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We used β-endorphin-deficient mice as a novel approach to confirm the physiological role that opioid peptides play in the development or regulation of the immune system. We found that mice lacking β-endorphin possessed an enhanced immune response, measured in terms of splenocyte proliferation and interleukin (IL)-2 mRNA levels, in vitro production of the splenic macrophage inflammatory cytokines IL-6 and Tumor Necrosis Factor (TNF)-α and plasma IL-6 following lipopolysaccharide (LPS) administration. β-Endorphin-deficient mice had attenuated increases of plasma ACTH and corticosterone levels in response to LPS. These results are consistent with a postulated inhibitory role of endogenous β-endorphin on the immune system at multiple levels. Fil: Refojo, Damian. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Kovalovsky, Damian. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina Fil: Young, Juan I.. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania Fil: Reul, Johannes M. H. M.. Max Planck Institute of Psychiatry; Alemania Fil: Low, Malcolm J.. Oregon Health and Science University; Estados Unidos Fil: Arzt, Eduardo Simon. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular. Laboratorio de Fisiología y Biología Molecular; Argentina

Immune system activation is often accompanied by alterations in the reproductive axis. Interleukin-1 (IL-1), a polypeptide cytokine, has been postulated as a chemical messenger between the immune and the neuroendocrine systems. Using superfused hypothalamic fragments explanted from intact male rats, we evaluated the effects of IL-1 (0.5 and 5 nM) on basal and N-methyl-D-aspartate (NMDA)-stimulated release of gonadotropin-releasing hormone (GnRH), and the associated modifications in the output of inhibitory amino acid neurotransmitters involved in the control of GnRH secretion. IL-1 did not modify basal GnRH release, but markedly restrained the stimulatory effect of NMDA on GnRH secretion. γ-Aminobutyric acid, glycine and taurine concentrations significantly increased in the superfusion medium only after pretreatment with the higher dose of IL-1 (p < 0.05). Our results indicate that this cytokine inhibits NMDA- stimulated GnRH release, affecting the activity and/or the release of hypothalamic excitatory and inhibitory amino acid neurotransmitters participating in the regulation of GnRH secretion.

Insulin-induced hypoglycemia (IIH), as with many other acute stressors, restrains the activity of the reproductive axis, reducing luteinizing hormone (LH) release. In adult ovariectomized, steroid-primed rats, we investigated the effect of IIH and of mediobasal intrahypothalamic perfusion with glucose (200 mg/dl) on pulsatile LH secretion. IIH led to a significant decrease in all pulsatility parameters studied using PC-Pulsar analysis, e.g. pulse amplitude and frequency, maximum and baseline LH levels (p < 0.05 versus control), and LH overall mean release (p < 0.01 versus control). Intrahypothalamic perfusion with glucose normalized LH pulse frequency, improved maximum and baseline levels, and partially ameliorated LH pulse amplitude and overall mean release. Thus, our results show that the glucoprivic cessation of LH release is restored, at least partially, by an adequate glucose supply to the hypothalamus; it is proposed, in view of these and previous results, that different mechanisms in the CNS may be involved in LH suppression observed during IIH.

Interleukin-1 (IL-1), a polypeptide cytokine, has been postulated as a chemical messenger between the immune and the neuroendocrine system. IL-1 receptors and immunopositive neurons have been visualized in the human and rat hypothalamus, suggesting that IL-1 can act as a neurotransmitter within the brain. In the hypothalamus IL-1 and the amino acid neurotransmitters are known to modulate several functions, such as fever, anorexia and the gonadal and adrenal axis. Since the hypothalamic actions of IL-1 on the amino acid neurotransmitter output are unknown, the aim of the present paper was to evaluate the effects of IL-1 on the hypothalamic release of both, the inhibitory taurine, glycine and GABA and the excitatory glutamate, amino acid neurotransmitters. Intact adult male rats were employed. The preoptic/mediobasal hypothalamic area was dissected and superfused with Earle’s balanced salt solution. Superfusate fractions were collected after a 60-min stabilization period. Following 60 min of basal release, IL-1 was added to the superfusion medium over 30 min. GABA, taurine and glycine release were significantly (p < 0.05) increased in the superfusion medium, while glutamate was not modified compared with the control group. These observations show that IL-1 increased GABA, taurine and glycine release. These effects indicate that this cytokine can affect the hypothalamic inhibitory amino acid output, which may help us to understand the mechanism by which IL-1 exerts its effects.

RNA interference (RNAi) can be induced in vitro either by application of synthetic short interfering RNAs (siRNAs), or by intracellular expression of siRNAs or short hairpin RNAs (shRNAs) from transfected vectors. The most widely used promoters for siRNA/shRNA expression are based on polymerase III (Pol III)-dependent transcription. We developed an alternative vector for siRNA/shRNA expression, using a mouse RNA polymerase I (Pol I) promoter. Pol I-dependent transcription serves in cells for production of ribosomal RNA (rRNA), and as such, is ubiquitously and stably active in different cell types. As Pol I-dependent transcription is highly species-specific, Pol I-based system provides an important biosafety advantage with respect to silencing of genes with unknown functions.