Microglia in Circumventricular Organs: The Pineal Gland Example.

The circumventricular organs (CVOs) are unique areas within the central nervous system. They serve as a portal for the rest of the body and, as such, lack a blood-brain barrier. Microglia are the primary resident immune cells of the brain parenchyma. Within the CVOs, microglial cells find themselves continuously challenged and stimulated by local and systemic stimuli, even under steady-state conditions. Therefore, CVO microglia in their typical state often resemble the activated microglial forms found elsewhere in the brain as they are responding to pathological conditions or other stressors. In this review, I focus on the dynamics of CVO microglia, using the pineal gland as a specific CVO example. Data related to microglia heterogeneity in both homeostatic and unhealthy environments are presented and discussed, including those recently generated by using advanced single-cell and single-nucleus technology. Finally, perspectives in the CVO microglia field are also included.Summary StatementMicroglia in circumventricular organs (CVOs) continuously adapt to react differentially to the diverse challenges they face. Herein, I discuss microglia heterogeneity in CVOs, including pineal gland. Further studies are needed to better understand microglia dynamics in these unique brain areas. .

Surgical Techniques and Nuances for Superior Cervical Ganglionectomy and Decentralization in Rats.

The sympathetic nervous system has been implicated in various physiological and pathological processes, including regulation of homeostatic functions, maintenance of the circadian rhythms, and neuronal disruption and recovery after injury. Of special interest is focus on the role of the superior cervical ganglion (SCG) in regulating the daily changes in pineal function. Removal of the superior cervical ganglion (SCGx) and decentralization have served as valuable microsurgical models to investigate the effects of surgical denervation on this gland or organ. In this chapter, we offer information about methodologies for performing SCGx along with decentralization and denervation procedures, including details about recommended equipment as well as tips that can improve these techniques.

Signaling within the pineal gland: A parallelism with the central nervous system.

The pineal gland (PG) derives from the neural tube, like the rest of the central nervous system (CNS). The PG is specialized in synthesizing and secreting melatonin in a circadian fashion. The nocturnal elevation of melatonin is a highly conserved feature among species which proves its importance in nature. Here, we review a limited set of intrinsic and extrinsic regulatory elements that have been shown or proposed to influence the PG's melatonin production, as well as pineal ontogeny and homeostasis. Intrinsic regulators include the transcription factors CREB, Pax6 and NeuroD1. In addition, microglia within the PG participate as extrinsic regulators of these functions. We further discuss how these same elements work in other parts of the CNS, and note similarities and differences to their roles in the PG. Since the PG is a relatively well-defined and highly specialized organ within the CNS, we suggest that applying this comparative approach to additional PG regulators may be a useful tool for understanding complex areas of the brain, as well as the influence of the PG in both health and disease, including circadian functions and disorders.

Differential response of pineal microglia to surgical versus pharmacological stimuli.

Microglial cells are one of the interstitial elements of the pineal gland (PG). We recently reported the pattern of microglia colonization and activation, and microglia-Pax6+ cell interactions during normal pineal ontogeny. Here, we describe the dynamics of microglia-Pax6+ cell associations and interactions after surgical or pharmacological manipulation. In adult rats, the superior cervical ganglia (SCG) were exposed, and either bilaterally excised (SCGx) or decentralized (SCGd). In the SCGx PGs, the density of Iba1+ microglia increased after surgery and returned to sham baseline levels 13 days later. Pineal microglia also responded to SCGd, a more subtle denervation. The number of clustered Iba1+ /PCNA+ /ED1+ microglia was higher 4 days after both surgeries compared to the sham-operated group. However, the number of Pax6+ /PCNA- cells and the percentage of Pax6+ cells contacted by and/or phagocytosed by microglia increased significantly only after SCGx. Separate groups of rats were treated with either bacterial lipopolysaccharides (LPS) or doxycycline (DOX) to activate or inhibit pineal microglia, respectively. Peripheral LPS administration caused an increase in the number of clustered Iba1+ /PCNA+ /ED1+ microglial cells, and in the percentage of Pax6+ cells associated with and/or engulfed by microglia. In the LPS-treated PGs, we also noted an increase in the number of PCNA+ cells that were Iba1- within the microglial cell clusters. The density of Pax6+ cells did not change after LPS treatment. DOX administration did not influence the parameters analyzed. These data suggest that pineal microglia are highly receptive cells capable of rapidly responding in a differential manner to surgical and pharmacological stimuli.

