Estudo morfológico e imunológico da encefalite induzida pelo vírus juruaçá em modelo murino

Muitos estudos têm sido realizados para o entendimento da neuropatogênese das encefalites virais a partir de trabalhos experimentais, porém, nenhum estudo experimental foi dedicado à compreensão da neuropatogênese de membros da família Picornaviridae isolados de morcegos na região amazônica. O vírus Juruaçá, um desses agentes, parcialmente caracterizado como membro da família Picornaviridae por Araújo e colaboradores (2006), causou lesões no encéfalo de camundongos neonatos com presença de gliose reativa, apesar de não provocar efeito citopático (ECP) em cultivos primários de células do sistema nervoso central (SNC), sugerindo que este agente viral seja responsável pela morte dos animais devido a uma intensa resposta imune. O objetivo desse trabalho foi investigar a resposta imune no SNC e alterações celulares causadas pelo vírus Juruaçá em camundongos albinos da linhagem BALB/c neonatos a partir de análises histopatológicas, de ativação microglial e da expressão de citocinas, óxido nítrico (NO) e espécies reativas de oxigênio (ROS). Para tanto, foram realizados processamento de amostras para histopatologia, ensaios imunoenzimáticos, imunohistoquímicos e de imunofluorescência, além de testes para quantificação de NO e ROS e análises estatísticas. Nossos resultados demonstraram que o vírus Juruaçá induz lesões por todo o encéfalo, com maior intensidade no parênquima cortical. Os testes imunohistoquímicos demonstraram a presença de antígenos virais e de micróglias reativas distribuídos por todo o encéfalo e região anterior da medula espinhal. Micróglias com aspecto ameboide, demonstrando intensa ativação, foram observadas principalmente no córtex cerebral, bulbo olfatório, núcleo olfatório anterior, prosencéfalo e diencéfalo próximo ao ventrículo lateral. A produção das citocinas anti-inflamatórias (IL-10, IL-4) diminuiu ao longo do tempo, enquanto que as pró-inflamatórias (IL-12, IL-6, IL-1β, TNF-α, IFN-γ) aumentaram significativamente a partir do 8º dia. Os ensaios para detecção de ROS demonstraram grande produção de radicais superóxido desde o 4º dia, já a produção de NO foi sempre menor nos animais infectados. Provavelmente, a ativação das células gliais, principalmente micróglias, e consequente produção de citocinas pró-inflamatórias e ROS promoveram uma ação devastadora sobre as células do SNC, que coincide com a intensificação dos sinais clínicos. Diante do exposto, ficou evidente que os nossos resultados indicam que o vírus Juruaçá é responsável por uma doença de cunho inflamatório que leva a óbito 100% de camundongos neonatos infectados.

Papel da serotonina no comportamento defensivo do paulistinha (Danio rerio Hamilton 1822) adulto: Diferenças entre modelos comportamentais, linhagens, e efeitos do estresse predatório agudo

