Confirmation of the presence of serotonergic cells in the fetal cerebral cortex in cultures and in situ.

BACKGROUND: Early appearance of serotonin in the fetal brain and its effects on brain morphogenesis support its neurotrophic role. OBJECTIVE: To determine the presence of serotonergic cells and the expression of tryptophan-5-hydroxylase (TPH), 5-hydroxytryptamine (5-HT), serotonin transporter (SERT), 5-HT1A receptor and Pet-1 during the development of the cerebral cortex, both in situ and in tissue cultures. MATERIAL AND METHODS: A descriptive, observational study was carried out in pregnant Wistar rats. The presence of the plug was regarded as the beginning of gestation. On days 13, 16 and 17, cesarean sections were performed to obtain the fetuses, and the brains were then immediately dissected to identify the presence of serotonergic cells, TPH, 5-HT, SERT, 5-HT1A and Pet-1 in tissue cultures and in situ by immunostaining detected on a confocal microscope. RESULTS: Serotonergic cells and terminals were observed in the midbrain on day 17 of gestation, and in neopallium cocultures on days 13 and 16. TPH, 5-HT, SERT and Pet-1 immunopositive cells were also observed in the neopallium on day 12 of culture. CONCLUSIONS: The presence of serotonergic cells and other elements of the serotonergic system in the early cerebral cortex was confirmed, which may be transient and participate in cortical maturation processes during brain development.

ANTECEDENTES: La aparición temprana de serotonina en el cerebro fetal y sus efectos en la morfogénesis cerebral apoyan su papel neurotrófico. OBJETIVO: Determinar la presencia de células serotoninérgicas y la expresión de triptófano-5-hidroxilasa (TPH), 5-hidroxitriptamina (5-HT), transportador de serotonina (SERT), receptor 5-HT1A y Pet-1 durante el desarrollo de la corteza cerebral, tanto in situ como en cultivo de tejidos. MATERIAL Y MÉTODOS: Se realizó estudio observacional descriptivo en ratas Wistar preñadas. La presencia del tapón se consideró el inicio de la gestación; en los días 13, 16 y 17 se practicaron cesáreas para obtener los fetos e inmediatamente se disecaron los cerebros para identificar células serotoninérgicas, TPH, 5-HT, SERT, 5-HT1A y Pet-1 en cultivo de tejido e in situ mediante inmunomarcaje detectado en un microscopio confocal. RESULTADOS: Células y terminales serotoninérgicas fueron observadas en el mesencéfalo el día 17 de gestación y en cocultivos de neopalio los días 13 y 16. También se observaron células inmunopositivas a TPH, 5-HT, SERT y Pet-1 en el neopalio en el día 12 del cultivo. CONCLUSIONES: Se confirmó la presencia de células serotoninérgicas y otros elementos del sistema serotoninérgico en la corteza cerebral temprana, la cual puede ser transitoria y participar en los procesos de maduración cortical durante el desarrollo cerebral.

Diabetes mellitus causes changes in the activity and expression of brain tryptophan-5-hydroxylases and in the number of serotonergic neurons, which do not return to normal with insulin treatment.

INTRODUCTION: Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. OBJECTIVES: To determine whether DM-induced changes in brain TPH1 or TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. METHODS: Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expression of TPH1 and TPH2 by West-ern blot, and the number of serotonergic neurons by immunohistochemistry. RESULTS: In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine, and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hy-droxytryptamine, TPH expression, or the number of serotonergic neurons. CONCLUSIONS: DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

INTRODUCCIÓN: La diabetes mellitus (DM) inhibe la biosíntesis de serotonina cerebral mediante cambios en la actividad y expresión de la triptófano-5-hidroxilasa (TPH). OBJETIVOS: Determinar si los cambios en la expresión de TPH1 o TPH2 cerebral y en el número de neuronas serotoninérgicas causados por la DM retornan a la normalidad en las ratas con diabetes tratadas con insulina. MÉTODOS: Ratas con diabetes inducida con estreptozotocina se dividieron en dos grupos: uno tratado con insulina y otro sin tratamiento. En el día 14, se obtuvieron tallos cerebrales para cuantificar niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH. La expresión de TPH1 y TPH2 fue mediante Western blot y el número de neuronas serotoninérgicas por inmu­nohistoquímica. RESULTADOS: En las ratas con diabetes se confirmó disminución de los niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH, así como una menor expresión de TPH1 y 2 y un menor número de neuronas serotoninérgicas. Cuando las ratas diabéticas fueron tratadas con insulina, el L-triptófano regreso a la normalidad, no así la 5-hidroxitriptamina, la expresión de TPH y el número de neuronas serotoninérgicas. CONCLUSIONES: La DM inhibe crónicamente la síntesis de 5-hidroxitriptamina cerebral mediante modificaciones en la expresión de TPH1 y TPH2 y disminución de las neuronas seroto­ninérgicas, que persisten a pesar del tratamiento con insulina.

Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions.

INTRODUCTION: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. OBJECTIVE: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). METHODS: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. RESULTS: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). CONCLUSIONS: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

INTRODUCCIÓN: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. OBJETIVO: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). MÉTODOS: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. RESULTADOS: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). CONCLUSIONES: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Modifications of plasma 5-HT concentrations during the allergic bronchoconstriction in guinea pigs.

Several contractile mediators involved in the antigen-induced airway obstruction have been identified, but the role of 5-HT (5-hydroxytryptamine or serotonin) has been scantily investigated. In this work, the potential role of 5-HT in the allergic bronchoconstriction was evaluated through a pharmacological approach and plasma 5-HT measurement in blood samples from the right and left ventricles of anesthetized guinea-pigs. Intravenous 5-HT caused a dose-dependent increase of the lung resistance in anesthetized, nonsensitized guinea pigs. Likewise, in sensitized animals the antigenic challenge with ovalbumin also caused a transient bronchoconstriction (356 ± 60% the basal value), which was largely inhibited by the blockade of serotonergic receptors with methiothepin plus tropisetron (134 ± 10%, P = .007). Sensitized animals tended to have plasma 5-HT concentrations higher than nonsensitized controls, and shortly after the peak of the allergic bronchoconstriction the 5-HT levels in the left ventricle (blood flowing out from lungs) tended to be higher than in the right ventricle (blood entering the lungs), although data dispersion precluded the obtaining of statistical significance. Interestingly, the degree of bronchoconstriction highly correlated with the concentrations of 5-HT found in the left ventricle and measured either in platelet-rich plasma (r = 0.97 P = .007) or platelet-poor plasma (r = 0.97, P = .006). After the obstructive response subsided these correlations were lost, but now the degree of bronchoconstriction turned to be correlated with 5-HT concentration in platelet concentrate (r = 0.76, P = .03). In conclusion, our results suggested that 5-HT is actively released from lungs during the antigenic challenge and that this autacoid is involved in the generation of the airway obstruction.

Serotonin and dopamine in the hypothalamus of control and malnourished mother rats during pregnancy and lactation and body composition of their offspring.

OBJECTIVE: To determine concentrations of serotonin and dopamine in the hypothalamus of undernourished rats and controls during pregnancy and lactation and body composition of their offspring. METHODS: Malnourished rats along with control rats were used during pregnancy and lactation. At birth of their offspring, control mothers nursed their young and malnourished rats and the undernourished mothers nursed their offspring and control pups. On days 5, 10, 15, and 21 of lactation (at the beginning and end of a feeding), L-tryptophan (L-Trp)-free, bound and total, plasma prolactin (PRL) and milk composition were determined. Serotonin and dopamine were measured in the hypothalamus. Body composition of offspring was determined. RESULTS: Increase of free L-Trp was confirmed in undernourished mothers. Furthermore, hypothalamic serotonin was elevated at the start of suckling and decreased at termination. There was also a decrease in dopamine in the hypothalamus at the beginning and end of suckling followed by an increase of plasma PRL that was greater in control mothers who breastfed malnourished offspring. Interestingly, undernourished offspring consumed more milk and showed a clear recovery of body composition with accumulation of body fat. DISCUSSION: Changes observed in hypothalamic neurotransmitters appear to be closely related to nutritional status and to the response and control of PRL production, possibly to adapt the offspring to the metabolic changes. It was also confirmed that on-demand feeding of undernourished offspring is the main factor involved in nutritional recovery and a predisposition to overweight in the recovered undernourished animals.

Disturbance of serotonergic neurotransmission in patients with postmyocardial infarction and depression.

