Purinergic P2 and glutamate NMDA receptor coupling contributes to osmotically driven excitability in hypothalamic magnocellular neurosecretory neurons.

KEY POINTS: Purinergic and glutamatergic signalling pathways play a key role in regulating the activity of hypothalamic magnocellular neurosecretory neurons (MNNs). However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Here, we report that ATP acts on purinergic P2 receptors in MNNs to potentiate in a Ca2+ -dependent manner extrasynaptic NMDAR function. The P2-NMDAR coupling is engaged in response to an acute hyperosmotic stimulation, contributing to osmotically driven firing activity in MNNs. These results help us to better understand the precise mechanisms contributing to the osmotic regulation of firing activity and hormone release from MNNs. ABSTRACT: The firing activity of hypothalamic magnocellular neurosecretory neurons (MNNs) located in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) is coordinated by the combined, fine-tuned action of intrinsic membrane properties, synaptic and extrasynaptic signalling. Among these, purinergic and glutamatergic signalling pathways have been shown to play a key role regulating the activity of MNNs. However, the precise cellular mechanisms by which ATP and glutamate act in concert to regulate osmotically driven MNN neuronal excitability remains unknown. Whole-cell patch-clamp recordings obtained from MNNs showed that ATP (100 µM) induced an increase in firing rate, an effect that was blocked by either 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]2-pyridinyl]azo]1,3-benzenedisulfonic acid tetrasodium salt (PPADS) (10 µM) or kynurenic acid (1 mm). While ATP did not affect the frequency or magnitude of glutamatergic excitatory postsynaptic currents (EPSCs), it induced an inward shift in the holding current that was prevented by PPADS or kynurenic acid treatment, suggesting that ATP enhances a tonic extrasynaptic glutamatergic excitatory current. We observed that ATP-potentiated glutamatergic receptor-mediated currents were evoked by focal application of L-glu (1 mm) and NMDA (50 µM), but not AMPA (50 µM). ATP potentiation of NMDA-evoked currents was blocked by PPADS (10 µM) and by chelation of intracellular Ca2+ with BAPTA (10 mm). Finally, we report that a hyperosmotic stimulus (mannitol 1%, +55 mOsm/kgH2 O) potentiated NMDA-evoked currents and increased MNN firing activity, effects that were blocked by PPADS. Taken together, our data support a functional excitatory coupling between P2 and extrasynaptic NMDA receptors in MNNs, which is engaged in response to an acute hyperosmotic stimulus.

Infertility treatment and autism risk using the Modified Checklist for Autism in Toddlers (M-CHAT).

STUDY QUESTION: Are toddlers conceived by fertility treatment at higher risk of failing a screening tool for autism spectrum disorders (ASD) than toddlers not conceived by treatment? SUMMARY ANSWER: Compared with children not conceived by infertility treatment, children conceived by any infertility treatment, ovulation induction with or without intrauterine insemination (OI/IUI), or assisted reproductive technologies (ART) appeared to have had higher odds of failing an ASD screening; however, results were inconclusive and need replication. WHAT IS KNOWN ALREADY: Although most of the studies which have examined risk of ASD after ART show no association, the results are mixed. Thus, further studies are needed to clarify this association. STUDY DESIGN SIZE, DURATION: The Upstate KIDS Study is a population-based, prospective cohort study of children born in New York State between 2008 and 2010. Children were screened for ASD using the Modified Checklist for Autism in Toddlers (M-CHAT) at ages 18 and 24 months. PARTICIPANTS/MATERIALS, SETTING, AND METHODS: The New York State live-birth registry was used to identify newborns conceived with and without fertility treatment with a 1:3 ratio, frequency matched on region of birth. At 18 and 24 months, 3183 and 3063 mothers, respectively, completed the M-CHAT questionnaire. The current analysis included 2586 singletons and 1296 twins with M-CHAT information at 18 and/or 24 months. Multivariable logistic regression with generalized estimating equations (GEE) was used to estimate odds ratios (aOR) and 95% confidence intervals (CI) after adjustment for covariates such as maternal age, education and plurality. MAIN RESULTS AND THE ROLE OF CHANCE: We found that 200 (5.2%) and 115 (3.0%) children failed the M-CHAT at 18 and 24 months, respectively. The associations between use of infertility treatment and failing the M-CHAT at 18 and/or 24 months were positive but inconclusive as they failed to exclude no association (18 months aOR 1.71, 95% CI: 0.81-3.61; 24 months aOR 1.78, 95% CI: 0.66-4.81; and both 18 and 24 months aOR 1.53, 95% CI: 0.78-2.99). The relationships between OI/IUI and ART with M-CHAT failure at 18 and/or 24 months were similar to those of using any fertility treatment. In vitro fertilization with intracytoplasmic sperm injection was not consistently positively or inversely associated with M-CHAT failure at each time point (18 months aOR 1.20, 95% CI: 0.51-2.83; 24 months aOR 0.93, 95% CI: 0.37-2.31; and both 18 and 24 months aOR 1.09, 95% CI: 0.50-2.60). LIMITATIONS REASONS FOR CAUTION: The M-CHAT is a screening tool used for ASD risk assessment, and therefore, M-CHAT failure does not indicate ASD diagnosis. In addition, we did not have power to detect associations of small magnitude. Finally, non-response to follow-up may bias the results. WIDER IMPLICATIONS OF THE FINDINGS: Despite lack of precision, the positive associations between ART and M-CHAT failure suggest that larger population-based studies with longer follow-up are needed. STUDY FUNDING/COMPETING INTEREST(S): Supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD; contracts HHSN275201200005C, HHSN267200700019C). The sponsor played no role in the study design, data collection, data analysis or interpretation, writing of the manuscript or decision to submit the article for publication. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Compromised blood-brain barrier permeability: novel mechanism by which circulating angiotensin II signals to sympathoexcitatory centres during hypertension.

