[Orexin A as mediator in the gut-brain dialogue]. / Orexina A como mediadora en el diálogo intestino-cerebro.

INTRODUCTION: The orexinergic system is one of the chemical mediators that modulate the gut-brain axis, given the involvement of hypothalamic orexin A (OXA) in gastrointestinal motility and secretion, and the presence of OXA in enteroendocrine cells of the intestinal mucosa and in primary afferent neurons of the mesenteric plexus, permitting its participation in gut-brain signaling. AIM: The source of OXA and the signal(s) triggering its peripheral release are not fully understood, and it is not known whether it acts on orexigenic receptors in peripheral tissues to meet physiological or pathological demands. The aim of this review is to address these questions in the light of new data indicating that OXA may have functions in the gut-brain axis that go beyond its participation in energy homeostasis. DEVELOPMENT: OXA in the enteric system protects against systemic and central inflammation, and hypothalamic OXA orchestrates numerous peripheral effects to suppress the systemic inflammatory response. For this reason, OXA may act as an immunomodulator in chronic inflammations or autoimmune diseases. OXA is also involved in the stress response, regulating physiological responses to emotional or stressful stimuli. CONCLUSIONS: OXA exerts anti-inflammatory and gastroprotective effects on the intestinal mucosa; however, it may increase the response to external and/or internal stress in individuals with chronic inflammation, exacerbating the gastrointestinal inflammation. Hence, pharmacologic interventions in the orexinergic system have been proposed to treat diseases in which intestinal hypersensitivity is combined with appetite loss, sleep disturbance, stress, and anxiety.

TITLE: Orexina A como mediadora en el diálogo intestino-cerebro.Introducción. Entre los mediadores químicos que modulan el eje intestino-cerebro debe incluirse el sistema orexinérgico, ya que la orexina A (OXA) hipotalámica interviene en la motilidad y en la secreción gastrointestinal. También está presente en las células enteroendocrinas de la mucosa intestinal y en las neuronas aferentes primarias del plexo mientérico, y puede intervenir en la señalización intestino-cerebro. Objetivo. No se conoce con exactitud la fuente ni la señal que originan la liberación de OXA periférica, ni tampoco si actúa en los receptores orexinérgicos de los tejidos periféricos ante demandas fisiológicas o patológicas. Esta revisión intenta analizar estas cuestiones a la luz de nuevos datos que indican que la OXA en el eje intestino-cerebro puede tener funciones más allá de su participación en la homeostasis energética. Desarrollo. La OXA en el sistema entérico protege de la inflamación sistémica y central, y en el hipotálamo orquesta numerosos efectos periféricos para suprimir la respuesta inflamatoria sistémica. Por ello, podría actuar como sustancia inmunomoduladora en inflamaciones crónicas o en enfermedades autoinmunitarias. La OXA también se relaciona con la respuesta de estrés, regulando las respuestas fisiológicas a estímulos emocionales o estresantes. Conclusiones. Aunque la OXA tiene efectos antiinflamatorios y gastroprotectores de la mucosa intestinal, en procesos de inflamación crónica podría incrementar la respuesta a estímulos estresantes, tanto externos como internos, y exacerbar la inflamación gastrointestinal. Por ello, se han propuesto intervenciones farmacológicas sobre el sistema orexinérgico como tratamiento para enfermedades en las que la hipersensibilidad intestinal coexiste con pérdida de apetito, alteraciones del sueño, estrés y ansiedad.

Evaluation of the foetal time to death in mice after application of direct and indirect euthanasia methods.

Directive 2010/63/EU on the protection of animals used for scientific purposes requires that the killing of mammal foetuses during the last third of their gestational period should be accomplished through effective and humane methods. The fact that murine foetuses are resistant to hypoxia-mediated euthanasia renders the current euthanasia methods ineffective or humane for the foetuses when these methods are applied to pregnant female mice. We have assessed the time to death of foetuses after performing either indirect (dam euthanasia) or direct (via intraplacental injection--a new approach to euthanasia) euthanasia methods in order to determine a euthanasia method that is appropriate, ethical and efficient for the killing of mouse foetuses. The respective times to death of foetuses after performing the three most commonly used euthanasia methods (namely cervical dislocation, CO2inhalation and intraperitoneal sodium pentobarbital administration) were recorded. Absence of foetal heartbeat was monitored via ultrasound. We consider that the most effective and humane method of foetal euthanasia was the one able to achieve foetal death within the shortest possible period of time. Among the indirect euthanasia methods assessed, the administration of a sodium pentobarbital overdose to pregnant female mice was found to be the fastest for foetuses, with an average post-treatment foetal death of approximately 29.8 min. As for the direct euthanasia method assessed, foetal time to death after intraplacental injection of sodium pentobarbital was approximately 14 min. Significant differences among the different mouse strains employed were found. Based on the results obtained in our study, we consider that the administration of a sodium pentobarbital overdose by intraplacental injection to be an effective euthanasia method for murine foetuses.

