Maristela O. Poletini

Release of norepinephrine in the preoptic area activates anteroventral periventricular nucleus neurons and stimulates the surge of luteinizing hormone.

The role of norepinephrine (NE) in regulation of LH is still controversial. We investigated the role played by NE in the positive feedback of estradiol and progesterone. Ovarian-steroid control over NE release in the preoptic area (POA) was determined using microdialysis. Compared with ovariectomized (OVX) rats, estradiol-treated OVX (OVX+E) rats displayed lower release of NE in the morning but increased release coincident with the afternoon surge of LH. OVX rats treated with estradiol and progesterone (OVX+EP) exhibited markedly greater NE release than OVX+E rats, and amplification of the LH surge. The effect of NE on LH secretion was confirmed using reverse microdialysis. The LH surge and c-Fos expression in anteroventral periventricular nucleus neurons were significantly increased in OVX+E rats dialyzed with 100 nm NE in the POA. After Fluoro-Gold injection in the POA, c-Fos expression in Fluoro-Gold/tyrosine hydroxylase-immunoreactive neurons increased during the afternoon in the A2 of both OVX+E and OVX+EP rats, in the locus coeruleus (LC) of OVX+EP rats, but was unchanged in the A1. The selective lesion of LC terminals, by intracerebroventricular N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, reduced the surge of LH in OVX+EP but not in OVX+E rats. Thus, estradiol and progesterone activate A2 and LC neurons, respectively, and this is associated with the increased release of NE in the POA and the magnitude of the LH surge. NE stimulates LH secretion, at least in part, through activation of anteroventral periventricular neurons. These findings contribute to elucidation of the role played by NE during the positive feedback of ovarian steroids.

Antagonism of Oxytocin Prevents Suckling- and Estradiol-Induced, But Not Progesterone-Induced, Secretion of Prolactin

In female rats, estradiol (E(2)) and suckling induce prolactin (PRL) secretion. This involves inhibition of hypothalamic dopaminergic tone and stimulation by a PRL-releasing hormone, possibly oxytocin (OT). Infusing an OT antagonist (OTA) i.v., we evaluated the role of OT on suckling- and E(2)-induced PRL secretion. Three days after parturition at 0900 h, lactating dams were fitted with 24-h osmotic minipumps filled with saline or OTA. On d 5 of lactation, pups were separated from their dams for 6 h. Immediately or 20 min after the resumption of suckling, dam trunk blood was collected. Also, ovariectomized (OVX) rats were treated with E(2) (OVE) and OTA at 1000 h on d 1. Blood samples were obtained from 1300 to 2100 h on d 2 for PRL measurements. Additionally, OVX rats were evaluated on d 2 after receiving progesterone (P(4)). OTA blocked suckling and E(2)-induced release of PRL but not that induced by E(2)+P(4). Pups from treated dams failed to gain weight when allowed to nurse for 20 min on d 5 but gained more than 7 g when nursed on d 7 of lactation, indicating that the OTA was active 48 h later. Western blot analysis showed that E(2) treatment increased OT receptors in the anterior pituitary when compared with OVX animals. No further increase was observed in response to the P(4), suggesting that the enhancing effect of P(4) on E(2)-induced PRL release may act through mechanisms independent of OT. These data demonstrate the role of OT in the control of suckling and steroid-induced PRL secretion.

Cervical stimulation activates A1 and locus coeruleus neurons that project to the paraventricular nucleus of the hypothalamus