Angiotensin II (AngII) is a pivotal peptide implicated in the regulation of blood pressure. In addition to its systemic vascular and renal effects, AngII acts centrally to modulate the activities of neuroendocrine and sympathetic neuronal networks, influencing in turn sympatho-humoral outflows to the circulation. Moreover, a large body of evidence supports AngII signalling dysregulation as a key mechanism contributing to exacerbated sympathoexcitation during hypertension. Due to its hydrophilic actions, circulating AngII does not cross the blood-brain barrier (BBB), signalling to the brain via the circumventricular organs which lack a tight BBB. In this review, we present and discuss recent studies from our laboratory showing that elevated circulating levels of AngII during hypertension result in disruption of the BBB integrity, allowing access of circulating AngII to critical sympathoexcitatory brain centres such as the paraventricular nucleus of the hypothalamus and the rostral ventrolateral medulla. We propose the novel hypothesis that AngII-driven BBB breakdown constitutes a complementary mechanism by which circulating AngII, working in tandem with the central renin-angiotensin system, further exacerbates sympatho-humoral activation during hypertension. These results are discussed within the context of a growing body of evidence in the literature supporting AngII as a pro-inflammatory signal, and brain microglia as key cell targets mediating central AngII actions during hypertension.

Use of ICSI in IVF cycles in women with tubal ligation does not improve pregnancy or live birth rates.

STUDY QUESTION: Does ICSI improve outcomes in ART cycles without male factor, specifically in couples with a history of tubal ligation as their infertility diagnosis? SUMMARY ANSWER: The use of ICSI showed no significant improvement in fertilization rate and resulted in lower pregnancy and live birth (LB) rates for women with the diagnosis of tubal ligation and no male factor. WHAT IS KNOWN ALREADY: Prior studies have suggested that ICSI use does not improve fertilization, pregnancy or LB rates in couples with non-male factor infertility. However, it is unknown whether couples with tubal ligation only diagnosis and therefore iatrogenic infertility could benefit from the use of ICSI during their ART cycles. STUDY DESIGN, SIZE, DURATION: Longitudinal cohort of nationally reported cycles in the Society for Assisted Reproductive Technology Clinic Outcomes Reporting System (SART CORS) of ART cycles performed in the USA between 2004 and 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: There was a total of 8102 first autologous fresh ART cycles from women with the diagnosis of tubal ligation only and no reported male factor in the SART database. Of these, 957 were canceled cycles and were excluded from the final analysis. The remaining cycles were categorized by the use of conventional IVF (IVF, n = 3956 cycles) or ICSI (n = 3189 cycles). The odds of fertilization, clinical intrauterine gestation (CIG) and LB were calculated by logistic regression modeling, and the adjusted odds ratios (AORs) with 95% confidence intervals were calculated by adjusting for the confounders of year of treatment, maternal age, race and ethnicity, gravidity, number of oocytes retrieved, day of embryo transfer and number of embryos transferred. MAIN RESULTS AND THE ROLE OF CHANCE: The main outcome measures of the study were odds of fertilization (2PN/total oocytes), clinical intrauterine gestation (CIG/cycle) and live birth (LB/cycle). The fertilization rate was higher in the ICSI versus IVF group (57.5% vs 49.1%); however, after adjustment this trend was no longer significant (AOR 1.14, 0.97-1.35). Interestingly, both odds of CIG (AOR 0.78, 0.70-0.86), and odds of LB were lower (AOR 0.77, 0.69-0.85) in the ICSI group. Plurality at birth, mean length of gestation and birth weight did not differ between the two groups. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study, therefore only the available parameters could be included, with parameters of interest such as smoking status not available for inclusion. Smoking status may have led practitioners to use ICSI to improve pregnancy and LB outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Studies have shown that in the USA there is an increasing usage of ICSI for non-male factor infertility despite a lack of evidence-based benefit. Our study corroborates this increasing use over the last 8 years, specifically in the tubal ligation only patient population. Even after adjusting for multiple confounders, the patients who underwent ICSI had no statistically significant improvement in fertilization rate and actually had a lower likelihood of achieving a clinical pregnancy and LB. Therefore, our data suggest that the use of ICSI in tubal ligation patients has no overall benefit. This study contributes to the body of evidence that the use of ICSI for non-male factor diagnosis does not improve ART outcomes over conventional IVF. STUDY FUNDING/COMPETING INTERESTS: None.