Modeling high pressure reactivity in unsaturated systems: application to dimethylacetylene.

A general model is introduced to study pressure-induced reactivity on unsaturated systems in the condensed state. The model is applied here to dimethylacetylene (DMA) in the solid phase II (C/2m) because it has been proposed that two DMA molecules can react to form tetramethyl-cyclobutadiene (TMCBD). The proposed reaction process has been modeled by studying the structural and electronic changes undergone by two DMA molecules as they approach each other preserving the crystal symmetry of phase II. Both monodeterminantal (MP2 and DFT) and multideterminantal (CASSCF and MRMP2) methodologies were used to check the reliability of our model in predicting the reactivity of the system under compression. In all cases, structural results are in agreement with low-temperature diffraction experiments for the solid phase II. Our model indicates that DMA is expected to form the TMCBD dimer at intermolecular distances close to 2 A. This value is in excellent agreement with previous calculations on the existence of long carbon-carbon bonds.

Effect of growth temperature and pH on the aminopeptidase activity of Pseudomonas putida, P. fluorescens and Flavobacterium odoratum; the 4-nitroaniline test is reliable.

No significant difference (p > 0.05) was observed in the specific aminopeptidase activity (SAA) developed by Pseudomonas fluorescens, P. putida and Flavobacterium odoratum either growing at pH 5.0-6.5 or at 7 and 12 degrees C. Nevertheless, a significant difference was found when comparing the SAA of these organisms. The SAA of F. odoratum was lower than those of pseudomonads. The 4-nitroaniline test is reliable to estimate the G(-) load of fresh food products.

Effects of a flavor-placement reversal test after different modalities of taste aversion learning.

Taste aversion learning is induced through two different behavioral procedures: a short-term or concurrent (two-daily flavors) and a long-term or sequential (one-daily flavor) procedure. For the concurrent group of animals, two gustatory/olfactory stimuli are presented separately but at the same time on a daily basis. One is paired with simultaneous intragastric administration of hypertonic NaCl and the other with physiological saline. For the sequential group, the two stimuli are presented on alternate days, one of them followed by intragastric injection of the aversive stimulus and the other by saline, both after a delay of 15 min. The two groups learned the task, but when they were subjected to a flavor-placement reversal test only the sequential group was successful in achieving it. In a second experiment, three groups of animals had to learn concurrent or sequential discrimination tasks (with either simultaneous or delayed administration of the visceral stimulus) using only spatial/proprioceptive cues. The data show that none of the groups learned them under these conditions. The results are discussed in terms of the different modalities of learning. Short-term and long-term taste aversion learning are different in the anatomical structures involved, the number of trials required for acquisition and, as shown in this paper, flexibility.

Aversiones gustativo-nutritivas desarrolladas en situaciones experimentales y clínicas: características y mecanismos biológicos implicado / Food aversions in clinical arad experimental context: characteristic and biological mechanisms involved

