Fmr1 exon 14 skipping in late embryonic development of the rat forebrain.

BACKGROUND: Fragile X syndrome, the major cause of inherited intellectual disability among men, is due to deficiency of the synaptic functional regulator FMR1 protein (FMRP), encoded by the FMRP translational regulator 1 (FMR1) gene. FMR1 alternative splicing produces distinct transcripts that may consequently impact FMRP functional roles. In transcripts without exon 14 the translational reading frame is shifted. For deepening current knowledge of the differential expression of Fmr1 exon 14 along the rat nervous system development, we conducted a descriptive study employing quantitative RT-PCR and BLAST of RNA-Seq datasets. RESULTS: We observed in the rat forebrain progressive decline of total Fmr1 mRNA from E11 to P112 albeit an elevation on P3; and exon-14 skipping in E17-E20 with downregulation of the resulting mRNA. We tested if the reduced detection of messages without exon 14 could be explained by nonsense-mediated mRNA decay (NMD) vulnerability, but knocking down UPF1, a major component of this pathway, did not increase their quantities. Conversely, it significantly decreased FMR1 mRNA having exon 13 joined with either exon 14 or exon 15 site A. CONCLUSIONS: The forebrain in the third embryonic week of the rat development is a period with significant skipping of Fmr1 exon 14. This alternative splicing event chronologically precedes a reduction of total Fmr1 mRNA, suggesting that it may be part of combinatorial mechanisms downregulating the gene's expression in the late embryonic period. The decay of FMR1 mRNA without exon 14 should be mediated by a pathway different from NMD. Finally, we provide evidence of FMR1 mRNA stabilization by UPF1, likely depending on FMRP.

Forebrain and Hindbrain Projecting-neurons Target the Post-inspiratory Complex Cholinergic Neurons.

The postinspiratory complex (PiCo) is a region located in the ventromedial medulla involved with the post-inspiratory activity. PiCo neurons are excitatory (VGlut2+) and express the enzyme choline acetyl transferase (ChAT+). Evidence also suggests that PiCo is coupled to two additional groups of neurons involved in breathing process, i.e. the pre-Bötzinger complex (preBötC, inspiration) and the retrotrapezoid nucleus (RTN, active expiration), composing all together, the hypothesized triple respiratory oscillator. Here, our main objective is to demonstrate the afferent connections to PiCo region. We mapped projecting-neurons to PiCo by injecting Fluorogold (FG) retrograde tracer into the PiCo of adult Long-Evans Chat-cre male rats. We reported extensive projections from periaqueductal grey matter and Kölliker-Fuse regions and mild projections from the nucleus of the solitary tract, ventrolateral medulla and hypothalamus. We also injected a cre-dependent vector expressing channelrhodopsin 2 (AAV5-ChR2) fused with enhanced mCherry into the PiCo of ChAT-cre rats to optogenetic activate those neurons and investigate the role of PiCo for inspiratory/postinspiratory activity. Both in urethane-anesthetized and unrestrained conscious rats the response of ChR2-transduced neurons to light induced an increase in postinspiratory activity. Our data confirmed that PiCo seems to be dedicated to postinspiratory activity and represent a site of integration for autonomic and motor components of respiratory and non-respiratory pathways.

Replay of innate vocal patterns during night sleep in suboscines.

Activation of forebrain circuitry during sleep has been variably characterized as 'pre- or replay' and has been linked to memory consolidation. The evolutionary origins of this mechanism, however, are unknown. Sleep activation of the sensorimotor pathways of learned birdsong is a particularly useful model system because the muscles controlling the vocal organ are activated, revealing syringeal activity patterns for direct comparison with those of daytime vocal activity. Here, we show that suboscine birds, which develop their species-typical songs innately without the elaborate forebrain-thalamic circuitry of the vocal learning taxa, also engage in replay during sleep. In two tyrannid species, the characteristic syringeal activation patterns of the song could also be identified during sleep. Similar to song-learning oscines, the burst structure was more variable during sleep than daytime song production. In kiskadees (Pitangus sulphuratus), a second vocalization, which is part of a multi-modal display, was also replayed during sleep along with one component of the visual display. These data show unambiguously that variable 'replay' of stereotyped vocal motor programmes is not restricted to programmes confined within forebrain circuitry. The proposed effects on vocal motor programme maintenance are, therefore, building on a pre-existing neural mechanism that predates the evolution of learned vocal motor behaviour.

Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.

The meninges shield the nervous system from diverse, rather harmful stimuli and pathogens from the periphery. This tissue is composed of brain endothelial cells (BECs) that express diverse ion channels and chemical-transmitter receptors also expressed by neurons and glial cells to communicate with each other. However, information about the effects of ATP and angiotensin II on BECs is scarce, despite their essential roles in blood physiology. This work investigated in vitro if BECs from the meninges from rat forebrain respond to ATP, angiotensin II and high extracellular potassium, with intracellular calcium mobilizations and its second messenger-associated pathways. We found that in primary BEC cultures, both ATP and angiotensin II produced intracellular calcium responses linked to the activation of inositol trisphosphate receptors and ryanodine receptors, which led to calcium release from intracellular stores. We also used RT-PCR to explore what potassium channel subunits are expressed by primary BEC cultures and freshly isolated meningeal tissue, and which might be linked to the observed effects. We found that BECs mainly expressed the inward rectifier potassium channel subunits Kir1.1, Kir3.3, Kir 4.1 and Kir6.2. This study contributes to the understanding of the functions elicited by ATP and angiotensin II in BECs from rat meninges. SIGNIFICANCE OF THE STUDY: Brain endothelial cells (BECs) express diverse ion channels and membrane receptors, which they might use to communicate with neurons and glia. This work investigated in vitro, if BECs from the rat forebrain respond to angiotensin II and ATP with intracellular calcium mobilizations. We found that these cells did respond to said substances with intracellular calcium mobilizations linked to inositol trisphosphate and ryanodine receptor activation, which led to calcium release from intracellular stores. These findings are important because they might uncover routes of active communication between brain cells and endothelial cells.

Neuroanatomy of the spinosaurid Irritator challengeri (Dinosauria: Theropoda) indicates potential adaptations for piscivory.

Spinosauridae, a theropod group characterized by elongated snouts, conical teeth, enlarged forelimbs, and often elongated neural spines, show evidence for semiaquatic adaptations and piscivory. It is currently debated if these animals represent terrestrial carnivores with adaptations for a piscivorous diet, or if they largely lived and foraged in aquatic habitats. The holotype of Irritator challengeri, a nearly complete skull from the late Early Cretaceous Santana Formation of northeastern Brazil, includes one of the few preserved spinosaurid braincases and can provide insights into neuroanatomical structures that might be expected to reflect ecological affinities. We generated digital models of the neuroanatomical cavities within the braincase, using computer tomography (CT) data. The cranial endocast of Irritator is generally similar to that of other non-maniraptoriform theropods, with weakly developed distinctions of hindbrain and midbrain features, relatively pronounced cranial flexures and relatively long olfactory tracts. The endosseous labyrinth has a long anterior semicircular canal, a posteriorly inclined common crus and a very large floccular recess fills the area between the semicircular canals. These features indicate that Irritator had the ability for fast and well-controlled pitch-down head movements. The skull table and lateral semicircular canal plane are strongly angled to one another, suggesting a downward angling of approximately 45° of the snout, which reduces interference of the snout with the field of vision of Irritator. These neuroanatomical features are consistent with fast, downward snatching movements in the act of predation, such as are needed for piscivory.

Morphological evolution of the vertebrate forebrain: From mechanical to cellular processes.

Although the cerebral hemispheres are among the defining characters of vertebrates, each vertebrate class is characterized by a different anatomical organization of this structure, which has become highly problematic for comparative neurobiology. In this article, we discuss some mechanisms involved in the generation of this morphological divergence, based on simple spatial constraints for neurogenesis and mechanical forces generated by increasing neuronal numbers during development, and the different cellular strategies used by each group to overcome these limitations. We expect this view to contribute to unify the diverging vertebrate brain morphologies into general, simple mechanisms that help to establish homologies across groups.

Search for appropriate reference genes for quantitative reverse transcription PCR studies in somite, prosencephalon and heart of early mouse embryo.

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb, Gapdh, Tbp, Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16-18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha, Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh, Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development.

Effects of a rat model of gestational hypothyroidism on forebrain dopaminergic, GABAergic, and serotonergic systems and related behaviors.

