Role of N-Methyl-D-Aspartate and Non-N-Methyl-D-Aspartate Receptors in the Cardiovascular Effects of L-Glutamate Microinjection Into the Hypothalamic Paraventricular Nucleus of Unanesthetized Rats

We report on the cardiovascular effects of L-glutamate (L-glu) microinjection into the hypothalamic paraventricular nucleus (PVN) as well as the mechanisms involved in their mediation. L-glu microinjection into the PVN caused dose-related pressor and tachycardiac responses in unanesthetized rats. These responses were blocked by intravenous (i.v.) pretreatment with the ganglion blocker pentolinium (PE; 5 mg/kg), suggesting sympathetic mediation. Responses to L-glu were not affected by local microinjection of the selective non-NMDA receptor antagonist NBQX (2 nmol) or by local microinjection of the selective NMDA receptor antagonist LY235959 (LY; 2 nmol). However, the tachycardiac response was changed to a bradycardiac response after treatment with LY235959, suggesting that NMDA receptors are involved in the L-glu heart rate response. Local pretreatment with LY235959 associated with systemic PE or dTyr(CH(2))(5)(Me)AVP (50 mu g/kg) respectively potentiated or blocked the response to L-glu, suggesting that L-glu responses observed after LY235959 are vasopressin mediated. The increased pressor and bradycardiac responses observed after LY + PE was blocked by subsequent i.v. treatment with the V(1)-vasopressin receptor antagonist dTyr(CH(2))(5)(Me)AVP, suggesting vasopressin mediation. The pressor and bradycardiac response to L-glu microinjection into the PVN observed in animals pretreated with LY + PE was progressively inhibited and even blocked by additional pretreatment with increasing doses of NBQX (2, 10, and 20 nmol) microinjected into the PVN, suggesting its mediation by local non-NMDA receptors. In conclusion, results suggest the existence of two glutamatergic pressor pathways in the PVN: one sympathetic pathway that is mediated by NMDA receptors and a vasopressinergic pathway that is mediated by non-NMDA receptors. (C) 2009 Wiley-Liss, Inc.

Cyhalothrin increased c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus in rats, and suppressed macrophage activity in an adrenal-dependent fashion

Synthetic type II pyrethroid insecticides, such as cyhalothrin at certain dosage levels, simultaneously induce stress-like symptoms and innate immunosuppressive effects in laboratory animals. The present study was designed to further analyze the stress-like effects induced by cyhalotrin and also investigate the role of Hypothalamus-Hypophysis-Adrenal (HHA) axis and Sympathetic Nervous Systems (SNS) and their effects on macrophage activity of rats. Results showed that cyhalothrin treatment (3.0 mg/kg/day. for 7 days) increased corticosterone serum levels and c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus (PVN) but induced no changes in c-fos expression at the basolateral amygdala (BLA). Both areas were related to HHA axis and SNS activations by stress. Further analysis showed that adrenalectomy partially abrogated the suppression effects of cyhalothrin on macrophage activity and that 6-OHDA-induced peripheral symphatectyomy had no effects on this innate immune cell activity. The present observed data support and reinforce the notion that cyhalotrin at this treatment schedule induces stress-like symptoms and suggest that other factors, beyond indirect neuroadaptative responses, are necessary for the suppression effects of insecticide on innate immune response. (C) 2008 Elsevier B.V. All rights reserved.

Activity of Hypothalamic Dopaminergic Neurones During the Day of Oestrus: Involvement in Prolactin Secretion

A secretory surge of prolactin occurs on the afternoon of oestrus in cycling rats. Pituitary prolactin is inhibited by dopamine. We evaluated the activity of the neuroendocrine dopaminergic neurones during oestrus and dioestrus, as determined by dopaminergic activity in the median eminence and neurointermediate lobe of the pituitary, as well as Fos-related antigen expression in tyrosine hydroxylase (TH)-immunoreactive (ir) neurones of the arcuate nucleus (ARC) and periventricular nucleus (Pe). During oestrus, the 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence decreased at 16.00 h, coinciding with the increase in plasma prolactin levels. Similarly, the expression of Fos-related antigen in TH-ir neurones of Pe and rostral-, dorsomedial- and caudal-ARC also decreased at 16.00 h. On dioestrus, 4-dihydroxyphenylacetic acid/dopamine ratio in the median eminence and Fos-related antigen expression in TH-ir neurones of Pe and rostral-ARC decreased at 18.00 h, whereas prolactin levels were unaltered. No variation in dopaminergic activity was found in the neurointermediate lobe of the pituitary on either oestrus or dioestrus. The number of TH-ir neurones in the ARC and parameters of dopaminergic activity were found to be generally lower on oestrus compared to dioestrus. The transitory decrease in the activity of neuroendocrine dopaminergic neurones temporally associated with the prolactin surge on the afternoon of oestrus suggests a role for dopamine in the generation of the oestrous prolactin surge.