The objective of this study was to assess the hypothesis that patients who develop depression after myocardial infarction (MI) have a lower level of brain serotonergic neurotransmission through measurement of plasma free fraction of L-tryptophan and intensity-dependence auditory-evoked potentials (IDAEPs). A cross-sectional study was carried out in 74 adults after MI. Thirty-four patients suffered from depression and 40 patients did not demonstrate depressive symptoms. We measured the free fraction, bound and total plasma L-tryptophan, and neutral amino acids as well as recording IDAEPs. Patients who developed depression after MI showed a significantly lower level in the free fraction of L-tryptophan and in the ratios of free fraction of L-tryptophan/total L-tryptophan and free fraction of L-tryptophan/neutral amino acids. It is noteworthy that the slope of the amplitude/stimulus intensity functions (ASF slope) of the N1/P2 component was significantly higher post-MI in depressed patients. Higher ASF slope of the N1/P2 component associated with a low free fraction of L-tryptophan in plasma reflect a low brain serotonergic neurotransmission. These findings suggest an important deterioration of brain serotonergic activity as a pathophysiological mechanism in post-MI patients for the development of clinical depression. Therefore, we propose these biochemical and electrophysiological procedures as noninvasive clinical indicators of brain serotonergic activity in these patients.

Evidence of 5-HT components in human sperm: implications for protein tyrosine phosphorylation and the physiology of motility.

Serotonin (5-hydroxytryptamine; C(10)H(12)N(2)O (5-HT)) is produced in the CNS and in some cells of peripheral tissues. In the mammalian male reproductive system, both 5-HT and tryptophan hydroxylase (TPH) have been described in Leydig cells of the testis and in principal cells of the caput epididymis. In capacitated hamster sperm, it has been shown that 5-HT promotes the acrosomal reaction. The aim of this work was to explore the existence of components of the serotoninergic system and their relevance in human sperm physiology. We used both immunocytochemistry and western blot to detect serotoninergic markers such as 5-HT, TPH1, MAO(A), 5-HT(1B), 5-HT(3), and 5HT(T); HPLC for TPH enzymatic activity; Computer Assisted Semen Analysis assays to measure sperm motility parameters and pharmacological approaches to show the effect of 5-HT in sperm motility and tyrosine phosphorylation was assessed by western blot. We found the presence of serotoninergic markers (5-HT, TPH1, MAO(A), 5-HT(1B), 5-HT(2A), 5-HT(3), 5-HT(T), and TPH enzymatic activity) in human sperm. In addition, we observed a significant increase in tyrosine phosphorylation and changes in sperm motility after 5-HT treatment. In conclusion, our data demonstrate the existence of components of a serotoninergic system in human sperm and support the notion for a functional role of 5-HT in mammalian sperm physiology, which can be modulated pharmacologically.

Triptófano-5-hidroxilasa, transportador de serotonina y receptores serotoninérgicos en miocardiopatía dilatada.

BACKGROUND: Cardiomyocytes synthesize, utilize and reuptake serotonin, which is involved in the paracrine and autocrine modulation of heart activity and in the pathophysiology of some cardiovascular diseases. OBJECTIVE: To determine the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter protein (SERT) and serotonergic receptors in hearts with dilated cardiomyopathy (DCM) compared to controls. METHOD: A comparative study was performed in six tissue blocks of the left ventricular free wall (LVWL) and inter-ventricular septum from patients who died of DCM and six who died of no cardiovascular diseases (controls). Five slices from each block were obtained to determine the expression of TPH1 and TPH2, SERT and serotonergic receptors with antibodies specific for immunofluorescence. Immunofluorescence was analyzed by Student's t-test, accepting a significance level of p < 0.05. RESULTS: An increase in TPH1, TPH2, 5-HT2A and 5-HT2B receptors expression were observed in dilated structures compared to controls (p < 0.05). For dilated inter-ventricular septum, the 5-HT4 receptor increased its expression (p < 0.05), and SERT in PLVI compared to controls (p < 0.05). CONCLUSIONS: These results suggest that the increases observed in the expression of TPH, SERT, and serotonergic receptors in hearts with DCM compared to controls could play an important role in the pathophysiology of MCD in humans.

ANTECEDENTES: Los cardiomiocitos sintetizan, utilizan y recapturan serotonina, la cual participa en la modulación parácrina y autócrina de la actividad del corazón y en la fisiopatología de algunas enfermedades cardiovasculares. OBJETIVO: Determinar la expresión de triptófano-5-hidroxilasa (TPH) 1 y 2, transportador de serotonina (SERT) y receptores serotoninérgicos en corazones con miocardiopatía dilatada (MCD) en comparación con controles. MÉTODO: Estudio comparativo en seis bloques de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular de pacientes fallecidos por MCD y seis que murieron por enfermedades no cardiovasculares. Se obtuvieron cinco cortes de cada bloque para determinar la expresión de TPH1 y TPH2, SERT y receptores serotoninérgicos con anticuerpos específicos por inmunofluorescencia. La inmunofluorescencia fue analizada por la t de Student, aceptando un nivel de significancia de p < 0.05. RESULTADOS: Se observó un aumento en la expresión de TPH1 y TPH2 y en los receptores 5-HT2A y 5-HT2B en las estructuras dilatadas en comparación con las controles (p < 0.05). El receptor 5-HT4 aumentó su expresión en el septum interventricular dilatado (p < 0.05) y el SERT en la PLVI en comparación con los controles (p < 0.05). CONCLUSIONES: Estos resultados sugieren que los aumentos observados en las expresiones de TPH, SERT y receptores serotoninérgicos en corazones con MCD en comparación con controles podrían desempeñar un papel importante en la fisiopatología de la MCD en los humanos.