Os transtornos de ansiedade apresentam a maior incidência na população mundial dentre os transtornos psiquiátricos, e a eficácia clínica das drogas ansiolíticas é baixa, em parte devido ao desconhecimento acerca das bases neuroquímicas desses transtornos. Para uma compreensão mais ampla e evolutivamente substanciada desses fenômenos, a utilização de espécies filogeneticamente mais antigas pode ser uma aproximação interessante no campo da modelagem comportamental; assim, sugerimos o uso do paulistinha (Danio rerio Hamilton 1822) na tentativa de compreender a modulação de comportamentos tipo-ansiedade pelo sistema serotonérgico. Demonstramos que os níveis extracelulares de serotonina no encéfalo de paulistinhas adultos expostos ao teste de preferência claro/escuro [PCE] (mas não ao teste de distribuição vertical eliciada pela novidade [DVN]) apresentam-se elevados em relação a animais manipulados mas não expostos aos aparatos. Além disso, os níveis teciduais de serotonina no rombencéfalo e no prosencéfalo são elevados pela exposição ao PCE, enquanto no mesencéfalo são elevados pela exposição ao DVN. Os níveis extracelulares de serotonina estão correlacionados negativamente com a geotaxia no DVN, e positivamente com a escototaxia, tigmotaxia e a avaliação de risco no PCE. O tratamento agudo com uma dose baixa de fluoxetina (2,5 mg/kg) aumenta a escototaxia, a tigmotaxia e a avaliação de risco no PCE, diminui a geotaxia e o congelamento e facilita a habituação no DVN. O tratamento com buspirona diminui a escototaxia, a tigmotaxia e o congelamento nas doses de 25 e 50 mg/kg no PCE, e diminui a avaliação de risco na dose de 50 mg/kg; no DVN, ambas as doses diminuem a geotaxia, enquanto somente a maior dose diminui o congelamento e facilita a habituação. O tratamento com WAY 100635 diminui a escototaxia nas doses de 0,003 e 0,03 mg/kg, enquanto somente a dose de 0,03 mg/kg diminui a tigmotaxia e a avaliação de risco no PCE. No DVN, ambas as doses diminuem a geotaxia, enquanto somente a menor dose facilita a habituação e aumenta o tempo em uma “base” (“homebase”). O tratamento com SB 224289 não produziu efeitos sobre a escototaxia, mas aumentou a avaliação de risco na dose de 2,5 mg/kg; no DVN, essa droga diminuiu a geotaxia e o nado errático nas doses de 2,5 e 5 mg/kg, enquanto a dose de 2,5 mg/kg aumentou a formação de “bases”. O tratamento com DL-para-clorofenilalanina (2 injeções de 300 mg/kg, separadas por 24 horas) diminuiu a escototaxia, a tigmotaxia e a avaliação de risco no PCE, aumentou a geotaxia e a formação de bases e diminuiu a habituação no DVN. Quando os animais são pré-expostos a uma “substância de alarme” co-específica, observa-se um aumento nos níveis extracelulares de serotonina associados a um aumento na escototaxia, congelamento e nado errático no PCE; os efeitos comportamentais e neuroquímicos foram bloqueados pelo pré tratamento com fluoxetina (2,5 mg/kg), mas não pelo pré-tratamento com WAY 100,635 (0,003 mg/kg). Animais da linhagem leopard apresentam maior escototaxia e avaliação de risco no PCE, assim como níveis teciduais elevados de serotonina no encéfalo; o fenótipo comportamental é resgatado pelo tratamento com fluoxetina (5 mg/kg). Esses dados sugerem que o sistema serotonérgico dessa espécie modula o comportamento no DVN e no PCE de forma oposta; que a resposta de medo produzida pela substância de alarme também parece aumentar a atividade do sistema serotonérgico, um efeito possivelmente mediado pelos transportadores de serotonina, e ao menos um fenótipo mutante de alta ansiedade também está associado a esses transportadores. Sugere-se que, de um ponto de vista funcional, a serotonina aumenta a ansiedade e diminui o medo em paulistinhas.

Role of the lateral parabrachial nucleus in the control of sodium appetite

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Tonic modulation of anxiety-like behavior by corticotropin-releasing factor (CRF) type 1 receptor (CRF1) within the medial prefrontal cortex (mPFC) in male mice: Role of protein kinase A (PKA)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Hydrogen peroxide attenuates the dipsogenic, renal and pressor responses induced by cholinergic activation of the medial septal area