In female rats, stimulation of the uterine cervix during mating induces two daily surges of prolactin. Inhibition of hypothalamic dopamine release and stimulation of oxytocin neurons in the paraventricular nucleus (PVN) are required for prolactin secretion. We aim to better understand how stimulation of the uterine cervix is translated into two daily prolactin surges. We hypothesize that noradrenergic neurons in the A1, A2, and locus coeruleus (LC) are responsible for conveying the peripheral stimulus to the PVN. In order to determine whether projections from these neurons to the PVN are activated by cervical stimulation (CS), we injected a retrograde tracer, Fluoro-Gold (FG), into the PVN of ovariectomized rats. Fourteen days after injection, animals were submitted to artificial CS or handling and perfused with a fixative solution. Brains were removed and sectioned from the A1, A2, and LC for c-Fos, tyrosine hydroxylase (TH), and FG triple-labeling using immunohistochemistry. CS increased the percentage of TH/FG+ double-labeled neurons expressing c-Fos in the A1 and LC. CS also increased the percentage of TH+ neurons expressing c-Fos within the A1 and A2, independent of their projections to the PVN. Our data reinforce the significant contributions of the A1 and A2 to carry sensory information during mating, and provide evidence of a functional pathway in which CS activates A1 and LC neurons projecting to the PVN, which is potentially involved in the translation of CS into two daily prolactin surges.

Central Clock Regulates the Cervically Stimulated Prolactin Surges by Modulation of Dopamine and Vasoactive Intestinal Polypeptide Release in Ovariectomized Rats

Background/Aims: Cervical stimulation induces a circadian rhythm of prolactin secretion and antiphase dopamine release. The suprachiasmatic nucleus (SCN) controls this rhythm, and we propose that it does so through clock gene expression within the SCN. Methods: To test this hypothesis, serial blood samples were taken from animals injected with an antisense deoxyoligonucleotide cocktail for clock genes (generated against the 5′ transcription start site and 3′ cap site of per1, per2, and clock mRNA) or with a random-sequence deoxyoligonucleotide in the SCN. To determine whether disruption of clock genes in the SCN compromises the neural mechanism controlling prolactin secretion, we sacrificed another group of rats (under the same treatments) at 12.00 or 17.00 h. Dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) were measured using HPLC/electrochemical detection in the median eminence as well as the intermediate and the neural lobe of the pituitary gland, and the DOPAC:dopamine ratio was used as an index of dopamine activity. Vasoactive intestinal polypeptide (VIP) content was determined in tissue punches of the SCN and paraventricular nucleus (PVN), an SCN efferent. Results: Treatment with clock gene antisense deoxyoligonucleotide cocktail abolished both the diurnal and nocturnal prolactin surges induced by cervical stimulation. This treatment abolished the antiphase relationship established by cervical stimulation between dopamine neuronal activity and prolactin secretion. Also, VIP content increased in the SCN and decreased in the PVN. Conclusion: These results suggest that the SCN clock determines the circadian rhythm of prolactin secretion in cervically stimulated rats by regulating dopamine neuronal activity and VIP inputs to the PVN.

Vasoactive intestinal polypeptide modulates the estradiol-induced prolactin surge by entraining oxytocin neuronal activity.

In female rats, estradiol is responsible for a circadian secretory prolactin (PRL) pattern which requires an intact suprachiasmatic nucleus (SCN). SCN outputs involved in this secretory profile remain elusive. Because oxytocin has been proposed to stimulate PRL secretion, we investigated whether the projections of vasoactive intestinal polypeptide (VIP) from the SCN to neurons producing oxytocin in the paraventricular and periventricular nuclei (PVN and PeVN, respectively) are responsible for timing PRL surges induced by estradiol (E(2)). E(2)-treated ovariectomized rats received an injection of antisense or random-sequence oligodeoxynucleotide for VIP in the SCN and blood samples were taken for PRL measurements by radioimmunoassay. Additionally, the percentage of oxytocin-positive neurons immunoreactive to FOS-related antigens was determined in the PVN and PeVN, as an index of neuronal activity. In the PVN, oxytocinergic neuronal activity increased in the early evening regardless of E(2) treatment, whereas E(2) induced an increase of activity in the PeVN. VIP antisense attenuated this increase observed in both neuronal populations. Additionally, in the PeVN, VIP antisense advanced this increase by 2 h (from 19:00 h to 17:00 h). This same effect was observed in the PRL surge that occurred at 17:00 h in the VIP antisense injected animals. Thus, the SCN influences the precise timing of the E(2)-induced PRL surge via VIP projections to oxytocinergic neurons of the PVN and PeVN.