Alterations in Metabolism and Diurnal Rhythms following Bilateral Surgical Removal of the Superior Cervical Ganglia in Rats.

Mammalian circadian rhythms are controlled by a master pacemaker located in the suprachiasmatic nuclei (SCN), which is synchronized to the environment by photic and nonphotic stimuli. One of the main functions of the SCN is to regulate peripheral oscillators to set temporal variations in the homeostatic control of physiology and metabolism. In this sense, the SCN coordinate the activity/rest and feeding/fasting rhythms setting the timing of food intake, energy expenditure, thermogenesis, and active and basal metabolism. One of the major time cues to the periphery is the nocturnal melatonin, which is synthesized and secreted by the pineal gland. Under SCN control, arylalkylamine N-acetyltransferase (AA-NAT)-the main enzyme regulating melatonin synthesis in vertebrates-is activated at night by sympathetic innervation that includes the superior cervical ganglia (SCG). Bilateral surgical removal of the superior cervical ganglia (SCGx) is considered a reliable procedure to completely prevent the nocturnal AA-NAT activation, irreversibly suppressing melatonin rhythmicity. In the present work, we studied the effects of SCGx on rat metabolic parameters and diurnal rhythms of feeding and locomotor activity. We found a significant difference between SCGx and sham-operated rats in metabolic variables such as an increased body weight/food intake ratio, increased adipose tissue, and decreased glycemia with a normal glucose tolerance. An analysis of locomotor activity and feeding rhythms showed an increased daytime (lights on) activity (including food consumption) in the SCGx group. These alterations suggest that superior cervical ganglia-related feedback mechanisms play a role in SCN-periphery phase coordination and that SCGx is a valid model without brain-invasive surgery to explore how sympathetic innervation affects daily (24 h) patterns of activity, food consumption and, ultimately, its role in metabolism homeostasis.

Cellular Basis of Pineal Gland Development: Emerging Role of Microglia as Phenotype Regulator.

The adult pineal gland is composed of pinealocytes, astrocytes, microglia, and other interstitial cells that have been described in detail. However, factors that contribute to pineal development have not been fully elucidated, nor have pineal cell lineages been well characterized. We applied systematic double, triple and quadruple labeling of cell-specific markers on prenatal, postnatal and mature rat pineal gland tissue combined with confocal microscopy to provide a comprehensive view of the cellular dynamics and cell lineages that contribute to pineal gland development. The pineal gland begins as an evagination of neuroepithelium in the roof of the third ventricle. The pineal primordium initially consists of radially aligned Pax6+ precursor cells that express vimentin and divide at the ventricular lumen. After the tubular neuroepithelium fuses, the distribution of Pax6+ cells transitions to include rosette-like structures and later, dispersed cells. In the developing gland all dividing cells express Pax6, indicating that Pax6+ precursor cells generate pinealocytes and some interstitial cells. The density of Pax6+ cells decreases across pineal development as a result of cellular differentiation and microglial phagocytosis, but Pax6+ cells remain in the adult gland as a distinct population. Microglial colonization begins after pineal recess formation. Microglial phagocytosis of Pax6+ cells is not common at early stages but increases as microglia colonize the gland. In the postnatal gland microglia affiliate with Tuj1+ nerve fibers, IB4+ blood vessels, and Pax6+ cells. We demonstrate that microglia engulf Pax6+ cells, nerve fibers, and blood vessel-related elements, but not pinealocytes. We conclude that microglia play a role in pineal gland formation and homeostasis by regulating the precursor cell population, remodeling blood vessels and pruning sympathetic nerve fibers.