El rechazo de los alimentos que se produce tras experiencias que implican malestar gastrointestinal (náuseas, vómitos, etc.) es un fenómeno biológicamente predispuesto que se observa frecuentemente tanto en la investigación animal como en nuestra propia vida diaria. Pero la adquisición de estas aversiones suele producirse y puede adoptar tintes dramáticos en el caso de pacientes oncológicos que sufren frecuentes vómitos y náuseas como consecuencia del tratamiento con quimioterapia o radioterapia. La calidad de vida de estas personas, ya de por sí comprometida por la propia enfermedad, puede verse seriamente mermada. En los últimos años se han realizado grandes esfuerzos por minimizar los efectos colaterales de esos tratamientos pero sus resultados no siempre han sido suficientemente eficaces. Parece necesario desarrollar nuevas líneas de investigación que proporcionen alternativas profilácticas factibles de ser llevadas a la práctica por su sencillez y por su inocuidad. La aplicación de los conocimientos y de los principios que rigen la formación de las aversiones gustativas aprendidas podía ser una de ellas. En este trabajo se analizan las características de estas aversiones gustativas/nutritivas entre las que destacan su rápida adquisición, su especificidad estimular y una amplia demora interestimular. Se examinan igualmente alguno de los sistemas neuroquímicos y anatómicos implicados en este proceso adquisitivo. Finalmente, se revisan algunas de las estrategias farmacológicas y conductuales derivadas de la investigación básica y encaminadas a la prevención del desarrollo de estas perturbadoras aversiones nutritivas El rechazo de los alimentos que se produce tras experiencias que implican malestar gastrointestinal (náuseas, vómitos, etc.) es un fenómeno biológicamente predispuesto que se observa frecuentemente tanto en la investigación animal como en nuestra propia vida diaria. Pero la adquisición de estas aversiones suele producirse y puede adoptar tintes dramáticos en el caso de pacientes oncológicos que sufren frecuentes vómitos y náuseas como consecuencia del tratamiento con quimioterapia o radioterapia. La calidad de vida de estas personas, ya de por sí comprometida por la propia enfermedad, puede verse seriamente mermada. En los últimos años se han realizado grandes esfuerzos por minimizar los efectos colaterales de esos tratamientos pero sus resultados no siempre han sido suficientemente eficaces. Parece necesario desarrollar nuevas líneas de investigación que proporcionen alternativas profilácticas factibles de ser llevadas a la práctica por su sencillez y por su inocuidad. La aplicación de los conocimientos y de los principios que rigen la formación de las aversiones gustativas aprendidas podía ser una de ellas. En este trabajo se analizan las características de estas aversiones gustativas/nutritivas entre las que destacan su rápida adquisición, su especificidad estimular y una amplia demora interestimular. Se examinan igualmente alguno de los sistemas neuroquímicos y anatómicos implicados en este proceso adquisitivo. Finalmente, se revisan algunas de las estrategias farmacológicas y conductuales derivadas de la investigación básica y encaminadas a la prevención del desarrollo de estas perturbadoras aversiones nutritivas (AU)

The role of the lateral parabrachial nuclei in concurrent and sequential taste aversion learning in rats.

The purpose of this study was to examine the role of the external lateral parabrachial subnucleus (PBNLe) in two different taste aversion learning (TAL) procedures. For the first, short-term (concurrent) TAL, two different-flavored stimuli were presented at the same time, one associated with simultaneous intragastric administration of an aversive product, hypertonic NaCl, and the other with saline. In the second, long-term (sequential/delayed) TAL, each gustatory stimulus was presented every other day and the intragastric products LiCl and saline were administered after a 15-min delay. Electrolytic lesions in the PBNLe blocked acquisition of concurrent TAL, in which the vagal visceral information is critical. But the same lesions failed to interrupt sequential TAL. This result was independent of the order in which the two tasks (concurrent and sequential) were presented. However, as found by other authors, the latter type of learning was impaired in the presence of larger lesions in this same area. This supports the existence of sensory information needed to establish sequential TAL in other subnuclei of the parabrachial complex. The results of these experiments suggest that the different modalities of TAL are anatomically specific.

Electrolytic lesions of the pedunculopontine nucleus disrupt concurrent learned aversion induced by NaCl.

Bilateral electrolytic lesions in the pedunculopontine nucleus (PPN) impair acquisition of short-term, or concurrent, Taste Aversion Learning (TAL) in rats. This type of TAL is characterized by the daily presentation of two different flavor stimuli at the same time, one associated with simultaneous intragastric administration of an aversive product (hypertonic NaCl) and the other with physiological saline. Sham-lesioned control animals learn this taste discrimination task, but both lesioned animals and control animals learn a long-term, or delayed, TAL task in which each gustatory stimulus is presented individually every other day and the intragastric products, LiCl (0.15 M) and physiological saline, are administered after a 15-min delay. These results are analyzed in the context of the cerebellar circuits involved in learning and in relation to the two TAL modalities described above.

Ethanol-induced c-fos expression in catecholamine- and neuropeptide Y-producing neurons in rat brainstem.