Association of metabolic syndrome with reduced central serotonergic activity.

The aim of this study was to determine the differences between two groups of adolescents with metabolic syndrome (MetS) and normal controls in relation to brain serotonergic activity through intensity-dependent auditory-evoked potentials (IDAEPs) and plasma free fraction of L-tryptophan. Eighteen adolescents with MetS and thirteen controls were studied. Free fraction, bound and total plasma L-tryptophan, glucose, cholesterol, triglycerides, HDL-cholesterol, albumin and IDAEPs were determined. Glycemia, triglycerides were significantly elevated, and HDL-cholesterol in plasma was significantly reduced. Free fraction and free fraction/total L-tryptophan ratio were decreased. The slope of the amplitude/stimulus intensity function of the N1/P2 component significantly increased in adolescents with MetS. Decrease of free fraction of L-tryptophan in plasma and increase of the slope of the N1/P2 component suggest a low brain serotonin tone. Cortex responses are regulated by serotonergic innervations and may show a different behavior in young patients with MetS. Therefore, the slope of the N1/P2 component along with the free fraction of L-tryptophan in plasma, indicate that in adolescents with MetS the state of serotonergic brain activity is depressed and possibly related to psychiatric disorders.

How intrauterine growth restriction due to nutritional stress changes the function of key proteins in brain serotonin metabolism during development.

This review aimed to describe and comment on how experimental intrauterine nutritional stress in animals produced some changes in tryptophan-5-hydroxylases (TPH) 1 and 2 in the brain and other key proteins such as plasma albumin, and how the intrauterine nutritional stress could produce long-lasting alterations in serotonin function in the brain of human infants.

El objetivo de esta revisión es describir y comentar cómo el estrés nutricional intrauterino experimental en animales produjo algunos cambios en las triptófano-5-hidroxilasas 1 y 2 en el cerebro y en otras proteínas clave, como la albúmina plasmática, y de qué manera el estrés nutricional intrauterino podría producir alteraciones duraderas en la función de la serotonina en el cerebro de lactantes.

Development of 5-HT(1B), SERT and thalamo-cortical afferents in early nutrionally restricted rats: an emerging explanation for delayed barrel formation.

Barrel formation is delayed in nutritionally restricted rats. The underlying cause of such delay is yet unclear. Because barrels appear upon the arrival of somatosensory thalamo-cortical afferents and the reorientation of the dendritic arborizations of cortical spiny stellate neurons, it is likely that at least one of these processes is altered by nutritional restriction. Also, the serotoninergic afferent system has been implicated in regulating barrel segregation and growth during early postnatal life. We then evaluated the pattern of immunostaining of the serotonin transporter (SERT) and of the serotonin receptor 1B (5-HT(1B)), as well as the growth and arrival time of somatosensory thalamo-cortical afferents, to infer the contribution of these elements in the delayed formation of barrels observed in nutritionally restricted rats. It was found that the rates of development and the segregation of thalamo-cortical fibers were normal in nutritionally restricted rats. SERT, but not 5-HT(1B) immunoreactivity, was decreased in the primary somatosensory cortex during barrel specification. The availability of both proteins in nutritionally restricted rats was lower than that observed in their well fed counterparts at later developmental times. It is concluded that the delayed formation of barrels observed in nutritionally restricted rats is due to a retarded reorientation of dendritic arbors of cortical neurons. This might happen as a secondary effect of decreasing the availability of SERT and/or increasing the availability of 5-HT(1B) receptor early in postnatal life.