We investigated the effects of maternal hypothyroidism on forebrain dopaminergic, GABAergic, and serotonergic systems and related behavior in adult rat offspring. Experimental gestational hypothyroidism (EGH) was induced by administering 0.02% methimazole (MMI) to pregnant rats from gestational day 9 to delivery. Neurotransmitter-related protein and gene expression were evaluated in offspring forebrain at postnatal day (P) 120. Exploratory behavior, contextual fear conditioning, locomotion, and 30-day reserpine Parkinson induction were assessed from P75-P120. Protein and gene expression assessments of medial prefrontal cortex showed group differences in dopaminergic, GABAergic, and serotonergic receptors, catabolic enzymes, and transporters. Striatum of MMI offspring showed an isolated decrease in the dopaminergic enzyme, tyrosine hydroxylase. MMI exposure increased GABA and dopamine receptor expression in amygdala. MMI offspring also had decreased state anxiety and poor contextual fear conditioning. We found that baseline locomotion was not changed, but reserpine treatment significantly reduced locomotion only in MMI offspring. Our results indicated that restriction of maternal thyroid hormones reduced dopaminergic, GABAergic, and serotoninergic forebrain components in offspring. Tyrosine hydroxylase deficiency in the striatum may underlie enhanced reserpine induction of Parkinson-like movement in these same offspring. Deficits across different neurotransmitter systems in medial prefrontal cortex and amygdala may underlie decreased state anxiety-like behavior and reduced fear conditioning in offspring, but no changes in trait anxiety-like behavior occurred with maternal MMI exposure. These findings strongly support the hypothesis that adequate delivery of maternal thyroid hormones to the fetus is crucial to the development of the central nervous system critical for emotion and motor regulation.

Simulation of the song motor pathway in birds: A single neuron initiates a chain of events that produces birdsong with realistic spectra properties.

Birdsong is a complex learned behavior regulated by Neuromuscular coordination of different muscle sets necessary for producing relevant sounds. We developed a heterogeneous and stochastically connected neural network representing the pathway from the high vocal center (HVC) to the robust nucleus of the arcopallium (RA) neurons that drive the muscles to generate sounds. We show that a single active neuron is sufficient to initiate a chain of spiking events that results to excite the entire network system. The network could synthesize realistic bird sounds spectra, with spontaneous generation of intermittent sound bursts typical of birdsong (song syllables). This study confirms experiments on animals and on humans, where results have shown that single neurons are responsible for the activation of complex behavior or are associated with high-level perception events.

Monoaminergic levels at the forebrain and diencephalon signal for the occurrence of mutualistic and conspecific engagement in client reef fish.

Social interactions are commonly found among fish as in mammals and birds. While most animals interact socially with conspecifics some however are also frequently and repeatedly observed to interact with other species (i.e. mutualistic interactions). This is the case of the (so-called) fish clients that seek to be cleaned by other fish (the cleaners). Clients face an interesting challenge: they raise enough motivation to suspend their daily activities as to selectively visit and engage in interactions with cleaners. Here we aimed, for the first time, to investigate the region-specific brain monoaminergic level differences arising from individual client fish when facing a cleaner (interspecific context) compared to those introduced to another conspecific (socio-conspecific context). We show that monoaminergic activity differences occurring at two main brain regions, the diencephalon and the forebrain, are associated with fish clients' social and mutualistic activities. Our results are the first demonstration that monoaminergic mechanisms underlie client fish mutualistic engagement with cleanerfish. These pathways should function as a pre-requisite for cleaning to occur, providing to clients the cognitive and physiological tools to seek to be cleaned.

First Natural Endocranial Cast of a Fossil Snake (Cretaceous of Patagonia, Argentina).

In this study, we describe a natural endocranial cast included in a partially preserved medium-sized skull of the Upper Cretaceous South American snake Dinilysia patagonica. The endocast is composed of sedimentary filling of the cranial cavity in which the posterior brain, the vessels, the cranial nerves, and the inner ear surrounded by delicate semicircular canals, are represented. It is simple in form, with little differentiation between the three main areas (Forebrain, Midbrain, and Hindbrain), and without flexures. The nervous system is well preserved. The posterior brain surface is smooth, except for two small prominences that make up the cerebellum. A large inner ear is preserved on the right side; it consists of a voluminous central mass, the vestibule, which occupies most of the space defined by the three semicircular canals. In particular, the lateral semicircular canal is very close to the vestibule. This characteristic, in combination with the medium to large body size of Dinilysia, its large skull and dorsally exposed orbits, and vertebrae bearing a rather high neural spine on a depressed neural arch, suggests that this snake would have had a semifossorial lifestyle. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 301:9-20, 2018. © 2017 Wiley Periodicals, Inc.

