Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of Nω-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Fine-tuning of defensive behaviors in the dorsal periaqueductal gray by atypical neurotransmitters

This paper presents an up-to-date review of the evidence indicating that atypical neurotransmitters such as nitric oxide (NO) and endocannabinoids (eCBs) play an important role in the regulation of aversive responses in the periaqueductal gray (PAG). Among the results supporting this role, several studies have shown that inhibitors of neuronal NO synthase or cannabinoid receptor type 1 (CB1) receptor agonists cause clear anxiolytic responses when injected into this region. The nitrergic and eCB systems can regulate the activity of classical neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) that control PAG activity. We propose that they exert a ‘fine-tuning’ regulatory control of defensive responses in this area. This control, however, is probably complex, which may explain the usually bell-shaped dose-response curves observed with drugs that act on NO- or CB1-mediated neurotransmission. Even if the mechanisms responsible for this complex interaction are still poorly understood, they are beginning to be recognized. For example, activation of transient receptor potential vanilloid type-1 channel (TRPV1) receptors by anandamide seems to counteract the anxiolytic effects induced by CB1 receptor activation caused by this compound. Further studies, however, are needed to identify other mechanisms responsible for this fine-tuning effect.

NAA and NAAG variation in neuronal activation during visual stimulation

N-acetyl-aspartyl-glutamate (NAAG) and its hydrolysis product N-acetyl-L-aspartate (NAA) are among the most important brain metabolites. NAA is a marker of neuron integrity and viability, while NAAG modulates glutamate release and may have a role in neuroprotection and synaptic plasticity. Investigating on a quantitative basis the role of these metabolites in brain metabolism in vivo by magnetic resonance spectroscopy (MRS) is a major challenge since the main signals of NAA and NAAG largely overlap. This is a preliminary study in which we evaluated NAA and NAAG changes during a visual stimulation experiment using functional MRS. The paradigm used consisted of a rest period (5 min and 20 s), followed by a stimulation period (10 min and 40 s) and another rest period (10 min and 40 s). MRS from 17 healthy subjects were acquired at 3T with TR/TE = 2000/288 ms. Spectra were averaged over subjects and quantified with LCModel. The main outcomes were that NAA concentration decreased by about 20% with the stimulus, while the concentration of NAAG concomitantly increased by about 200%. Such variations fall into models for the energy metabolism underlying neuronal activation that point to NAAG as being responsible for the hyperemic vascular response that causes the BOLD signal. They also agree with the fact that NAAG and NAA are present in the brain at a ratio of about 1:10, and with the fact that the only known metabolic pathway for NAAG synthesis is from NAA and glutamate.

Mapping and signaling of neural pathways involved in the regulation of hydromineral homeostasis

Several forebrain and brainstem neurochemical circuitries interact with peripheral neural and humoral signals to collaboratively maintain both the volume and osmolality of extracellular fluids. Although much progress has been made over the past decades in the understanding of complex mechanisms underlying neuroendocrine control of hydromineral homeostasis, several issues still remain to be clarified. The use of techniques such as molecular biology, neuronal tracing, electrophysiology, immunohistochemistry, and microinfusions has significantly improved our ability to identify neuronal phenotypes and their signals, including those related to neuron-glia interactions. Accordingly, neurons have been shown to produce and release a large number of chemical mediators (neurotransmitters, neurohormones and neuromodulators) into the interstitial space, which include not only classic neurotransmitters, such as acetylcholine, amines (noradrenaline, serotonin) and amino acids (glutamate, GABA), but also gaseous (nitric oxide, carbon monoxide and hydrogen sulfide) and lipid-derived (endocannabinoids) mediators. This efferent response, initiated within the neuronal environment, recruits several peripheral effectors, such as hormones (glucocorticoids, angiotensin II, estrogen), which in turn modulate central nervous system responsiveness to systemic challenges. Therefore, in this review, we shall evaluate in an integrated manner the physiological control of body fluid homeostasis from the molecular aspects to the systemic and integrated responses.

Silibinin improves palmitate-induced insulin resistance in C2C12 myotubes by attenuating IRS-1/PI3K/Akt pathway inhibition

The present study investigated the effect of silibinin, the principal potential anti-inflammatory flavonoid contained in silymarin, a mixture of flavonolignans extracted from Silybum marianum seeds, on palmitate-induced insulin resistance in C2C12 myotubes and its potential molecular mechanisms. Silibinin prevented the decrease of insulin-stimulated 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose) uptake and the downregulation of glutamate transporter type 4 (GLUT4) translocation in C2C12 myotubes induced by palmitate. Meanwhile, silibinin suppressed the palmitate-induced decrease of insulin-stimulated Akt Ser473 phosphorylation, which was reversed by wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase (PI3K). We also found that palmitate downregulated insulin-stimulated Tyr632 phosphorylation of insulin receptor substrate 1 (IRS-1) and up-regulated IRS-1 Ser307 phosphorylation. These effects were rebalanced by silibinin. Considering several serine/threonine kinases reported to phosphorylate IRS-1 at Ser307, treatment with silibinin downregulated the phosphorylation of both c-Jun N-terminal kinase (JNK) and nuclear factor-κB kinase β (IKKβ), which was increased by palmitate in C2C12 myotubes mediating inflammatory status, whereas the phosphorylation of PKC-θ was not significantly modulated by silibinin. Collectively, the results indicated that silibinin prevented inhibition of the IRS-1/PI3K/Akt pathway, thus ameliorating palmitate-induced insulin resistance in C2C12 myotubes.

Genetic purity certificate in seeds of hybrid maize using molecular markers

One of the main features that confer high quality to the seed is its genetic purity, in which one of the major causes of contamination is the self-pollination of the female parent. Up to date, there is no accurate and fast methods for detecting such contamination. Thus, this work was carried out to certify the genetic purity in seeds of hybrid maize using different biochemical and DNA-based markers. Two single-cross hybrids and their parental lines derived from the maize breeding program at UFLA were evaluated by isoenzymatic pattern of alcohol dehydrogenase (ADH), esterase (EST), acid phosphatase (ACP), glutamate-oxaloacetate transaminase (GOT), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphoglucomate dehydrogenase (PGDH), catalase (CAT) and ß-glucosidade (ßGLU) and by microsatellites markers. The enzymatic systems that were able to distinguish the hybrids from their parental line were the catalase, the isocitrate dehydrogenase and the esterase. The esterase showed a Mendelian segregation pattern for UFLA 8/3 hybrid, that enables a safer genetic purity certificate. Microsatellites were able to differentiate the hybrid lines and the respective parental lines. Moreover, this technique was fast, precise and without environment effects. For microsatellites, the amplification pattern was identical when young leaves or seeds were used as DNA source. The possibility of using seeds as DNA source would accelerate and facilitate the role process of the genetic purity analysis.