Cholinergic activation of the medial septal area (MSA) with carbachol produces thirst, natriuresis, antidiuresis and pressor response. In the brain, hydrogen peroxide (H2O2) modulates autonomic and behavioral responses. In the present study, we investigated the effects of the combination of carbachol and H2O2 injected into the MSA on water intake, renal excretion, cardiovascular responses and the activity of vasopressinergic and oxytocinergic neurons in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Furthermore, the possible modulation of carbachol responses by H2O2 acting through K+ATP channels was also investigated. Male Holtzman rats (280–320 g) with stainless steel cannulas implanted in the MSA were used. The pre-treatment with H2O2 in the MSA reduced carbachol-induced thirst (7.9 ± 1.0, vs. carbachol: 13.2 ± 2.0 ml/60 min), antidiuresis (9.6 ± 0.5, vs. carbachol: 7.0 ± 0.8 ml/120 min,), natriuresis (385 ± 36, vs. carbachol: 528 ± 46 μEq/120 min) and pressor response (33 ± 5, vs. carbachol: 47 ± 3 mmHg). Combining H2O2 and carbachol into the MSA also reduced the number of vasopressinergic neurons expressing c-Fos in the PVN (46.4 ± 11.2, vs. carbachol: 98.5 ± 5.9 c-Fos/AVP cells) and oxytocinergic neurons expressing c-Fos in the PVN (38.5 ± 16.1, vs. carbachol: 75.1 ± 8.5 c-Fos/OT cells) and in the SON (57.8 ± 10.2, vs. carbachol: 102.7 ± 7.4 c-Fos/OT cells). Glibenclamide (K+ATP channel blocker) into the MSA partially reversed H2O2 inhibitory responses. These results suggest that H2O2 acting through K+ATP channels in the MSA attenuates responses induced by cholinergic activation in the same area.

Effect of lithium on behavioral disinhibition induced by electrolytic lesion of the median raphe nucleus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Induced reproduction and early development histology of Oscar Astronotus ocellatus (Agassiz, 1831)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mapping brain Fos immunoreactivity in response to water deprivation and partial rehydration: influence of sodium intake

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Iptakalim: A Potential Antipsychotic Drug with Novel Mechanisms?

Iptakalim is a novel putative adenosine triphosphate (ATP)-sensitive potassium (KATP) channel opener. In the brain, iptakalim is thought to act on the neuronal and astrocytic plasma membrane and/or mitochondrial KATP channels. Because iptakalim demonstrates an action on the regulation of dopamine and glutamate release in the forebrain regions, we examined its potential antipsychotic efficacy in several preclinical tests. First, we show that iptakalim is effective in reducing amphetamine- and phencyclidine-induced hyperlocomotion as well as selectively disrupting conditioned avoidance responding. Next, we show that combined iptakalim and amphetamine treatment produces a reduction on prepulse inhibition of acoustic startle and this combined drug effect is also found with haloperidol, but not with clozapine. Finally, we show that iptakalim and clozapine preferentially increase c-Fos expression in the medial prefrontal cortex, nucleus accumbens and lateral septal nucleus, whereas haloperidol induces a greater increase in the nucleus accumbens, the dorsolateral striatum and lateral septal nucleus. Collectively, our findings indicate that iptakalim is likely to be a potential antipsychotic drug with distinct mechanisms of action. This study also suggests that neuronal and astrocytic plasma membrane and/or mitochondrial KATP channels may be a novel target that deserves attention for antipsychotic drug development. Future research using other sensitive tests is needed to confirm this property of iptakalim.

Analysis of the NMDA in Focal Cerebral Ischemia in Rats

NMDAR (N-methyl-D-aspartate receptor) is one subtype of ionotrophic glutamate receptor which is extensively distributed in the central nervous system (CNS). In the mammalian CNS, NMDAR serves prominent roles in the pathophysiologic process of cerebral ischemia. This study aimed to investigate the pattern of expression of protein and gene of the excitatory neurotransmitter NMDAR in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. 120 rats were randomly divided into 6 groups (20 animals each): control - no surgery; sham - simulation of surgery; ischemic - focal ischemia for 1 hour, without reperfusion; ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. Ten animals from each experimental group were used to establish the volume of infarct. Transient focal cerebral ischemia was obtained in rats by occlusion of the middle cerebral artery with an intraluminal suture. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the protein and gene NMDA were evaluated by immunohistochemistry and quantitative real-time PCR, respectively. Increases in the protein and gene NMDA receptor in the ischemics areas were observed and these increases were reduced by hypothermia and ketoprofen. The increase in the NMDA receptor protein and gene expression observed in the ischemic animals was reduced by neuroprotection (hypothermia and ketoprofen). The NMDA receptor increases in the ischemic area suggests that the NMDA mediated neuroexcitotoxicity plays an important role in cell death and that the neuroprotective effect of both, hypothermia and ketoprofen is directly involved with the NMDA.