Confirmación de la presencia de células serotoninérgicas en la corteza cerebral fetal en cultivos e in situ / Confirmation of the presence of serotonergic cells in the fetal cerebral cortex in cultures and in situ

Resumen Antecedentes: La aparición temprana de serotonina en el cerebro fetal y sus efectos en la morfogénesis cerebral apoyan su papel neurotrófico. Objetivo: Determinar la presencia de células serotoninérgicas y la expresión de triptófano-5-hidroxilasa (TPH), 5-hidroxitriptamina (5-HT), transportador de serotonina (SERT), receptor 5-HT1A y Pet-1 durante el desarrollo de la corteza cerebral, tanto in situ como en cultivo de tejidos. Material y métodos: Se realizó estudio observacional descriptivo en ratas Wistar preñadas. La presencia del tapón se consideró el inicio de la gestación; en los días 13, 16 y 17 se practicaron cesáreas para obtener los fetos e inmediatamente se disecaron los cerebros para identificar células serotoninérgicas, TPH, 5-HT, SERT, 5-HT1A y Pet-1 en cultivo de tejido e in situ mediante inmunomarcaje detectado en un microscopio confocal. Resultados: Células y terminales serotoninérgicas fueron observadas en el mesencéfalo el día 17 de gestación y en cocultivos de neopalio los días 13 y 16. También se observaron células inmunopositivas a TPH, 5-HT, SERT y Pet-1 en el neopalio en el día 12 del cultivo. Conclusiones: Se confirmó la presencia de células serotoninérgicas y otros elementos del sistema serotoninérgico en la corteza cerebral temprana, la cual puede ser transitoria y participar en los procesos de maduración cortical durante el desarrollo cerebral.

Abstract Background: Early appearance of serotonin in the fetal brain and its effects on brain morphogenesis support its neurotrophic role. Objective: To determine the presence of serotonergic cells and the expression of tryptophan-5-hydroxylase (TPH), 5-hydroxytryptamine (5-HT), serotonin transporter (SERT), 5-HT1A receptor and Pet-1 during the development of the cerebral cortex, both in situ and in tissue cultures. Material and methods: A descriptive, observational study was carried out in pregnant Wistar rats. The presence of the plug was regarded as the beginning of gestation. On days 13, 16 and 17, cesarean sections were performed to obtain the fetuses, and the brains were then immediately dissected to identify the presence of serotonergic cells, TPH, 5-HT, SERT, 5-HT1A and Pet-1 in tissue cultures and in situ by immunostaining detected on a confocal microscope. Results: Serotonergic cells and terminals were observed in the midbrain on day 17 of gestation, and in neopallium cocultures on days 13 and 16. TPH, 5-HT, SERT and Pet-1 immunopositive cells were also observed in the neopallium on day 12 of culture. Conclusions: The presence of serotonergic cells and other elements of the serotonergic system in the early cerebral cortex was confirmed, which may be transient and participate in cortical maturation processes during brain development.

Serotonin concentration, synthesis, cell origin, and targets in the rat caput epididymis during sexual maturation and variations associated with adult mating status: morphological and biochemical studies.

The caput epididymis of some mammals contains large quantities of serotonin whose origin, targets, and physiological variations have been poorly studied. We combined morphological and biochemical techniques to begin approaching these aspects of serotonin in the rat caput epididymis. Serotonin immunostaining was detected in mast, epithelial, and neuroendocrine cells. Epithelial cells displayed immunoreactivity to 5HT(1A), 5HT(2A,) and 5HT(3) serotonin receptors. Endothelial and mast cells labeled positive for 5HT(1B) serotonin receptors and spermatozoa displayed 5HT(2A) and 5HT(3) serotonin receptor immunoreactivity. Epithelial, endothelial, and mast cells stained positive for serotonin transporters. Only epithelial cells showed tryptophan hydroxylase immunoreactivity; this enzyme catalyzes the limiting step in the serotonin synthetic pathway. In addition, Western blot analyses of caput homogenates documented the presence of 2 protein bands ( approximately 51 kd and approximately 48 kd) that were immunoreactive for tryptophan hydroxylase. Chromatographic analyses documented the presence of tryptophan hydroxylase in the caput, and showed that both its activity and serotonin availability increased with sexual maturation and decreased following p-chlorophenylalanine treatment, an inhibitor of tryptophan hydroxylase activity. Interestingly, serotonin concentration and tryptophan hydroxylase activity tended to be higher in breeding males than in those with no mating experience. We think that these results support the existence of a local serotoninergic system in the rat caput epididymis that might regulate some aspects of male reproductive function.