Oxidative stress and Na,K-ATPase activity differential regulation in brainstem and forebrain of Wistar Audiogenic rats may lead to increased seizure susceptibility.

The Wistar Audiogenic Rat (WAR) is a well-characterized seizure-prone, inbred rodent strain that, when acutely stimulated with high-intensity sounds, develops brainstem-dependent tonic-clonic seizures that can evolve to limbic-like, myoclonic (forebrain) seizures when the acoustic stimuli are presented chronically (audiogenic kindling). In order to investigate possible mechanisms underlying WAR susceptibility to seizures, we evaluated Na,K-ATPase activity, Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and oxidative stress markers in whole forebrain and whole brainstem samples of naïve WAR, as compared to samples from control Wistar rats. We also evaluated the expression levels of α1 and α3 isoforms of Na,K-ATPase in forebrain samples. We observed increased Na,K-ATPase activity in forebrain samples and increased oxidative stress markers (lipid peroxidation, glutathione peroxidase and superoxide dismutase) in brainstem samples of WAR. The Ca-ATPase activity, Mg-ATPase activity, lipid membrane composition and expression levels of α1 and α3 isoforms of Na,K-ATPase were unaltered. In view of previous data showing that the membrane potentials from naïve WAR's neurons are less negative than that from neurons from Wistar rats, we suggest that Na,K-ATPase increased activity might be involved in a compensatory mechanism necessary to maintain WAR's brains normal activity. Additionally, ongoing oxidative stress in the brainstem could bring Na,K-ATPase activity back to normal levels, which may explain why WAR's present increased susceptibility to seizures triggered by high-intensity sound stimulation.

Selenite restores Pax6 expression in neuronal cells of chronically arsenic-exposed Golden Syrian hamsters.

Arsenic is a worldwide environmental pollutant that generates public health concerns. Various types of cancers and other diseases, including neurological disorders, have been associated with human consumption of arsenic in drinking water. At the molecular level, arsenic and its metabolites have the capacity to provoke genome instability, causing altered expression of genes. One such target of arsenic is the Pax6 gene that encodes a transcription factor in neuronal cells. The aim of this study was to evaluate the effect of two antioxidants, α-tocopheryl succinate (α-TOS) and sodium selenite, on Pax6 gene expression levels in the forebrain and cerebellum of Golden Syrian hamsters chronically exposed to arsenic in drinking water. Animals were divided into six groups. Using quantitative real-time reverse transcriptase (RT)-PCR analysis, we confirmed that arsenic downregulates Pax6 expression in nervous tissues by 53 ± 21% and 32 ± 7% in the forebrain and cerebellum, respectively. In the presence of arsenic, treatment with α-TOS did not modify Pax6 expression in nervous tissues; however, sodium selenite completely restored Pax6 expression in the arsenic-exposed hamster forebrain, but not the cerebellum. Although our results suggest the use of selenite to restore the expression of a neuronal gene in arsenic-exposed animals, its use and efficacy in the human population require further studies.

Temperature manipulation of neuronal dynamics in a forebrain motor control nucleus.