GABAergic signaling in the rat pineal gland.

Pinealocytes secrete melatonin at night in response to norepinephrine released from sympathetic nerve terminals in the pineal gland. The gland also contains many other neurotransmitters whose cellular disposition, activity, and relevance to pineal function are not understood. Here, we clarify sources and demonstrate cellular actions of the neurotransmitter γ-aminobutyric acid (GABA) using Western blotting and immunohistochemistry of the gland and electrical recording from pinealocytes. GABAergic cells and nerve fibers, defined as containing GABA and the synthetic GAD67, were identified. The cells represent a subset of interstitial cells while the nerve fibers were distinct from the sympathetic innervation. The GABAA receptor subunit α1 was visualized in close proximity of both GABAergic and sympathetic nerve fibers as well as fine extensions among pinealocytes and blood vessels. The GABAB 1 receptor subunit was localized in the interstitial compartment but not in pinealocytes. Electrophysiology of isolated pinealocytes revealed that GABA and muscimol elicit strong inward chloride currents sensitive to bicuculline and picrotoxin, clear evidence for functional GABAA receptors on the surface membrane. Applications of elevated potassium solution or the neurotransmitter acetylcholine depolarized the pinealocyte membrane potential enough to open voltage-gated Ca(2+) channels leading to intracellular calcium elevations. GABA repolarized the membrane and shut off such calcium rises. In 48-72-h cultured intact glands, GABA application neither triggered melatonin secretion by itself nor affected norepinephrine-induced secretion. Thus, strong elements of GABA signaling are present in pineal glands that make large electrical responses in pinealocytes, but physiological roles need to be found.

Expression and cellular localization of the transcription factor NeuroD1 in the developing and adult rat pineal gland.

Circadian rhythms govern many aspects of mammalian physiology. The daily pattern of melatonin synthesis and secretion is one of the classic examples of circadian oscillations. It is mediated by a class of neuroendocrine cells known as pinealocytes which are not yet fully defined. An established method to evaluate functional and cytological characters is through the expression of lineage-specific transcriptional regulators. NeuroD1 is a basic helix-loop-helix transcription factor involved in the specification and maintenance of both endocrine and neuronal phenotypes. We have previously described developmental and adult regulation of NeuroD1 mRNA in the rodent pineal gland. However, the transcript levels were not influenced by the elimination of sympathetic input, suggesting that any rhythmicity of NeuroD1 might be found downstream of transcription. Here, we describe NeuroD1 protein expression and cellular localization in the rat pineal gland during development and the daily cycle. In embryonic and perinatal stages, protein expression follows the mRNA pattern and is predominantly nuclear. Thereafter, NeuroD1 is mostly found in pinealocyte nuclei in the early part of the night and in cytoplasm during the day, a rhythm maintained into adulthood. Additionally, nocturnal nuclear NeuroD1 levels are reduced after sympathetic disruption, an effect mimicked by the in vivo administration of α- and ß-adrenoceptor blockers. NeuroD1 phosphorylation at two sites, Ser(274) and Ser(336) , associates with nuclear localization in pinealocytes. These data suggest that NeuroD1 influences pineal phenotype both during development and adulthood, in an autonomic and phosphorylation-dependent manner.

Hypoxic preconditioning differentially affects GABAergic and glutamatergic neuronal cells in the injured cerebellum of the neonatal rat.

In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult--showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization--were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are protected by a preconditioning hypoxia while the latter are not.

NeuroD1 is required for survival of photoreceptors but not pinealocytes: results from targeted gene deletion studies.