Cambios en la actividad y expresión de las triptófano-5-hidroxilasas cerebrales y en el número de neuronas serotoninérgicas por la diabetes mellitus, que no retornan a la normalidad con la insulina / Diabetes mellitus causes changes in brain tryptophan-5-hydroxylases activity and expression and in the number of serotonergic neurons, which do not return to normal with insulin treatment

Resumen Introducción: La diabetes mellitus (DM) inhibe la biosíntesis de serotonina cerebral mediante cambios en la actividad y expresión de triptófano-5-hidroxilasa (TPH). Objetivos: Determinar si los cambios en la expresión de TPH1 y TPH2 cerebral y en el número de neuronas serotoninérgicas causados por la DM retornan a la normalidad en ratas con diabetes tratadas con insulina. Métodos: Ratas con diabetes inducida con estreptozotocina se dividieron en dos grupos uno tratado con insulina y otro sin tratamiento. En el día 14, se obtuvieron tallos cerebrales para cuantificar niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH. La expresión de TPH1 y TPH2 fue mediante Western blot y el número de neuronas serotoninérgicas por inmunohistoquímica. Resultados: En las ratas con diabetes se confirmó disminución de los niveles de L-triptófano, 5-hidroxitriptamina y la actividad de la TPH, así como menor expresión de TPH1 y TPH2 y menor número de neuronas serotoninérgicas. Cuando las ratas diabéticas fueron tratadas con insulina, el L-triptófano regresó a la normalidad, no así la 5-hidroxitriptamina, la expresión de TPH ni el número de neuronas serotoninérgicas. Conclusiones: La DM inhibe crónicamente la síntesis de 5-hidroxitriptamina cerebral mediante modificaciones en la expresión de TPH1 y TPH2 y disminución de las neuronas serotoninérgicas, que persisten a pesar del tratamiento con insulina.

Abstract Introduction: Diabetes mellitus (DM) inhibits brain serotonin biosynthesis through changes in tryptophan-5-hydroxylase (TPH) activity and expression. Objectives: To determine whether DM-induced changes in brain TPH1 and TPH2 expression and in the number of serotonergic neurons return to normal in diabetic rats treated with insulin. Methods: Rats with streptozotocin-induced diabetes were divided in two groups: one treated with insulin and the other without treatment. On day 14, brain stems were obtained in order to quantify L-tryptophan and 5-hydroxytryptamine levels, as well as to determine TPH activity. The expressión of TPH1 and THP2 by Western blot, and the number of serotonergic neurons by immunohistochemistry. Results: In diabetic rats, a decrease in the levels of L-tryptophan, 5-hydroxytryptamine and TPH activity was confirmed, as well as lower TPH1 and TPH2 expression and lower numbers of serotonergic neurons. When diabetic rats were treated with insulin, L-tryptophan returned to normal, but not 5-hydroxytryptamine, TPH expression, or the number of serotonergic neurons. Conclusions: DM chronically inhibits the synthesis of brain 5-hydroxytryptamine through changes in TPH1 and TPH2 expression and a decrease in the number of serotonergic neurons, which persist despite insulin treatment.

La miocardiopatía hipertrófica induce cambios en la expresión de la triptófano-5-hidroxilasa, el transportador de serotonina y receptores serotoninérgicos / Hypertrophic cardiomyopathy induces changes in the tryptophan-5-hydroxylase, serotonin transporter and serotonergic receptors expressions

Resumen Introducción: Los cardiomiocitos poseen la maquinaria bioquímica capaz de sintetizar, utilizar y recapturar serotonina. Objetivo: Determinar si la miocardiopatía hipertrófica (MCH) induce cambios en la expresión de la triptófano-5-hidroxilasa (TPH) 1 y 2, el transportador de serotonina (SERT) y los receptores serotoninérgicos (RS). Métodos: Estudio transversal de cinco bloques de tejido de corazones con MCH y cinco bloques de corazones de control. Se obtuvieron cinco cortes de la pared libre del ventrículo izquierdo (PLVI) y del septum interventricular (SIV) de cada bloque, para determinar la expresión de TPH1 y TPH2, SERT y RS con anticuerpos por inmunofluorescencia. La inmunofluorescencia fue evaluada mediante t de WELCH, con nivel de significación de p < 0.05. Resultados: La PLVI y el SIV de los corazones con MCH mostraron aumento de la expresión de TPH1 y TPH2, así como de los receptores 5-HT2A y 5-HT2B en comparación con los controles (p < 0.01). El receptor 5-HT4 y SERT aumentaron en el SIV de los corazones con MCH (p < 0.01). Conclusiones: Se demostró aumento de las expresiones de TPH, SERT y RS en los cardiomiocitos de los corazones con MCH en comparación con los controles, lo cual podría participar en la fisiopatología de la MCH en los humanos.