Different neuronal types within brain motor areas contribute to the generation of complex motor behaviors. A widely studied songbird forebrain nucleus (HVC) has been recognized as fundamental in shaping the precise timing characteristics of birdsong. This is based, among other evidence, on the stretching and the "breaking" of song structure when HVC is cooled. However, little is known about the temperature effects that take place in its neurons. To address this, we investigated the dynamics of HVC both experimentally and computationally. We developed a technique where simultaneous electrophysiological recordings were performed during temperature manipulation of HVC. We recorded spontaneous activity and found three effects: widening of the spike shape, decrease of the firing rate and change in the interspike interval distribution. All these effects could be explained with a detailed conductance based model of all the neurons present in HVC. Temperature dependence of the ionic channel time constants explained the first effect, while the second was based in the changes of the maximal conductance using single synaptic excitatory inputs. The last phenomenon, only emerged after introducing a more realistic synaptic input to the inhibitory interneurons. Two timescales were present in the interspike distributions. The behavior of one timescale was reproduced with different input balances received form the excitatory neurons, whereas the other, which disappears with cooling, could not be found assuming poissonian synaptic inputs. Furthermore, the computational model shows that the bursting of the excitatory neurons arises naturally at normal brain temperature and that they have an intrinsic delay at low temperatures. The same effect occurs at single synapses, which may explain song stretching. These findings shed light on the temperature dependence of neuronal dynamics and present a comprehensive framework to study neuronal connectivity. This study, which is based on intrinsic neuronal characteristics, may help to understand emergent behavioral changes.

P2 purinergic receptor antagonists disrupt maternal behavior in lactating rats.

The involvement of purinergic signaling in several brain functions has been recognized, but the modulation on maternal behavior by the purinergic system is not established, even though there are functional interactions between the purinergic and oxytocinergic systems. Therefore, the aim of our study was to investigate whether central administration of P2 receptor antagonists affected the maternal behavior of lactating rats and c-Fos immunoreactivity in the forebrain. On day 7 of lactation, female rats were treated with vehicle (5µL; i.c.v.), suramin (9.4-75.0µg/5µL; i.c.v.) or PPADS (9.4-75.0µg/5µL; i.c.v.) 30min before the experiment began. The maternal behavior was evaluated during the 30min following suramin or PPADS administration. In addition, c-Fos-positive nuclei were counted in the medial preoptic area (MPOA) and in the bed nucleus of the stria terminalis (BNST), and neurons that were double-labeled for c-Fos/OT were counted in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus of lactating rats. The results show that P2 receptor antagonists decreased maternal care and decreased neuronal activation in the MPOA and BNST and activation of oxytocinergic neurons in hypothalamic nuclei. Our results indicate that the purinergic system modulates maternal behavior and neuronal activation induced by suckling during lactation.

Evaluation of mitochondrial respiratory function in highly glycolytic glioma cells reveals low ADP phosphorylation in relation to oxidative capacity.

High-grade gliomas are aggressive and intensely glycolytic tumors. In the present study, we evaluated the mitochondrial respiratory function of glioma cells (T98G and U-87MG) and fresh human glioblastoma (GBM) tissue. To this end, measurements of oxygen consumption rate (OCR) were performed under various experimental conditions. The OCR of T98G and U-87MG cells was well coupled to ADP phosphorylation based on the ratio of ATP produced per oxygen consumed of ~2.5. In agreement, the basal OCR of GBM tissue was also partially associated with ADP phosphorylation. The basal respiration of intact T98G and U-87MG cells was not limited by the supply of endogenous substrates, as indicated by the increased OCR in response to a protonophore. These cells also displayed a high affinity for oxygen, as evidenced by the values of the partial pressure of oxygen when respiration is half maximal (p 50). In permeabilized glioma cells, ADP-stimulated OCR was only approximately 50% of that obtained in the presence of protonophore, revealing a significant limitation in oxidative phosphorylation (OXPHOS) relative to the activity of the electron transport system (ETS). This characteristic was maintained when the cells were grown under low glucose conditions. Flux control coefficient analyses demonstrated that the impaired OXPHOS was associated with the function of both mitochondrial ATP synthase and the adenine nucleotide translocator, but not the phosphate carrier. Altogether, these data indicate that the availability and metabolism of respiratory substrates and mitochondrial ETS are preserved in T98G and U-87MG glioma cells even though these cells possess a relatively restrained OXPHOS capability.

Brucella abortus-activated microglia induce neuronal death through primary phagocytosis.

Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis.

Deep brain stimulation of the dorsal raphe inhibits avoidance and escape reactions and activates forebrain regions related to the modulation of anxiety/panic.