Detection of human papillomavirus in the oral cavities of persons with Fanconi anemia.

OBJECTIVE: We conducted a cross-sectional study to describe the prevalence and correlates of type-specific human papillomavirus (HPV) DNA in the oral cavities of persons with Fanconi anemia. MATERIALS AND METHODS: Oral swabs were collected from 67 participants with Fanconi anemia and tested for 27 HPV genotypes using polymerase chain reaction-based methods. RESULTS: Participants were a mean of 18.6 (standard deviation, 10.0) years of age (range 4-47 years). The prevalence of oral HPV infection was 7.5%, and the prevalence of high-risk HPV infection was 6.0%. HPV type 16 was not detected in any samples. Prevalence was higher in adults than in children (13.3% vs 2.7% in those ≥18 vs <18 years of age). Among adults, prevalence was higher in males than in females (25.0% vs 9.1%, respectively). CONCLUSIONS: Prevalence of oral HPV infection in persons with Fanconi anemia was comparable to estimates from other studies in the general population. However, in contrast to previous studies, we did not identify HPV type 16 (the type found in most HPV-related head and neck cancers) in any participants.

Imbalanced K+ and Ca2+ subthreshold interactions contribute to increased hypothalamic presympathetic neuronal excitability in hypertensive rats.

Despite the importance of brain-mediated sympathetic activation in the morbidity and mortality of patients with high blood pressure, the precise cellular mechanisms involved remain largely unknown. We show that an imbalanced interaction between two opposing currents mediated by potassium (I(A)) and calcium (I(T)) channels occurs in sympathetic-related hypothalamic neurons in hypertensive rats. We show that this imbalance contributes to enhanced membrane excitability and firing activity in this neuronal population. Knowledge of how these opposing ion channels interact in normal and disease states increases our understanding of underlying brain mechanisms contributing to the high blood pressure condition.

Molecular characterization of T-type Ca(2+) channels responsible for low threshold spikes in hypothalamic paraventricular nucleus neurons.

The hypothalamic paraventricular nucleus (PVN) is composed of functionally heterogeneous cell groups, possessing distinct electrophysiological properties depending on their functional roles. Previously, T-type Ca(2+) dependent low-threshold spikes (LTS) have been demonstrated in various PVN neuronal types, including preautonomic cells. However, the molecular composition and functional properties of the underlying T-type Ca(2+) channels have not been characterized. In the present study, we combined single cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry and patch-clamp recordings to identify subtypes of T-type Ca(2+) channels expressed in PVN cells displaying LTS (PVN-LTS), including identified preautonomic neurons. LTS appeared at the end of hyperpolarizing pulses either as long-lasting plateaus or as short-lasting depolarizing humps. LTS were mediated by rapidly activating and inactivating T-type Ca(2+) currents and were blocked by Ni(2+). Single cell RT-PCR and immunohistochemical studies revealed Cav3.1 (voltage-gated Ca(2+) channel) as the main channel subunit detected in PVN-LTS neurons. In conclusion, these data indicate that Cav3.1 is the major subtype of T-type Ca(2+) channel subunit that mediates T-type Ca(2+) dependent LTS in PVN neurons.