Kisspeptin Regulates Prolactin Release through Hypothalamic Dopaminergic Neurons

Prolactin (PRL) is tonically inhibited by dopamine (DA) released from neurons in the arcuate and periventricular nuclei. Kisspeptin plays a pivotal role in LH regulation. In rodents, kisspeptin neurons are found mostly in the anteroventral periventricular and arcuate nuclei, but the physiology of arcuate kisspeptin neurons is not completely understood. We investigated the role of kisspeptin in the control of hypothalamic DA and pituitary PRL secretion in adult rats. Intracerebroventricular kisspeptin-10 (Kp-10) elicited PRL release in a dose-dependent manner in estradiol (E2)-treated ovariectomized rats (OVX+E2), whereas no effect was found in oil-treated ovariectomized rats (OVX). Kp-10 increased PRL release in males and proestrous but not diestrous females. Associated with the increase in PRL release, intracerebroventricular Kp-10 reduced Fos-related antigen expression in tyrosine hydroxylase-immunoreactive (ir) neurons of arcuate and periventricular nuclei in OVX+E2 rats, with no effect in OVX rats. Kp-10 also decreased 3,4-dihydroxyphenylacetic acid concentration and 3,4-dihydroxyphenylacetic acid-DA ratio in the median eminence but not striatum in OVX+E2 rats. Double-label immunofluorescence combined with confocal microscopy revealed kisspeptin-ir fibers in close apposition to and in contact with tyrosine hydroxylase-ir perikarya in the arcuate. In addition, Kp-10 was not found to alter PRL release from anterior pituitary cell cultures regardless of E2 treatment. We provide herein evidence that kisspeptin regulates PRL release through inhibition of hypothalamic dopaminergic neurons, and that this mechanism is E2 dependent in females. These findings suggest a new role for central kisspeptin with possible implications for reproductive physiology. (Endocrinology 151: 3247-3257, 2010)

Chronic supplementation of beta-hydroxy-beta methylbutyrate (HM beta) increases the activity of the GH/IGF-I axis and induces hyperinsulinemia in rats

Objective: Beta-hydroxy-beta-methylbutyrate (HM beta) is a metabolite of leucine widely used for improving sports performance. Although limp is recognized to promote anabolic or anti-catabolic effects on protein metabolism, the impact of its long-term use on skeletal muscle and/or genes that control the skeletal protein balance is not fully known. This study aimed to investigate whether chronic HM beta treatment affects the activity of GH/IGF-I axis and skeletal muscle IGF-I and myostatin mRNA expression. Design: Rats were treated with HK beta (320 mg/kg BW) or vehicle, by gavage, for 4 weeks, and killed by decapitation. Blood was collected for evaluation of serum insulin, glucose and IGF-I concentrations. Samples of pituitary, liver, extensor digitorum longus (EDL) and soleus muscles were collected for total RNA or protein extraction to evaluate the expression of pituitary growth hormone (GH) gene (mRNA and protein), hepatic insulin-like growth factor I (IGF-I) mRNA, skeletal muscle IGF-I and myostatin mRNA by Northern blotting/real time-PCR, or Western blotting. Results: Chronic HM beta treatment increased the content of pituitary GH mRNA and GH, hepatic IGF-I mRNA and serum IGF-I concentration. No changes were detected on skeletal muscle IGF-I and myostatin mRNA expression. However, the HIM-treated rats although normoglycemic, exhibited hyperinsulinemia. Conclusions: The data presented herein extend the body of evidence on the potential role of HM beta-treatment in stimulating GH/IGF-I axis activity. In spite of this effect, HM beta supplementation also induces an apparent insulin resistance state which might limit the beneficial aspects of the former results, at least in rats under normal nutritional status and health conditions. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons

Studies m hum ins and rodents indicate that a minimum amount of stored energy is required for normal pubertal development The adipocyte-derived hormone leptin is a key metabolic signal to the neuroendocrine reproductive axis Humans and mice lacking leptin or the leptin receptor (LepR) (ob/ob and db/db mice, respectively) are infertile and fail to enter puberty Leptin administration to leptin-deficient subjects and ob/ob mice induces puberty and restores fertility, but the exact site or sites of leptin action are unclear Here, we found that genetic deletion of LepR selectively from hypothalamic Kiss1 neurons m mice had no effect on puberty or fertility, indicating that direct leptin signaling m Kiss1 neurons is not required for these processes However, bilateral lesions of the ventral premammillary nucleus (PMV) of ob/ob mice blunted the ability of exogenous leptin to induce sexual maturation Moreover, unilateral reexpression of endogenous LepR m PMV neurons was sufficient to induce puberty and improve fertility m female LepR-null mice This LepR reexpression also normalized the increased hypothalamic GnRH content characteristic of leptin-signaling deficiency These data suggest that the PMV is a key site for leptin's permissive action at the onset of puberty and support the hypothesis that the multiple actions of leptin to control metabolism and reproduction at e anatomically dissociated

Finite Element Analysis and Optimization of a Single-Axis Acoustic Levitator

A finite element analysis and a parametric optimization of single-axis acoustic levitators are presented. The finite element method is used to simulate a levitator consisting of a Langevin ultrasonic transducer with a plane radiating surface and a plane reflector. The transducer electrical impedance, the transducer face displacement, and the acoustic radiation potential that acts on small spheres are determined by the finite element method. The numerical electrical impedance is compared with that acquired experimentally by an impedance analyzer, and the predicted displacement is compared with that obtained by a fiber-optic vibration sensor. The numerical acoustic radiation potential is verified experimentally by placing small spheres in the levitator. The same procedure is used to optimize a levitator consisting of a curved reflector and a concave-faced transducer. The numerical results show that the acoustic radiation force in the new levitator is enhanced 604 times compared with the levitator consisting of a plane transducer and a plane reflector. The optimized levitator is able to levitate 3, 2.5-mm diameter steel spheres with a power consumption of only 0.9 W.

Single Axis Controlled Hybrid Magnetic Bearing for Left Ventricular Assist Device: Hybrid Core and Closed Magnetic Circuit

In previous studies, we presented main strategies for suspending the rotor of a mixed-flow type (centrifugal and axial) ventricular assist device (VAD), originally presented by the Institute Dante Pazzanese of Cardiology (IDPC), Brazil. Magnetic suspension is achieved by the use of a magnetic bearing architecture in which the active control is executed in only one degree of freedom, in the axial direction of the rotor. Remaining degrees of freedom, excepting the rotation, are restricted only by the attraction force between pairs of permanent magnets. This study is part of a joint project in development by IDPC and Escola Politecnica of Sao Paulo University, Brazil. This article shows advances in that project, presenting two promising solutions for magnetic bearings. One solution uses hybrid cores as electromagnetic actuators, that is, cores that combine iron and permanent magnets. The other solution uses actuators, also of hybrid type, but with the magnetic circuit closed by an iron core. After preliminary analysis, a pump prototype has been developed for each solution and has been tested. For each prototype, a brushless DC motor has been developed as the rotor driver. Each solution was evaluated by in vitro experiments and guidelines are extracted for future improvements. Tests have shown good results and demonstrated that one solution is not isolated from the other. One complements the other for the development of a single-axis-controlled, hybrid-type magnetic bearing for a mixed-flow type VAD.

Gabaergic mechanisms of hypothalamic nuclei in the expression of conditioned fear

The amygdala, the dorsal periaqueductal gray (dPAG), and the media] hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN). the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses. (C) 2008 Elsevier Inc. All rights reserved.