Abstract Introduction: Cardiomyocytes have a biochemical machinery with the capacity to synthesize, utilize and reuptake serotonin. Objective: To determine whether hypertrophic cardiomyopathy (HCM) induces changes in the expression of tryptophan-5-hydroxylase (TPH) 1 and 2, serotonin transporter (SERT) and serotonergic receptors (SR). Methods: Cross-sectional study of five tissue blocks from hearts with HCM and five controls. Five sections of the left ventricular free wall (LVFW) and interventricular septum (IVS) were obtained from each block to determine the expression of TPH1 and TPH2, SERT and SRs by immunofluorescence with specific antibodies. Immunofluorescence was evaluated by WELCH t-test, with a level of significance of p < 0.05. Results: LVFW and IVS of hearts with HCM showed an increase in the expression of TPH1 and TPH 2 and 5-HT2A and 5-HT2B receptors in comparison with controls (p < 0.01). The 5-HT4 receptor and SERT showed an increase in the IVS of hearts with HCM (p < 0.01). Conclusions: This study demonstrated an increased expression of TPH, SERT and SRs in cardiomyocytes from hearts with HCM in comparison with controls, which could be involved in the pathophysiology of HCM in humans.

Effects of (-)-epicatechin on frontal cortex DAPC and dysbindin of the mdx mice.

INTRODUCTION: Multiple components of the dystrophin-associated protein complex (DAPC) are expressed in numerous tissues including the brain. Members of the DAPC and dysbindin are abnormally expressed in the brain of Duchenne Muscular Dystrophy (DMD) patients, which has been associated with cognitive impairments. However, little is known about the expression pattern of individual members of the DAPC in animal models of DMD and their relationship with dysbindin. METHODS: Ten mdx mice were randomly allocated into a control and intervention group [(-)-epicatechin (Epi) 1mg/kg/day for four weeks] and results compared to a wild-type mice. After sacrifice, brain pre-frontal cortices were collected for Western blotting and immunoprecipitation assays, and sagittal sections processed for immunohistochemistry. RESULTS: Epi promotes a partial recovery of DAPC members [α1-Syntrophin, sarcoglycans (SG), dystrophin 71 (Dp71)], dysbindin, and utrophin protein levels. Epi also appears to restore the association of DAPC between dysbindin, and utrophin with Dp71 and ε-SG. Co-immunostaining evidence increased protein levels of dysbindin, dystrophin, and ε-SG and their colocalization. CONCLUSIONS: Altogether, results suggest that Epi is capable of restoring pre-frontal cortex DAPC and dysbindin levels of mdx mice towards that of healthy brains. The functional implications of such studies warrant further investigation.

Neither increased nor decreased availability of cortical serotonin (5HT) disturbs barrel field formation in isocaloric undernourished rat pups.

Serotonin (5HT) is expressed transiently in primary sensory areas of the rat neocortex during the establishment of the thalamo-cortical topography and somatotopy. The precise role of 5HT during the specification of neocortical areas is still uncertain. We evaluated the effects of increasing and decreasing cortical serotonin concentrations on the specification of the barrel cortex using a rat model of isocaloric undernutrition. This manipulation increases brain 5HT levels during brain development. Undernourished animals were also treated with p-clorophenylalanine; an inhibitor of 5HT synthesis. Barrels representing the head were readily seen at postnatal day 5 in control and p-clorophenylalanine treated rats. In contrast, undernourished rats treated or not with p-clorophenylalanine showed no barrels representing the head but until postnatal day 7. Chromatographic analyses demonstrated that the concentration of cortical 5HT increased by 50% in undernourished pups during barrel field formation. Control and undernourished animals treated with p-clorophenylalanine had a significant reduction (90%) of 5HT in the cortex. The overall geometry of the barrel field and of individual barrels was similar among animal groups. Our results support that 5HT plays a small role in triggering and timing barrel field somatotopy.

[Anatomopathological findings during development of diabetic cardiomyopathy in rats]. / Hallazgos anatomopatológicos durante el desarrollo de la miocardiopatía diabética en la rata.

BACKGROUND: The diabetic cardiomyopathy occurs in both type 1 and type 2 diabetes mellitus. Hyperglycemia and associated metabolic changes participate in the pathogenesis of this disease. OBJECTIVE: To characterizes various pathological changes occurring during the development of diabetic cardiomyopathy in rats. METHODS: Diabetic rats were used for streptozotocin administration. At 7, 14, 21 and 30 days after toxic administration, the heart was obtained and placed in a Hartman solution and 4% p-formaldehyde. Five-micrometer thick sections were stained with hematoxylin-eosin, Masson trichrome and immunocytochemistry using anti-ß-tubulin antibody. RESULTS: At 14 days after application of streptozotocin, dilated sinusoids with endothelial lining in the myocardium and collagen deposits in the cardiac interstitium and between the Purkinje fibers were observed. At 21 days there was a slight decrease of the arteriolar lumen due to hyperplasia of the medial layer. It is important to note that cardiac sinusoids as well as collagen deposits became more evident at 30 day of the study, as well as a major derangement of the microtubular system of the cardiomyocytes. CONCLUSIONS: Cardiac sinusoids representing fetal vascular pattern and interstitial fibrosis in the myocardium and the microtubular derangement of cardiomyocytes support the fact that the pathophysiological mechanism of diabetic cardiomyopathy begins in the coronary microcirculation due to changes in cardiac metabolism, contributing to the development of myocardial dysfunction in diabetes.

