demonstrated by their immunoreactivity, reveals an intricate network that may be related to the similarities and differences reported between rodents and primates in the literature.

Efferent projections of the suprachiasmatic nucleus based on the distribution of vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) immunoreactive fibers in the hypothalamus of Sapajus apella

The suprachiasmatic nucleus (SCN), which is considered to be the master circadian clock in mammals, establishes biological rhythms of approximately 24 h that several organs exhibit. One aspect relevant to the study of the neurofunctional features of biological rhythmicity is the identification of communication pathways between the SCN and other brain areas. As a result, SCN efferent projections have been investigated in several species, including rodents and a few primates. The fibers originating from the two main intrinsic fiber subpopulations, one producing vasoactive intestinal peptide (VIP) and the other producing arginine vasopressin (AVP), exhibit morphological traits that distinguish them from fibers that originate from other brain areas. This distinction provides a parameter to study SCN efferent projections. In this study, we mapped VIP (VIP-ir) and AVP (AVP-ir) immunoreactive (ir) fibers and endings in the hypothalamus of the primate Sapajus apella via immunohistochemical and morphologic study. Regarding the fiber distribution pattern, AVP-ir and VIP-ir fibers were identified in regions of the tuberal hypothalamic area, retrochiasmatic area, lateral hypothalamic area, and anterior hypothalamic area. VIP-ir and AVP-ir fibers coexisted in several hypothalamic areas; however, AVP-ir fibers were predominant over VIP-ir fibers in the posterior hypothalamus and medial periventricular area. This distribution pattern and the receiving hypothalamic areas of the VIP-ir and AVP-ir fibers, which shared similar morphological features with those found in SCN, were similar to the patterns observed in diurnal and nocturnal animals. This finding supports the conservative nature of this feature among different species. Morphometric analysis of SCN intrinsic neurons indicated homogeneity in the size of VIP-ir neurons in the SCN ventral portion and heterogeneity in the size of two subpopulations of AVP-ir neurons in the SCN dorsal portion. The distribution of fibers and morphometric features of these neuronal populations are described and compared with those of other species in the present study.

Correlation between sleep and quality of life in cerebral palsy

PURPOSE To investigate and correlate the sleep pattern and quality of life in individuals with cerebral palsy (CP) compared to healthy controls. METHODS Seventy-eight subjects (aged 4-18 years, both genders) comprised two groups: CP, composed of 43 individuals with CP, and control, composed of 35 individuals without neurodevelopmental disorders. General Sleep Habits Questionnaire, Sleep Disturbance Scale for Children, sleep diary, Child Health Questionnaire, and Childrens Quality of Life Scale were used. RESULTS This study identified that 60.5% individuals with CP had sleep disorders. The respiratory disorders (25.6%) and the sleep hyperhidrosis (34.9%) were the most common disturbances in the group with CP. Moreover, 23.2% individuals of the group with CP reported awaking in the middle of the night and 37.2% of them snore, both percentages were higher than those in the control group. The sleep diary showed that individuals in the group with CP spend more time to initiate sleep (around 21 minutes). The group with CP also showed deficits in all parameters analyzed by the Child Health Questionnaire, except in family activity and the sleep-wake pattern, and quality of life showed negative correlation in several respects. CONCLUSION The altered pattern of sleep in individuals with CP directly affects their physical and emotional well-being.

Retinal projections and neurochemical characterization of the pregeniculate nucleus of the common marmoset (Callithrix jacchus)

In mammals, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) are the main components of the circadian timing system. The SCN is the site of the endogenous biological clock that generates rhythms and synchronizes them to environmental cues. The IGL is a key structure that modulates SCN activity and is responsible for the transmission of non-photic information to the SCN, thus participating in the integration between photic and non-photic stimuli. Both the SCN and IGL receive projections of retinal ganglion cells and the IGL is connected to the SCN through the geniculohypothalamic tract. Little is known about these structures in the primate brain and the pregeniculate nucleus (PGN) has been suggested to be the primate equivalent of the rodent IGL. The aim of this study was to characterize the PGN of a primate, the common marmoset (Callithrix jacchus), and to analyze its retinal afferents. Here, the marmoset PGN was found to be organized into three subsectors based on neuronal size, pattern of retinal projections, and the distribution of neuropeptide Y-, GAD-, serotonin-, enkephalin- and substance P-labeled terminals. This pattern indicates that the marmoset PGN is equivalent to the IGL. This detailed description contributes to the understanding of the circadian timing system in this primate species considering the importance of the IGL within the context of circadian regulation.