Molecular analysis of CYP21A2 can optimize the follow-up of positive results in newborn screening for congenital adrenal hyperplasia

Neonatal screening for congenital adrenal hyperplasia (CAH) is useful in diagnosing salt wasting form (SW). However, there are difficulties in interpreting positive results in asymptomatic newborns. The main objective is to analyze genotyping as a confirmatory test in children with neonatal positive results. Patients comprised 23 CAH children and 19 asymptomatic infants with persistently elevated 17-hydroxyprogesterone (17OHP) levels. CYP21A2 gene was sequenced and genotypes were grouped according to the enzymatic activity of the less severe allele: A1 null, A2 < 2%, B 3-7%, C > 20%. Twenty-one children with neonatal symptoms and/or 17OHP levels > 80 ng/ml carried A genotypes, except two virilized girls (17OHP < 50 ng/ml) without CAH genotypes. Patients carrying SW genotypes (A1, A2) and low serum sodium levels presented with neonatal 17OHP > 200 ng/ml. Three asymptomatic boys carried simple virilizing genotypes (A2 and B): in two, the symptoms began at 18 months; another two asymptomatic boys had nonclassical genotypes (C). The remaining 14 patients did not present CAH genotypes, and their 17OHP levels were normalized by 14 months of age. Molecular analysis is useful as a confirmatory test of CAH, mainly in boys. It can predict clinical course, identify false-positives and help distinguish between clinical forms of CAH.

Stereotactic disconnection of hypothalamic hamartoma to control seizure and behavior disturbance: case report and literature review

An 18-year-old boy with refractory epilepsy and aggressiveness associated to a hypothalamic hamartoma was submitted to a stereotactically guided lesion by thermocoagulation. The target was based on magnetic resonance (MR) images merged with computed tomography scan images taken on the day of surgery while patient was on a stereotactic frame. In order to reveal structures not discernible in MR images, the Schaltenbrand digital brain atlas was merged onto the patient`s images. Target and trajectory of the depth electrode were chosen based on three-dimensional imaging reconstructions. A surgical plan was devised to disconnect the hypothalamic hamartoma from the hypothalamus, medial forebrain bundle, fasciculus princeps, and dorsal longitudinal fasciculus. Our target was placed at the inferior portion of the posterolateral component of the hamartoma, bordering the normal hypothalamus. The patient evolved with marked lessening of aggressiveness. Seizure frequency was reduced from several seizures per day to less than one tonic-clonic seizure during sleep per month and only two episodes suggestive of partial complex seizures during daytime. These results have remained consistent over a 24-month postoperative follow-up. Functional neuroanatomy of hypothalamic connections involved in seizure propagation and aggressive behavior was reviewed.

The actual incidence of congenital adrenal hyperplasia in Brazil may not be as high as inferred - An estimate based on a public neonatal screening program in the state of Goias

The incidence of 21-hydroxylase deficiency (CYP21 D) congenital adrenal hyperplasia (CAH) in Brazil is purportedly one of the highest in the world (1:7,533). However, this information is not based on official data. The aim of this study was to determine the incidence of CYP21 D CAH in the state of Goias, Brazil, based on the 2005 results of government-funded mandatory screening. Of the live births during this period, 92.95% were screened by heel-prick capillary 17 alpha-hydroxyprogesterone (17-OHP). Of these, 82,343 were normal, 28 were at high risk for CAH and 232 at low risk for CAH. Eight cases, all from the high risk group, were confirmed. Eight asymptomatic children at 6-18 months of age still have high 17-OHP levels and await diagnostic definition. Based on the number of confirmed CYP21 D CAH cases among the 82,603 screened, the estimated annual incidence of the disease was 1:10,325, lower than the previously reported rate in Brazil.

Increased Transactivation Associated with SOX3 Polyalanine Tract Deletion in a Patient with Hypopituitarism

Backgound and Aims: Correct gene dosage of SOX3 is critical for the development of the hypothalamo-pituitary axis. Both overdosage of SOX3, as a result of gene duplication, and loss of function resulting from expansion of the first polyalanine (PA) tract are associated with variable degrees of hypopituitarism, with or without mental retardation. The aim of this study was to further investigate the contribution of SOX3 in the etiology of hypopituitarism and the mechanisms involved in the phenotypic variability. Methods: We screened 154 patients with congenital hypopituitarism and an undescended posterior pituitary for mutations in SOX3 and variability in the length of the first PA tract. In addition, 300 patients with variable septooptic dysplasia were screened for variability of the PA tract. Results: We report a novel 18-base pair deletion (p.A243_A248del6, del6PA) in a female patient with hypopituitarism resulting in a 2-fold increase in transcriptional activation in vitro, compared with wild-type SOX3. We also identified a previously reported seven-alanine expansion (p.A240_A241ins7, +7PA) in two male siblings with isolated GH deficiency and a distinct phenotype, in addition to the nonsynonymous variant p.R5Q in an unrelated individual; this appears to have no functional effect on the protein. In contrast to +7PA, del6PA maintained its ability to repress beta-catenin mediated transcription in vitro. Conclusion: This is the first study to report that PA tract deletions associated with hypopituitarism have functional consequences in vitro, possibly due to increased activation of SOX3 target genes. In addition, we have expanded the phenotypic spectrum associated with PA tract expansion (+7PA) mutations to include panhypopituitarism or isolated GH deficiency, with or without mental retardation. (J Clin Endocrinol Metab 96: E685-E690, 2011)