BACKGROUND: Previous studies have used c-Fos-like immunoreactivity (cFLI) to examine the neuroanatomical location of cells that are activated in response to ethanol administration. However, the use of cFLI alone fails to reveal the phenotypical identity of cells. In the present study we used double-labeling procedures to identify the neurochemical phenotype of neurons that showed ethanol-induced cFLI in the rat brainstem. METHODS: Individual groups of rats received intraperitoneal injection of ethanol (1.5 g/kg or 3.5 g/kg) or isotonic saline (23 ml/kg). To assess the specificity of cFLI induced by ethanol, we injected other rats with the drug lithium chloride (LiCl; 76 mg/kg). Two hours after injection, rats were killed and their brains were processed for immunohistochemistry. RESULTS: Both doses of ethanol promoted cFLI in several brainstem regions, including the nucleus of the solitary tract (NTS), the locus coeruleus (LC), and the ventrolateral medulla (VLM). Although LiCl caused significant cFLI in the NTS, this drug promoted only minimal cFLI in the VLM and no significant activation in the LC. We found that a significant proportion of tyrosine hydroxylase (TH)-positive neurons coexpressed ethanol-induced cFLI in the VLM (approximately 75-85%), the NTS (approximately 65-75%), and the LC (approximately 30-65%). Additionally, a significant proportion of neuropeptide Y (NPY)-producing neurons in the VLM coexpressed ethanol-induced cFLI (approximately 60-75%). On the other hand, LiCl promoted activation of TH-positive neurons in the VLM and the NTS but failed to stimulate cFLI in TH-producing neurons in the LC or in NPY-producing neurons of the VLM. CONCLUSIONS: Neurons in the rat brainstem that show ethanol-induced c-Fos expression produce catecholamines and NPY. This research demonstrates the usefulness of double-labeling immunohistochemistry procedures for identifying the neurochemical identity of neurons that are activated after ethanol administration.

[Validation of half-night polysomnography for sleep apnea/hypoapnea syndrome]. / Validación de estudios polisomnográficos de mitad de la noche en el síndrome de apneas/hipoapneas durante el sueño.

UNLABELLED: A diagnosis of sleep apnea/hypopnea syndrome (SAHS) is based on clinical signs and nighttime polysomnograms. Brief polysomnography has been proposed as an alternative to all-night recording. OBJECTIVES: The aim of this study was to determine whether a polysomnograms obtained during the first half of the night is sufficient for establishing a diagnosis of SAHS and to determine the correlation between polysomnographic variables recorded during the first four hours (half the study time) with those recorded over the full eight hours (full study time), as well as to determine diagnostic agreement. DESIGN: Thirty-five patients suspected of having SAHS were studied prospectively. Baseline polysomnograms were scored blindly by two independent observers following standard methods. A diagnosis of SAHS was made according to guidelines of the Spanish Society of Pneumology and Chest Surgery. During the first half of the night and up to the end of each recording period we gathered neurophysiological and respiratory variables and diagnostic impressions. RESULTS: The correlation between variables (sleep stage, overall AHI, REM-AHI, non-REM-AHI and sleep efficiency) recorded in the first half of the night and throughout the night was significant (p < 0.05) by both Pearson's correlation coefficient (r) and by the intraclass correlation coefficient (ICC). In 33 of 35 patients (94.3%) diagnostic agreement was achieved (95% CI 80.84-99.30); when SAHS was severe, agreement was 100%. CONCLUSION: Based on these results, we conclude that for patients with a diagnosis of severe SAHS during the first half of the night, data recorded during the second half can be considered supplementary.

Retention of concurrent taste aversion learning after electrolytic lesioning of the interpositus-dentate region of the cerebellum.

Lesions in the interpositus-dentate region of the cerebellum impair short-term, or concurrent, TAL. In this type of learning, animals must discriminate between two flavor stimuli presented at the same time, one of which is associated with an aversive product. The task is learned by the control animals, and within this group the animals that acquire it adequately enough (15/22, 70% criterion) retain the learned taste discrimination when they are subjected to it again after being lesioned in the interpositus-dentate region. These results suggest that the deep nuclei are essential in the concurrent TAL acquisition process, but not in its retention.

Validación de estudios polisomnográficos de mitad de la noche en el síndrome de apneas/hipoapneas durante el sueño / Validation of half-night polysomnograms for sleep apnea syndrome

