Involvement of hypothalamic paraventricular nucleus non-N-methyl-d-aspartate receptors in the pressor response to noradrenaline microinjected into the bed nucleus of the stria terminalis of unanesthetized rats

Microinjection of noradrenaline into the bed nucleus of the stria terminalis (BST) has been reported to cause a pressor response in unanesthetized rats, which was shown to be mediated by acute vasopressin release into the systemic circulation. In the present study we verified the involvement of magnocellular neurons of the hypothalamic paraventricular (PVN) or supraoptic (SON) nuclei and the local neurotransmitter involved in the pressor response to noradrenaline microinjection into the BST. The PVN pretreatment with the non-selective neurotransmission blocker CoCl(2) (1 nmol/100 nL) inhibited the noradrenaline-evoked pressor response. However, responses were not affected by SON treatment with CoCl(2). Further experiments were carried out to test if glutamatergic neurotransmission in the PVN mediates the pressor response evoked by noradrenaline microinjection into the BST. Pretreatment of the PVN with the selective N-methyl-d-aspartate (NMDA) receptor antagonist LY235959 (2 nmol/100 nL) did not affect the noradrenaline-evoked pressor response. However, PVN pretreatment with the selective non-NMDA receptor antagonist NBQX (2 nmol/100 nL) significantly reduced the pressor response to noradrenaline microinjection into the BST. In conclusion, our results suggest that pressor responses to noradrenaline microinjection into the BST are mediated by PVN magnocellular neurons without involvement of SON neurons. They also suggest that a glutamatergic neurotransmission through non-NMDA glutamate receptors in the PVN mediates the response.

Focal CO2 dialysis in raphe obscurus does not stimulate ventilation but enhances the response to focal CO2 dialysis in the retrotrapezoid nucleus

Simultaneous inhibition of the retrotrapezoid nucleus (RTN) and raphe obscurus (ROb) decreased the systemic CO2 response by 51%, an effect greater than inhibition of RTN (- 24%) or ROb (0%) alone, suggesting that ROb modulates chemoreception by interaction with the RTN (19). We investigated this interaction further by simultaneous dialysis of artificial cerebrospinal fluid equilibrated with 25% CO2 in two probes located in or adjacent to the RTN and ROb in conscious adult male rats. Ventilation was measured in a whole body plethysmograph at 30 C. There were four groups (n = 5): 1) probes correctly placed in both RTN and ROb (RTN-ROb); 2) one probe correctly placed in RTN and one incorrectly placed in areas adjacent to ROb (RTN-peri-ROb); 3) one probe correctly placed in ROb and one probe incorrectly placed in areas adjacent to RTN (peri-RTN-ROb); and 4) neither probe correctly placed (peri-RTN-peri-ROb). Focal simultaneous acidification of RTN-ROb significantly increased ventilation ((V) over dot E) up to 22% compared with baseline, with significant increases in both breathing frequency and tidal volume. Focal acidification of RTN-peri-ROb increased (V) over dot E significantly by up to 15% compared with baseline. Focal acidification of ROb and peri-RTN had no significant effect. The simultaneous acidification of regions just outside the RTN and ROb actually decreased (V) over dot E by up to 11%. These results support a modulatory role for the ROb with respect to central chemoreception at the RTN.

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.

Ionotropic glutamatergic receptors in the rostral medullary raphe modulate hypoxia and hypercapnia-induced hyperpnea

It has been suggested that the medullary raphe (MR) plays a key role in the physiological responses to hypoxia and hypercapnia. We assessed the role of ionotropic glutamate receptors in the rostral MR (rMR) in the respiratory responses to hypoxia and hypercapnia by measuring pulmonary ventilation (V(E)) and body temperature (Tb) of male Wistar rats before and after microinjecting Kynurenic acid (KY, an ionotropic glutamate receptors antagonist, 0.1 mM) into the rMR followed by 60 min of hypoxia (7% O(2)) or hypercapnia exposure (7% CO(2)). Compared to the control group, the ventilatory response to hypoxia was attenuated in animals treated with KY intra-rMR, however the ventilatory response to hypercapnia increased significantly. No differences in Tb among groups were observed during hypoxia or hypercapnia. These data suggest that the glutamate acting on ionotropic receptors in the rMR exerts an excitatory modulation on hyperventilation induced by hypoxia but an inhibitory modulation on the hypercapnia-induced hyperpnea. (C) 2010 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.