Intermittent activation of peripheral chemoreceptors in awake rats induces Fos expression in rostral ventrolateral medulla-projecting neurons in the paraventricular nucleus of the hypothalamus.

Despite the well-established sympathoexcitation evoked by chemoreflex activation, the specific sub-regions of the CNS underlying such sympathetic responses remain to be fully characterized. In the present study we examined the effects of intermittent chemoreflex activation in awake rats on Fos-immunoreactivity (Fos-ir) in various subnuclei of the paraventricular nucleus of the hypothalamus (PVN), as well as in identified neurosecretory preautonomic PVN neurons. In response to intermittent chemoreflex activation, a significant increase in the number of Fos-ir cells was found in autonomic-related PVN subnuclei, including the posterior parvocellular, ventromedial parvocellular and dorsal-cap, but not in the neurosecretory magnocellular-containing lateral magnocellular subnucleus. No changes in Fos-ir following chemoreflex activation were observed in the anterior PVN subnucleus. Experiments combining Fos immunohistochemistry and neuronal tract tracing techniques showed a significant increase in Fos-ir in rostral ventrolateral medulla (RVLM)-projecting (PVN-RVLM), but not in nucleus of solitarii tract (NTS)-projecting PVN neurons. In summary, our results support the involvement of the PVN in the central neuronal circuitry activated in response to chemoreflex activation, and indicate that PVN-RVLM neurons constitute a neuronal substrate contributing to the sympathoexcitatory component of the chemoreflex.

Expression of GABAB receptors in magnocellular neurosecretory cells of male, virgin female and lactating rats.

GABA is one of the key neurotransmitters that regulate the firing activity of neurones in the supraoptic (SON) and paraventricular (PVN) nuclei. In the present study, we used immunohistochemical techniques to study the distribution and subcellular localisation of metabotropic GABA(B) receptors in magnocellular neurones in the SON and PVN. Robust GABA(B) receptor immunoreactivity (GABA(B)R; both subunit 1 and subunit 2 of the heterodimer), was observed in the SON and PVN. At the light microcope level, GABA(B)R immonoreactivity displayed a clustered pattern localised both intracytoplasmically and at the plasma membrane. Densitometry analysis indicated that GABA(B)R immunoreactivity was significantly more intense in vasopressin cells than in oxytocin cells, both in male, virgin female and lactating rats, and was denser in males than in virgin females. Light and electron microscope studies indicated that cytoplasmic GABA(B)R was localised in various organelles, including the Golgi, early endosomes and lysosomes, suggesting the cycling of the receptor within the endocytic and trafficking pathways. Some smaller clusters at the level of the cell plasma membrane were apposed to glutamic acid decarboxylase 67 immunoreactive boutons, and appeared to be colocalised with gephyrin, a constituent protein of the postsynaptic density at inhibitory synapses. The presence of GABA(B)R immunoreactivity at synaptic and extrasynaptic sites was supported by electron microscopy. These results provide anatomical evidence for the expression of postsynaptic GABA(B) receptors in magnocellular neurosecretory cells.

Unique CD8+ T cell-rich lymphoid aggregates in human uterine endometrium.

Using confocal scanning laser microscopy of viable tissue sections, we have demonstrated organized lymphoid aggregates (LA), that have a unique structure, in the stratum basalis of uterine endometrium. These LA consist of a core of B cells surrounded by more numerous T cells and an outer halo of monocytes/ macrophages. The T cells in the LA were almost exclusively CD8+CD4-. These CD8+ LA, in terms of both their T cell and B cell components, were either small or absent during the early proliferative stage of the menstrual cycle, significantly larger in size at mid-cycle and during the secretory phase, and absent in post-menopausal women, suggesting that their development is hormonally influenced. This new finding of a menstrual cycle-dependent, phenotypically unique, organized immune cell structure may lead to new insights into the mechanisms by which the endometrium accepts a semiallogeneic graft while providing resistance to infectious organisms.

Neuroendocrine-autonomic integration in the paraventricular nucleus: novel roles for dendritically released neuropeptides.