El diagnóstico del síndrome de apneas/hipoapneas del sueño (SAHS), se establece en función de manifestaciones clínicas y registros polisomnográficos (PSG) nocturnos. Como alternativa a la PSG nocturna completa, se han propuesto estudios nocturnos abreviados. Objetivos: Determinar si la corrección de la PSG de la primera mitad de la noche es suficiente para establecer el diagnóstico de SAHS y analizar la correlación existente entre las variables polisomnográficas de los registros de 4 h (mitad del estudio) frente a los de ocho horas de duración (estudio completo), así como determinar la coincidencia diagnóstica. Diseño: Se estudian de forma prospectiva 35 pacientes con sospecha clínica de SAHS, a los que se realiza un estudio polisomnográfico basal nocturno, corregido de forma ciega por dos observadores diferentes, según los métodos estándar. El diagnóstico de SAHS, se realiza según la normativa de la Sociedad Española de Neumología y Cirugía Torácica (SEPAR). En la primera mitad y al final del estudio PSG se recogen variables neurofisiológicas, respiratorias y la impresión diagnóstica. Resultados: La correlación encontrada entre las diferentes variables consideradas (estadios de sueño, índice de apnea/hipoapnea [IAH] global, IAH REM, IAH no REM y eficiencia del sueño) entre la mitad (MN) y el estudio PSG completo (TN) es significativa para un valor de p < 0,05, tanto al aplicar el coeficiente de correlación de Pearson (r) como al aplicar el coeficiente de correlación intraclase (CCI). La coincidencia diagnóstica se produjo en 33 de los 35 pacientes estudiados (94,3 por ciento; intervalo de confianza [IC] del 95 por ciento: 80,84-99,30), siendo del 100 por ciento en el caso de los SAHS severos. Conclusión: Sobre la base de los resultados obtenidos, concluimos que los pacientes con diagnóstico de SAHS severo en la primera mitad de la noche son subsidiarios de estudios de titulación en la segunda mitad de la noche. (AU)

Inferior olive lesions impair concurrent taste aversion learning in rats.

Taste aversion learning can be established according to two different procedures, concurrent and sequential. For the concurrent task, two different taste stimuli are offered at the same time, one associated with simultaneous intragastric administration of an aversive stimulus and the other associated with physiological saline. This discrimination is learned by sham-lesioned control animals and by animals with lesions in the cerebellar cortex but not by rats lesioned in the inferior olive. At the same time, animals with lesions in the inferior olive and sham-lesioned animals achieve sequential learning when the gustatory stimuli are offered individually during each daily session. The results obtained show that electrolytic lesions in the inferior olive impair acquisition of concurrent learning and are analyzed in terms of an anatomical system consisting of the vagus nerve, inferior olive, and cerebellum, which differentiates between the two modalities of taste aversion learning, concurrent and sequential.

Bilateral lesions in the cerebellar interpositus-dentate region impair taste aversion learning in rats.

Taste Aversion Learning (TAL) has been induced through two different behavioral procedures: a short-term o concurrent (two-daily flavors) and a long-term (one-daily flavor) procedure. For the first, two gustatory/olfactory stimuli are presented separately but at the same time on a daily basis. One of the flavors is paired with simultaneous intragastric administration of hypertonic NaCl and the other is paired with physiological saline. In the long-term procedure, the two stimuli are presented on alternate days, one of them followed by intragastric injection of the aversive stimulus, and the other by saline. The subjects for both types of tests were animals that had been lesioned in the interpositus-dentate region of the cerebellum. The experiments show that the lesions disrupt short-term TAL, but have no effect on long-term TAL. The results are discussed in terms of the role of the cerebellum in relation to TAL and the different anatomical substrates of both learning modalities.

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Thyroid hormones regulate the onset of osmotic activity of rat liver mitochondria after birth.

The effect of thyroid hormone deprivation on the osmotic activity of liver mitochondria from early newborn rats was studied. Experimentally induced hypothyroidism prevented the increase in the osmotic activity of mitochondria observed immediately after birth. Osmotic activity was restored by T4 and T3 treatment to hypothyroid newborns but not when this treatment was supplemented with cycloheximide. Under the same circumstances, streptomycin had no effect. Hypothyroidism abolished the change in the slope of the osmotic curve (plot of inverse absorbance of mitochondrial suspensions incubated in sucrose solutions vs. inverse sucrose concentration) observed in mitochondria from euthyroid newborns at 110-120 mOsm sucrose, suggesting that hypothyroidism prevents the formation of tight physical connections between mitochondrial outer and inner membranes. Thyroid hormone deprivation increased the passive permeability of the mitochondrial inner membrane to protons, resulting in a decreased respiratory control ratio. Hypothyroidism prevented the sharp decrease in the affinity of mitochondria for ATP observed in euthyroid newborns immediately after birth. These results corroborate our previous suggestion (Endocrinology, 1995, 136:4448) that, during the early neonatal period, thyroid hormones control the synthesis of some nucleus-coded protein(s) involved in the assembly of F0,F1-ATPase.

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline.

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.