Serotonergic neurons in the nucleus raphe obscurus contribute to interaction between central and peripheral ventilatory responses to hypercapnia

Serotonergic (5-HT) neurons in the nucleus raphe obscurus (ROb) are involved in the respiratory control network. However, it is not known whether ROb 5-HT neurons play a role in the functional interdependence between central and peripheral chemoreceptors. Therefore, we investigated the role of ROb 5-HT neurons in the ventilatory responses to CO(2) and their putative involvement in the central-peripheral CO(2) chemoreceptor interaction in unanaesthetised rats. We used a chemical lesion specific for 5-HT neurons (anti-SERT-SAP) of the ROb in animals with the carotid body (CB) intact or removed (CBR). Pulmonary ventilation (V (E)), body temperature and the arterial blood gases were measured before, during and after a hypercapnic challenge (7% CO(2)). The lesion of ROb 5-HT neurons alone (CB intact) or the lesion of 5-HT neurons of ROb+CBR did not affect baseline V (E) during normocapnic condition. Killing ROb 5-HT neurons (CB intact) significantly decreased the ventilatory response to hypercapnia (p < 0.05). The reduction in CO(2) sensitivity was approximately 15%. When ROb 5-HT neurons lesion was combined with CBR (anti-SERT-SAP+CBR), the V (E) response to hypercapnia was further decreased (-31.2%) compared to the control group. The attenuation of CO(2) sensitivity was approximately 30%, and it was more pronounced than the sum of the individual effects of central (ROb lesion; -12.3%) or peripheral (CBR; -5.5%) treatments. Our data indicate that ROb 5-HT neurons play an important role in the CO(2) drive to breathing and may act as an important element in the central-peripheral chemoreception interaction to CO(2) responsiveness.

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)

Role of preoptic opioid receptors in the body temperature reduction during hypoxia

Evidence indicates that endogenous opioids play a role in body temperature (Tb) regulation in mammals but no data exist about the involvement of the specific opioid receptors, mu, kappa and delta, in the reduction of Tb induced by hypoxia. Thus, we investigated the participation of these opioid receptors in the anteroventral preoptic region (AVPO) in hypoxic decrease of Th. To this end, Th of unanesthetized Wistar rats was monitored by temperature data loggers before and after intra-AVPO microinjection of the selective kappa-opioid receptor antagonist nor-binaltorphimine dihydrochloride (nor-BNI; 0.1 and 1.0 mu g/100 nL/animal), the selective mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) cyclic (CTAP; 0.1 and 1.0 mu g/100 nL/animal), and the selective delta-opioid receptor antagonist Naltrindole (0.06 and 0.6 mu g/100 nL/animal) or saline (vehicle, 100 nu animal), during normoxia and hypoxia (7% inspired O(2)). Under normoxia, no effect of opioid antagonists on Th was observed. Hypoxia induced Th to reduce in vehicle group, a response that was inhibited by the microinjection intra-AVPO of nor-BNI. In contrast, CTAP and Naltrindole did not change Th during hypoxia but caused a longer latency for the return of Th to the normoxic values just after low O(2) exposure. Our results indicate the kappa-opioid receptor in the AVPO is important for the reduction of Th during hypoxia while the mu and delta receptors are involved in the increase of Th during normoxia post-hypoxia. (C) 2009 Elsevier B.V. All rights reserved.