Unique Alterations of an Ultraconserved Non-Coding Element in the 3 ' UTR of ZIC2 in Holoprosencephaly

Coding region alterations of ZIC2 are the second most common type of mutation in holoprosencephaly (HPE). Here we use several complementary bioinformatic approaches to identify ultraconserved cis-regulatory sequences potentially driving the expression of human ZIC2. We demonstrate that an 804 bp element in the 3' untranslated region (3'UTR) is highly conserved across the evolutionary history of vertebrates from fish to humans. Furthermore, we show that while genetic variation of this element is unexpectedly common among holoprosencephaly subjects (6/528 or >1%), it is not present in control individuals. Two of six proband-unique variants are de novo, supporting their pathogenic involvement in HPE outcomes. These findings support a general recommendation that the identification and analysis of key ultraconserved elements should be incorporated into the genetic risk assessment of holoprosencephaly cases.

Ether-A -go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether A go-go (Eag) gene encodes the voltage-gated potassium (K+) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K+ channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K+ channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K+-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K+ channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Clinical findings in patients with GLI2 mutations - phenotypic variability

Mutations in the human GLI2 gene were first reported in association with defective anterior pituitary formation, panhypopituitarism, and forebrain anomalies represented by typical holoprosencephaly (HPE) and holoprosencephaly-like (HPE-L) phenotypes and postaxial polydactyly. Subsequently, anophthalmia plus orbital anomalies, heminasal aplasia, branchial arch anomalies and polydactyly have also been incorporated into the general phenotype. Here we described six Brazilian patients with phenotypic manifestations that range from isolated cleft lip/palate with polydactyly, branchial arch anomalies to semi-lobar holoprosencephaly. Novel sequence variants were found in the GLI2 gene in patients with marked involvement of the temporomandibular joint (TMJ), a new clinical finding observed with mutations of this gene. Clinical, molecular and genetic aspects are discussed.

Both alpha(1)- and beta(1)-adrenoceptors in the bed nucleus of the stria terminalis are involved in the expression of conditioned contextual fear

BACKGROUND AND PURPOSE The bed nucleus of the stria terminalis (BNST) is a limbic structure that is involved in the expression of conditioned contextual fear. Among the numerous neural inputs to the BNST, noradrenergic synaptic terminals are prominent and some evidence suggests an activation of this noradrenergic neurotransmission in the BNST during aversive situations. Here, we have investigated the involvement of the BNST noradrenergic system in the modulation of behavioural and autonomic responses induced by conditioned contextual fear in rats. EXPERIMENTAL APPROACH Male Wistar rats with cannulae bilaterally implanted into the BNST were submitted to a 10 min conditioning session (6 footshocks, 1.5 ma/ 3 s). Twenty-four hours later freezing and autonomic responses (mean arterial pressure, heart rate and cutaneous temperature) to the conditioning box were measured for 10 min. The adrenoceptor antagonists were administered 10 min before the re-exposure to the aversive context. KEY RESULTS L-propranolol, a non-selective beta-adrenoceptor antagonist, and phentolamine, a non-selective a-adrenoceptor antagonist, reduced both freezing and autonomic responses induced by aversive context. Similar results were observed with CGP20712, a selective beta 1-adrenoceptor antagonist, and WB4101, a selective a1-antagonist, but not with ICI118,551, a selective beta 2-adrenoceptor antagonist or RX821002, a selective a2-antagonist. CONCLUSIONS AND IMPLICATIONS These findings support the idea that noradrenergic neurotransmission in the BNST via a1- and beta 1-adrenoceptors is involved in the expression of conditioned contextual fear.

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of pi opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT2 agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons. (C) 2012 Elsevier Inc. All rights reserved.