Neuroendocrine Regulation and Homeostasis

This special issue of the Journal of Neuroendocrinology comprisesfive review articles, centred on the theme of the neuroendocrineregulation of homeostasis, and provides a snapshot of the sessionsat the 2013 International Workshop in Neuroendocrinology (IWNE).The workshop was held in Santa Clara Resort, in the town ofDourado, S~ao Paulo, Brazil, from 4–7 August 2013 and emergedfrom the unification of two independent neuroendocrine meetingsheld in August 2011: The First US–Latin American Workshop inNeuroendocrinology (Vi~na del Mar, Chile) and the First BrazilianInternational Symposium on Integrative Neuroendocrinology (Dourado,Brazil). The small, intimate venue not only facilitated scientificinteractions between Neuroendocrinologists from the United Statesand South America, but also provided graduate students and post-doctoral fellows with the opportunity to interact with each otherand with established investigators. It provided the unique opportu-nity for 11 competitively chosen young investigators to discusstheir research findings as an oral or poster presentation in aninformal setting. Several of these young investigators spearheadedthe writing and compilation of the review articles, with input fromthe established investigators.Stress coping mechanisms have evolved over phyla and play animportant role in the survival of the species. In adaptive responseto stress, living organisms redirect their physiological functionstowards the maintenance of homeostasis, by mobilising energystores and suppressing diverse processes such as feeding, bodyrepair, reproduction and behaviours. Glucocorticoids, the end prod-ucts of the stress-stimulated hypothalamic-pituitary-adrenal (HPA)axis, exert effects on multiple organ systems to regulate metabolic,cardiovascular, immune, behavioural and neuroendocrine activitiesto maintain homeostasis. Sex differences in stress reactivity as aresult of the activational effects of gonadal steroids are wellknown. The HPA and hypothalamic-pituitary-gonadal axes are inti-mately intertwined, wherein activation of the former affects thefunction of the latter and vice versa. In addition, the adaptive stressresponse is determined by genetic, environmental, nutritional anddevelopmental factors, which dictate the characteristics of themobilised defences against stressors throughout the lifespan. Thereviews in this special issue attempt to summarise current researchon various aspects related to the central theme of the neuroendocrineregulation of homeostasis.The first review by Uchoa et al. (1) discusses the current state ofknowledge about the regulation of the HPA axis by glucocorticoids,and elaborates the role of glucocorticoids in neural plasticity,immunomodulation and feeding behaviours. Toufexis et al. (2) out-line the complex interplay between the reproductive and stressaxes. Based on studies from rodents to primates, the review high-lights how stressors, during development and adulthood, disruptgonadal hormone-mediated neuroendocrine and behaviouralresponses and impair reproductive health and emotional wellbeing.The review by Boersma et al. (3) discusses how environmentalperturbations such as stress and nutrition during development canlead to epigenetic alterations and maladaptive consequences (i.e.obesity and heightened stress sensitivity) in adulthood. Amaralet al. (4) emphasise the importance of circadian rhythms in themaintenance of physiological homeostasis and discuss the impor-tance of hormonal and neural cues in the synchronisation of thecircadian timing system to the light/dark cycle. No discussion onneuroendocrine regulation can be complete without due consider-ation of the environmental effects of oestrogens on homeostasis.The review by Cruz et al. (5) discusses how neonatal exposure toenvironmental oestrogens can modify epigenetic programming andproduce alterations in reproductive function in adulthood.The topics covered in this issue on neuroendocrine regulation ofstress have important implications for the day-to-day lives of allliving organisms. Although neuroendocrinologists have begun tounravel the multifaceted and integrative mechanisms involved insuch regulation, we have barely touched the tip of the iceberg.Application of newer concepts and tools will potentially propel thefield into revealing the nuts and bolts biology of how stress exposurecan disrupt homeostatic functions, leading to pathophysiology.