GABAergic antagonist blocks the reduction of tonic immobility behavior induced by activation of 5-HT(2) receptors in the basolateral nucleus of the amygdala in guinea pigs

Tonic immobility (TI) is a temporary state of profound motor inhibition induced by situations that generate intense fear, with the objective of protecting an animal from attacks by predators. A preliminary study by our group demonstrated that microinjection into the basolateral nucleus of the amygdala (BLA) of an agonist to 5-HT(1A) and 5-HT(2) receptors promoted a decrease in TI duration. In the current study, the effects of GABAergic stimulation of the BLA and the possible interaction between GABA(A) and 5-HT(2) receptors on TI modulation were investigated. Observation revealed that GABAergic agonist muscimol (0.26 nmol) reduced the duration of TI episodes, while microinjection of the GABAergic antagonist bicuculline (1 nmol) increased TI duration. Additionally, microinjection of 5-HT(2) agonist receptors (alpha-methyl-5-HT, 0.32 nmol) into the BLA decreased TI duration, an effect reversed by pretreatment with bicuculline (at the dose that had no effect per se, 0.2 nmol). Moreover, the activation of GABA(A) and 5-HT(2) receptors in the BLA did not alter the spontaneous motor activity in the open field test. These experiments demonstrated that the activation of GABA(A) and 5-HT(2) receptors of the BLA possibly produce a reduction in unconditioned fear that decreases the TI duration in guinea pigs, but this is not due to increased spontaneous motor activity, which could affect a TI episode nonspecifically. Furthermore, these results suggest an interaction between GABAergic and serotoninergic mechanisms mediated by GABA(A) and 5-HT(2) receptors. In addition, the GABAergic circuit of the BLA presents a tonic inhibitory influence on TI duration. (C) 2009 Elsevier Inc. All rights reserved.

Effects of estrogen receptor alpha and beta gene deletion on estrogenic induction of progesterone receptors in the locus coeruleus in female mice

Locus coeruleus (LC) is involved in the LHRH regulation by gonadal steroids. We investigated the expression of progesterone and estrogen receptors (PR; ER) in LC neurons of ER alpha (alpha ERKO) or ER beta (beta ERKO) knockout mice, and their wild-type (alpha WT and beta WT). Immunocytochemical studies showed that LC expresses PR and both ERs, although ER beta was more abundant. Estradiol benzoate (EB) decreased ER alpha-positive cells in WT and beta ERKO mice, and progesterone caused a further reduction, whereas none of the steroids influenced ER beta expression. ER beta deletion increased ER alpha while ER alpha deletion did not alter ER beta expression. In both WT mice, EB increased PR expression, which was diminished by progesterone. These steroid effects were also observed in alpha ERKO animals but to a lesser extent, suggesting that ER alpha is partially responsible for the estrogenic induction of PR in LC. Steroid effects on PR in beta ERKO mice were similar to those in the alpha ERKO but to a lesser extent, probably because PR expression was already high in the oil-treated group. This expression seems to be specific of LC neurons, since it was not observed in other areas studied, the preoptic area and ventromedial nucleus of hypothalamus. These findings show that LC in mice expresses alpha ER, beta ER, and PR, and that a balance between them may be critical for the physiological control of reproductive function.

Neonatal handling reduces the number of cells in the medial preoptic area of female rats

Early-life events may induce alterations in neuronal function in adulthood. A crucial aspect in studying long-lasting effects induced by environmental interventions imposed to the animal several weeks before is finding a stable change that could be causally related to the phenotype observed in adulthood. In order to explain an adult trait, it seems necessary to look back to early life and establish a temporal line between events. The neonatal handling procedure is an experimental tool to analyze the long-lasting impact of early-life events. Aside from the neuroendocrine response to stress, neonatal handling also alters the functionality of the hypothalamus-pituitary-gonad (HPG) axis. Reductions in ovulation and surge of the luteinizing hormone (LH) on the proestrous day were shown in female rats. Considering the importance of the medial preoptic area (MPA) for the control of ovulation, the present study aimed to verify the effects of neonatal handling on the numerical density and cell size in the MPA in 11-day-old and 90-day-old female rats. Cellular proliferation was also assessed using BrdU (5-bromo-2`-deoxyuridine) in 11-day-old pups. Results showed that neonatal handling induces a stable reduction in the number of cells and in the size of the cell soma, which were lower in handled females than in nonhandled ones at both ages. Cellular proliferation in the MPA was also reduced 24 h after the last manipulation. The repeated mother-infant disruption imposed by the handling procedure ""lesioned"" the MPA. The dysfunction in the ovulation mechanisms induced by the handling procedure could be related to that neuronal loss. The study also illustrates the impact of an environmental intervention on the development of the brain. (C) 2008 Elsevier B.V. All rights reserved