NeuroD1 encodes a basic helix-loop-helix transcription factor involved in the development of neural and endocrine structures, including the retina and pineal gland. To determine the effect of NeuroD1 knockout in these tissues, a Cre/loxP recombination strategy was used to target a NeuroD1 floxed gene and generate NeuroD1 conditional knockout (cKO) mice. Tissue specificity was conferred using Cre recombinase expressed under the control of the promoter of Crx, which is selectively expressed in the pineal gland and retina. At 2 months of age, NeuroD1 cKO retinas have a dramatic reduction in rod- and cone-driven electroretinograms and contain shortened and disorganized outer segments; by 4 months, NeuroD1 cKO retinas are devoid of photoreceptors. In contrast, the NeuroD1 cKO pineal gland appears histologically normal. Microarray analysis of 2-month-old NeuroD1 cKO retina and pineal gland identified a subset of genes that were affected 2-100-fold; in addition, a small group of genes exhibit altered differential night/day expression. Included in the down-regulated genes are Aipl1, which is necessary to prevent retinal degeneration, and Ankrd33, whose protein product is selectively expressed in the outer segments. These findings suggest that NeuroD1 may act through Aipl1 and other genes to maintain photoreceptor homeostasis.

NeuroD1: developmental expression and regulated genes in the rodent pineal gland.

NeuroD1/BETA2, a member of the bHLH transcription factor family, is known to influence the fate of specific neuronal, endocrine and retinal cells. We report here that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during the day and night, and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identifies 127 transcripts that are down-regulated (>twofold, p < 0.05) and 16 that are up-regulated (>twofold, p < 0.05). According to quantitative RT-PCR, the most dramatically down-regulated gene is kinesin family member 5C ( approximately 100-fold) and the most dramatically up-regulated gene is glutamic acid decarboxylase 1 ( approximately fourfold). Other impacted transcripts encode proteins involved in differentiation, development, signal transduction and trafficking. These findings represent the first step toward elucidating the role of NeuroD1 in the rodent pinealocyte.

Bilateral enucleation and captivity influence the reproductive cycle of male Viscacha (Lagostomus maximus maximus).

The viscacha (Lagostomus maximus maximus) is a seasonal rodent living in the Southern Hemisphere. The adult males exhibit an annual reproductive cycle characterized by a gonadal regression period during winter. In this study, we investigated the effects of bilateral enucleation and captivity on their annual reproductive cycle. Testicular volume relative to body weight was recorded monthly in intact and bilaterally enucleated animals placed under natural photoperiod, water, and food ad lib. and constant temperature. Testes and accessory organs were evaluated by qualitative and quantitative light microscopic studies. The intact animals showed an annual reproductive cycle with complete gonadal atrophy in the first year. In the second year, testicular regression was observed but attenuated in regard to that recorded in the first winter period, indicating that adaptive changes might be involved. Bilateral enucleation in the viscacha dampened and extended the period of its annual reproductive cycle. The results suggest that both conditions, constant captivity and enucleation, produced stimulatory effects on the reproductive system of this rodent. Furthermore, local control mechanisms might be responsible for the morphological differences observed in testes, epididymis, and seminal vesicles from both groups, which exhibited similar levels of serum testosterone. Finally, an intact retinohypothalamic-pineal axis and/or photoperiodic input would be necessary to maintain the reproductive cycle amplitude and timing in viscacha.

Automedicaçäo em odontologia / Self-medication in dentistry

A utilizaçäo de medicamentos sem a orientaçäo profissional adequada é perigosa, e näo respeita as diferenças individuais de cada paciente. Através de 141 questionários aplicados juntamente com a anamneses, foi estudada a incidência de automedicaçäo em pacientes com odontalgia que procuraram os serviços de pronto atendimento odontológico em Curitiba. As drogas e práticas mais comuns para o alívio da dor foram identificadas, a indicaçäo e a posologia desses produtos avaliadas, bem como a ocorrência de associaçäo de medicamentos. A automedicaçäo é muito frequente na Odontologia, assim como na Medicina. Em nosso estudo o índice encontrado foi de 78 por cento, sendo que as mulheres se medicaram mais que os homens. Os medicamentos mais utilizados foram os analgésicos, com destaque para a dipirona, já em desuso em outros países. Observamos que 1/4 dos pacientes usaram produtos tópicos, sendo alguns de aplicaçäo duvidosa e sem comprovaçäo científica