One of the main neurochemical systems associated with anxiety/panic is the serotonergic system originating from the dorsal raphe nucleus (DR). Previous evidence suggests that the DR is composed of distinct subpopulations of neurons, both morphologically and functionally distinct. It seems that mainly the dorsal region of the DR (DRD) regulates anxiety-related reactions, while lateral wings DR (lwDR) serotonin (5-HT) neurons inhibit panic-related responses. In this study we used the technique of deep brain stimulation (DBS) to investigate the role played by the DRD and lwDR in defense. Male Wistar rats were submitted to high-frequency stimulation (100µA, 100Hz) in one of the two DR regions for 1h and immediately after tested in the avoidance or escape tasks of the elevated T-maze (ETM). In clinical terms, these responses have been related to generalized anxiety and panic disorder, respectively. After being submitted to the ETM, animals were placed in an open field for locomotor activity assessment. An additional group of rats was submitted to DBS of the DRD or the lwDR and used for quantification of c-Fos immunoreactive (Fos-ir) neurons in brain regions related to the modulation of defense. Results showed that stimulation of the DRD decreased avoidance latencies, an anxiolytic-like effect. DRD stimulation also led to increases in Fos-ir in the medial amygdala, lateral septum and cingulate cortex. DBS applied to the lwDR increased escape latencies, a panicolytic-like effect. This data highlights the importance of raphe topography and the potential benefit of the DBS technique for the treatment of anxiety-related disorders.

Defensive behaviors and prosencephalic neurogenesis in pigeons (Columba livia) are affected by environmental enrichment in adulthood.

Neurogenesis in the adult brain appears to be phylogenetically conserved across the animal kingdom. In pigeons and other adult non-oscine birds, immature neurons are observed in several prosencephalic areas, suggesting that neurogenesis may participate in the control of different behaviors. The mechanisms controlling neurogenesis and its relevance to defensive behaviors in non-oscine birds remain elusive. Herein, the contribution of the environment to behavior and neurogenesis of pigeons was investigated. Adult pigeons (Columba livia, n = 6/group), housed in standard (SE) or enriched environment (EE) for 42 days, were exposed to an unfamiliar environment (UE) followed by presentation to a novel object (NO). Video recordings of UE+NO tests were analyzed and scored for latency, duration and frequency of angular head movements, peeping, grooming, immobility and locomotion. Twenty-four hours later, pigeons were submitted to the tonic immobility test (TI) and number of trials for TI and TI duration were scored, followed by euthanasia 2 h later. Brains were immunohistochemically processed to reveal doublecortin (DCX), a marker for newborn neurons. Compared to those housed in SE, the pigeons housed in EE responded to a NO with more immobility. In addition, the pigeons housed in EE presented longer TI, more DCX-immunoreactive (DCX-ir) cells in the hippocampus and fewer DCX-ir cells in the lateral striatum than those housed in SE. There was no correlation between the number of DCX-ir cells and the scores of immobility in behavioral tests. Together, these data suggest that enrichment favored behavioral inhibition and neurogenesis in the adult pigeons through different, parallel mechanisms.

Association between respiratory and postural adaptations and self-perception of school-aged children with mouth breathing in relation to their quality of life

Objective: To investigate the respiratory and postural adaptations associated with mouth and nasal breathing and to evaluate the associations of such adaptations in mouth breathers' self-perceived quality of life. Method: Cross-sectional study with mouth breathers (initial n=116 and final n=48) and nasal breathers (initial n=131 and final n=24) from elementary school, aged between 7 and 14 years. Chest expansion, using cirtometry, the breathing pattern and the use of accessory muscles, by means of clinical evaluations and photogrammetry, and flexibility tests were evaluated in both groups. Subsequently, the mouth breathers were asked to complete the quality of life questionnaire. Statistical tests: Chi-square, odds ratio, Mann-Whitney, and binomial tests were first applied followed by logistic regressions. Results: Thoracic breathing (p=0.04), using of accessory muscles (p=0.03) and reductions in flexibility (p=0.001) increased the chances of an individual being a mouth breather when compared to nasal breathers. Subsequently, using of accessory muscles decreased the chances of snoring among mouth breathers (p=0.03); the presence of shoulder asymmetry reduced the chances of experiencing quiet sleep (p=0.05) and increased the chances of coughing or being tired when playing or running (p=0.008). Finally, forward head position reduced the chances of waking up at night (p=0.04) and experiencing shortness of breath (p=0.05). Conclusions: Respiratory and postural adaptations increased the chances of individuals persisting with mouth breathing. Additionally, these adaptations could be associated with mouth breathers' self-perceived quality of life. .