Novel Heterozygous Nonsense GLI2 Mutations in Patients with Hypopituitarism and Ectopic Posterior Pituitary Lobe without Holoprosencephaly

Context: GLI2 is a transcription factor downstream in Sonic Hedgehog signaling, acting early in ventral forebrain and pituitary development. GLI2 mutations were reported in patients with holoprosencephaly (HPE) and pituitary abnormalities. Objective: The aim was to report three novel frameshift/nonsense GLI2 mutations and the phenotypic variability in the three families. Setting: The study was conducted at a university hospital. Patients and Methods: The GLI2 coding region of patients with isolated GH deficiency (IGHD) or combined pituitary hormone deficiency was amplified by PCR using intronic primers and sequenced. Results: Three novel heterozygous GLI2 mutations were identified: c. 2362_2368del p. L788fsX794 (family 1), c. 2081_2084del p. L694fsX722 (family 2), and c. 1138 G > T p. E380X (family 3). All predict a truncated protein with loss of the C-terminal activator domain. The index case of family 1 had polydactyly, hypoglycemia, and seizures, and GH, TSH, prolactin, ACTH, LH, and FSH deficiencies. Her mother and seven relatives harboring the same mutation had polydactyly, including two uncles with IGHD and one cousin with GH, TSH, LH, and FSH deficiencies. In family 2, a boy had cryptorchidism, cleft lip and palate, and GH deficiency. In family 3, a girl had hypoglycemia, seizures, excessive thirst and polyuria, and GH, ACTH, TSH, and antidiuretic hormone deficiencies. Magnetic resonance imaging of four patients with GLI2 mutations and hypopituitarism showed a hypoplastic anterior pituitary and an ectopic posterior pituitary lobe without HPE. Conclusion: We describe three novel heterozygous frameshift or nonsense GLI2 mutations, predicting truncated proteins lacking the activator domain, associated with IGHD or combined pituitary hormone deficiency and ectopic posterior pituitary lobe without HPE. These phenotypes support partial penetrance, variable polydactyly, midline facial defects, and pituitary hormone deficiencies, including diabetes insipidus, conferred by heterozygous frameshift or nonsense GLI2 mutations. (J Clin Endocrinol Metab 95: E384-E391, 2010)

Texture Image Analysis in Differentiating Malignant from Benign Adrenal Cortical Tumors in Children and Adults

Objective: To investigate the possible role of chromatin texture parameters, nuclear morphology, DNA ploidy and clinical functional status in discriminating benign from malignant adrenocortical tumors (ACT). Patients and Methods: Forty-eight cases of clinically benign (n=40) and clinically malignant (n=8) ACT with a minimum of 5-years` follow-up were evaluated for chromatin texture parameters (run length, standard deviation, configurable run length, valley, slope, peak and other 21 Markovian features that describe the distribution of the chromatin in the nucleus), nuclear morphology (nuclear area, nuclear perimeter, nuclear maximum and minumum diameter, nuclear shape), and DNA ploidy. Nuclear parameters were evaluated in Feulgen-stained 5 mu m paraffin-sections analyzed using a CAS 200 image analyzer. Results: Since ACTs present different biological features in children and adults, patients were divided into two groups: children (<= 15 years) and adults (>15 years). In the group of children DNA ploidy presented a marginal significance (p=0.05) in discriminating ACTs. None of the parameters discriminated between malignant and benign ACT in the adult group. Conclusion: ACTs are uncommon and definitive predictive criteria for malignancy remain uncertain, particularly in children. Our data point to DNA content evaluated by image analysis as a new candidate tool for this challenging task. Texture image analysis did not help to differentiate malignant from benign adrenal cortical tumors in children and adults.