Temporal relationship between the auditory brainstem response and focal responses of auditory nerve root and cochlear nucleus during development in the tammar wallaby (Macropus eugenii)

Thirty-two pouch-young tammar wallabies were used to discover the generators of the auditory brainstem response (ABR) during development by the use of simultaneous ABR and focal brainstem recordings. A click response from the auditory nerve root (ANR) in the wallaby was recorded from postnatal day (PND) 101, when no central auditory station was functional, and coincided with the ABR, a simple positive wave. The response of the cochlear nucleus (CN) was detected from PND 110, when the ABR had developed 1 positive and 1 negative peak. The dominant component of the focal ANR response, the N-1 wave, coincided with the first half of the ABR P wave, and that of the focal CN response, the N-1 wave, coincided with the later two thirds. In older animals, the ANR response coincided with the ABR's N-1, wave, while the CN response coincided with the ABR's P-2, N-2 and P-3 waves, with its contribution to the ABR P-2 dominant. The protracted development of the marsupial auditory system which facilitated these correlations makes the tammar wallaby a particularly suitable model. Copyright (C) 2001 S. Karger AG, Basel.

Transcripts of the MHM region on the chicken Z chromosome accumulate as non-coding RNA in the nucleus of female cells adjacent to the DMRT1 locus

The male hypermethylated (MHM) region, located near the middle of the short arm of the Z chromosome of chickens, consists of approximately 210 tandem repeats of a BamHI 2.2-kb sequence unit. Cytosines of the CpG dinucleotides of this region are extensively methylated on the two Z chromosomes in the male but much less methylated on the single Z chromosome in the female. The state of methylation of the MHM region is established after fertilization by about the 1-day embryonic stage. The MHM region is transcribed only in the female from the particular strand into heterogeneous, high molecular-mass, non-coding RNA, which is accumulated at the site of transcription, adjacent to the DMRT1 locus, in the nucleus. The transcriptional silence of the MHM region in the male is most likely caused by the CpG methylation, since treatment of the male embryonic fibroblasts with 5-azacytidine results in hypo-methylation and active transcription of this region. In ZZW triploid chickens, MHM regions are hypomethylated and transcribed on the two Z chromosomes, whereas MHM regions are hypermethylated and transcriptionally inactive on the three Z chromosomes in ZZZ triploid chickens, suggesting a possible role of the W chromosome on the state of the MHM region.

Soft biomaterials as a substitute for the Nucleus Pulposus

One of the largest health problems faced worldwide, when evaluated by direct (clinical) as well indirect cost (absenteeism), is the degeneration of the intervertebral disc (IVD) that leads to back pain and, potentially disability and individual´s quality of life decreasing. The intervertebral disc is a mechanical and biological complex structure, formed by a tough outer layer of fibrocartilage called Annulus Fibrosus (AF),which surrounds a soft, elastic and gelatinous core called Nucleus Pulposus (NP). These two structures are completed by two upper and lower encasing layer called Vertebral Endplates (VEP). The degeneration of the IVD is marked by the dehydration of the Nucleus Pulposus, reducing the hydrostatic pressure inside the nucleus, resulting in a loss of capability to support compressive forces, during the active period, and to regain height during the resting period. This situation will compromise the role of shock absorber by the NP and transfers these forces to the AF. This transfer will result in cracks on the AF, deteriorating the IVD, allowing the ingrowth of vessels and nerves. This project was based on the developing a protocol to test suitable NP replacements, in hope to future assessment of discrete mechanical values and characteristics for an NP replacement. For this, Nucleus pulposus samples from goat, encapsulated Hydromed gel denominated “Raviolis” and Chitosan gels, produced via wet route using an ammonium environment, were confined compressed. Chitosan was rheologically tested and swelling capability of all the three type of materials was assessed. Results showed that the Nucleus Pulposus and “Raviolis” have similar mechanical behavior, being able to swell and “build up” hydrostatic pressure after a compression stage, while the Chitosan gel did not showed that ability. Therefore, “Raviolis” are a more suitable candidate to replace the NP than Chitosan gels. It was also observed that confined compression is the key test to perform on any possible candidate to replace the NP.