Analyses of melatonin, cytokines, and sleep in chronic renal failure

PurposeInflammation and oxidative stress are involved in the process of chronic renal failure (CRF). CRF patients show indication of sleep disturbances, and the melatonin rhythm, which modulates sleep, is abnormal in these patients; however, it is still unclear whether inflammation could be related to the blockage of melatonin production and sleep disturbances in this population. The aim of this study was to characterize and correlate sleep, the melatonin rhythm, and the levels of the inflammatory cytokines tumor necrosis factor (TNF) and interleukin (IL)-6 in patients with CRF and controls.MethodsSleep was evaluated by the “Sleep Quality Index Pittsburgh” (PSQI) questionnaire, and melatonin and cytokine contents in saliva and blood samples, respectively, were analyzed by ELISA.ResultsThe CRF group scored higher on the global PSQI, which indicates a lower sleep quality and a higher prevalence of sleep disorders, than the control group. The CRF individuals also showed lower melatonin content than the control groups, both during the day and at night, and lacked rhythmicity in melatonin production. The CRF group also showed higher contents of TNF and IL-6 than the control group and a negative correlation between TNF and melatonin content.ConclusionThese results suggest that the sleep disorders observed in the CRF group were probably related to the low production of melatonin observed in this population. The high level of TNF, as previously demonstrated in other pathologies, is probably involved in this blockage of melatonin production in CRF.

Correlations between behavior, memory, sleep-wake and melatonin in Williams-Beuren syndrome.

Williams-Beuren syndrome (WBS), a neurodevelopmental disorder caused by a microdeletion on chromosomic region 7q11.23, presents with peculiar behavioral and neurocognitive phenotypes that are marked by apparently preserved social and communicative abilities, which contrasts with low overall cognitive and particularly visuospatial performance. In addition, parents often report complaints of sleep disorders and behavioral problems of unknown cause. Sleep is a biological phenomenon that is modulated by the plasma concentration of melatonin and with influence on behavioral aspects and memory. Thus, this study sought to investigate the behavior, memory and the presence of sleep disorders in WBS and to correlate these factors with each other and with the plasma melatonin content. We used the Child Behavior Checklist for ages 6-18 (CBCL), the digit subtest of the Wechsler scale for auditory memory, the visual sequential memory subtest of the Illinois Test of Psycholinguistic Abilities (ITPA) and the Sleep Disturbance Scale for Children (SDSC). Determination of urinary aMT6s, an indirect measure of plasma melatonin content, was held for 72h by ELISA, and the analysis of the circadian rhythm of this content was performed by the Cosinor method. The results of the CBCL showed that 87% of the WBS group presented with a clinical score on the overall competence and total behavioral problems. Furthermore, the behavioral problems that were most frequently reported by parents were anxiety and problems of thought. All individuals with WBS presented with impairments in auditory memory and 47% with impairments in visual sequential memory; 65% of the WBS group presented with an indicative of at least one sleep disorder, where respiratory, initiation and maintenance of sleep (DIMS) and hyperhidrosis were the most frequent disorders. The night time aMT6s levels were lower in individuals with WBS when compared with controls; 53% of the WBS group did not present with circadian rhythm variations in aMT6s levels. In addition, there was a negative correlation between the scores of auditory memory and the total score of sleep disorders and between the DIMS and nocturnal aMT6s content. In conclusion, in the present study, individuals with WBS showed a high frequency of behavioral and memory problems, sleep disturbances and no rhythm variation in aMT6s levels. The low melatonin content may be related with sleep disorders in this population, which, in turn, can have an adverse effect on specific cognitive skills such as memory.

5-HT1B receptor in the suprachiasmatic nucleus of the common marmoset (Callithrix jacchus)

Serotonin (5-HT) is involved in the fine adjustments at several brain centers including the core of the mammal circadian timing system (CTS) and the hypothalamic suprachiasmatic nucleus (SCN). The SCN receives massive serotonergic projections from the midbrain raphe nuclei, whose inputs are described in rats as ramifying at its ventral portion overlapping the retinohypothalamic and geniculohypothalamic fibers. In the SCN, the 5-HT actions are reported as being primarily mediated by the 5-HT1 type receptor with noted emphasis for 5-HT(1B) subtype, supposedly modulating the retinal input in a presynaptic way. In this study in a New World primate species, the common marmoset (Callithrix jacchus), we showed the 5-HT(1B) receptor distribution at the dorsal SCN concurrent with a distinctive location of 5-HT-immunoreactive fibers. This finding addresses to a new discussion on the regulation and synchronization of the circadian rhythms in recent primates.