Antecedentes: la miocardiopatía diabética ocurre en ambos tipos de diabetes mellitus y en su patogenia intervienen la hiperglucemia y los cambios metabólicos asociados. Objetivo: caracterizar los diferentes cambios patológicos que aparecen durante la evolución de la miocardiopatía diabética en la rata. Material y métodos: estudio transversal comparativo en dos grupos de ratas diabéticas por la administración de estreptozotocina. A los 14, 21 y 30 días de la administración del tóxico se obtuvieron los corazones, que se colocaron en p-formaldehído al 4%. Se efectuaron cortes de 5 µm y se tiñeron con hematoxilina-eosina, tricrómica de Masson e inmunocitoquímica con anticuerpos anti ß-tubulina. Resultados: a los 14 días de la aplicación de la estreptozotocina se observaron en el miocardio sinusoides dilatadas y depósito de colágena entre las fibras de Purkinje e intersticio cardiaco. A los 21 días disminuyó la luz arteriolar por hiperplasia de la capa media. A los 30 días del estudio se hicieron más evidentes los sinusoides cardiacos y los depósitos de colágena y un importante desarreglo del sistema microtubular de los cardiomiocitos. Conclusiones: los sinusoides cardiacos, que representan un patrón vascular fetal y la fibrosis intersticial en el miocardio y el desarreglo microtubular de los cardiomiocitos, apoyan el hecho de que el mecanismo fisiopatológico de la miocardiopatía diabética se inicia en la microcirculación coronaria debido a cambios en el metabolismo cardiaco que contribuyen a la disfunción miocárdica durante el estado diabético.

Increased expression of tryptophan-5-hydroxylase 1, but not 2, in brainstem as a result of intrauterine malnutrition.

The aim of this study was to determine whether intrauterine malnutrition (IUM) produces a change in the expression of tryptophan-5-hydroxylase (TPH) 1 and/or 2 as the primary mechanism to explain the observed chronic cerebral acceleration of the synthesis of 5-hydroxytryptamine (5-HT). We used an IUM model and controls with ages of 1, 15 and 21 days. The brainstem was obtained to determine L-tryptophan, 5-HT and TPH activity. Expression of TPH1 and TPH2 via specific antibodies for each was also evaluated by immunocytochemistry and Western blot. Malnourished offspring had a significant elevation of L-Trp, TPH activity and 5-HT in the brainstem. Both isoforms (1 and 2) of TPH were expressed from birth in both groups; however, TPH1 expression was significantly higher in offspring with IUM in relation to the controls. Importantly, these malnourished offspring showed reduced expression of TPH2 compared to controls. It was confirmed that IUM produces an increase in 5-HT in the brainstem and also showed increased expression of TPH1 at birth, with decreased expression of TPH2. These findings together allow us to propose that chronic elevation of synthesis of 5-HT observed in the brain of the offspring with IUM is probably due to a change in the expression and activity of TPH1 induced from fetal life.

Dihydropteridine reductase activity in the brainstem of intrauterine growth-restricted rats.

The aim of this study was to determine whether intrauterine growth restriction produces an increase of dihydropteridine reductase activity as a compensatory mechanism that maintains the necessary concentration of cofactor, tetrahydrobiopterin, during accelerated brain serotonin biosynthesis. Intrauterine growth-restricted offspring and controls were used. On days 1, 10, 15 and 21 of life, the brainstem was dissected and l-tryptophan, serotonin, tryptophan-5-hydroxylase and dihydropteridine reductase activities were determined. Intrauterine growth-restricted pups showed a significant increase of l-tryptophan, 5-hydroxytryptamine, tryptophan-5-hydroxylase and also dihydropteridine activity in the brainstem in comparison to normal pups. These results confirm that intrauterine growth restriction produces an increase of serotonin biosynthesis in the brainstem. This is accompanied by an increase in dihydropteridine activity that appears to be a compensatory mechanism to maintain sufficient tetrahydrobiopterin for the donation of electrons during the accelerated synthesis of brain serotonin in intrauterine growth-restricted rats.