Communication between pairs of neurones in the central nervous system typically involves classical 'hard-wired' synaptic transmission, characterised by high temporal and spatial precision. Over the last two decades, however, knowledge regarding the repertoire of communication modalities used in the brain has notably expanded to include less conventional forms, characterised by a diffuse and less temporally precise transfer of information. These forms are best suited to mediate communication among entire neuronal populations, now recognised to be a fundamental process in the brain for the generation of complex behaviours. In response to an osmotic stressor, the hypothalamic paraventricular nucleus (PVN) generates a multimodal homeostatic response that involves orchestrated neuroendocrine (i.e. systemic release of vasopressin) and autonomic (i.e. sympathetic outflow to the kidneys) components. The precise mechanisms that underlie interpopulation cross-talk between these two distinct neuronal populations, however, remain largely unknown. The present review summarises and discusses a series of recent studies that have identified the dendritic release of neuropeptides as a novel interpopulation signalling modality in the PVN. A current working model is described in which it is proposed that the activity-dependent dendritic release of vasopressin from neurosecretory neurones in the PVN acts in a diffusible manner to increase the activity of distant presympathetic neurones, resulting in an integrated sympathoexcitatory population response, particularly within the context of a hyperosmotic challenge. The cellular mechanism underlying this novel form of intercellular communication, as well as its physiological and pathophysiological implications, is discussed.

Carbon monoxide and nitric oxide interactions in magnocellular neurosecretory neurones during water deprivation.

Nitric oxide (NO) and carbon monoxide (CO) are diffusible gas messengers in the brain. Previously, we have shown their independent involvement in central fluid/electrolyte homeostasis control. In the present study, we investigated a possible functional interaction between NO/CO in the regulation of vasopressin (VP) and oxytocin (OT) magnocellular neurosecretory cells (MNCs) activity in euhydrated (EU) and dehydrated [48-h water-deprived (48WD)] rats. Using brain slices from EU and 48WD rats, we measured, by immunohistochemistry, the expression of neuronal NO synthase (nNOS, which synthesises NO) and haeme-oxygenase (HO-1, which synthesises CO) in the hypothalamic supraoptic nucleus (SON). In addition, we used patch-clamp electrophysiology to investigate whether regulation of SON MNC firing activity by endogenous CO was dependent on NO bioavailability and GABAergic inhibitory synaptic function. We found a proportion of OT and VP SON MNCs in EU rats to co-express both of HO-1 and nNOS (33.2 ± 2.9% and 15.3 ± 1.4%, respectively), which was increased in 48WD rats (55.5 ± 0.9% and 21.0 ± 1.7%, respectively, P < 0.05 for both). Inhibition of endogenous HO activity [chromium mesoporphyrin IX chloride (CrMP) 20 µm] induced MNC membrane hyperpolarisation and decreased firing activity, and these effects were blunted by previous blockade of endogenous NOS activity (l-NAME, 2 mm) or blockade of inhibitory GABA function [Picrotoxin (Sigma-Aldrich, St Louis, MO, USA), 50 µm]. No significant changes in SON NO bioavailability (4,5 diaminofluorescein diacetate fluorescence) were observed after CrMP treatment. Taken together, our results support a state-dependent functional inter-relationship between NO and CO in MNCs, in which CO acts as an excitatory gas molecule, whose effects are largely dependent on interactions with the inhibitory SON signals NO and GABA.

Immunoglobulin and secretory component regulation in the rat uterus at the time of decidualization.

To determine the effect of decidualization on uterine immunoglobulins (Igs), we measured the levels of IgG, IgA, and secretory component (SC) after induction of artificial decidual cell reactions (DCR) in hormonally primed ovariectomized rats. When progesterone-treated (2.5 mg/day, 3 days) rats received an intraluminal instillation of oil or a needle scratch stimulation in one uterine horn, the stimulated horn had an increase in wet weight and cytoplasmic protein relative to the contralateral horn. Under these conditions, IgG levels increased 10-fold in the lumen of the stimulated horn. This response was selective for IgG, because induction of DCR had no effect on accumulation of IgA or SC in the stimulated horn. The progesterone-induced accumulation of IgG after DCR was further enhanced by estradiol. The addition of a small amount of estradiol (0.2 microgram) on day 3 of a 4-day progesterone pretreatment resulted in further increases in both the wet weight of the stimulated horn and the concentration of IgG in the lumen. The amount of IgG in the lumen of the stimulated horn was 5-fold greater than that in the stimulated horn after progesterone alone. Levels of IgA and SC, however, remained unchanged with this treatment. These results indicate that movement of IgG into the uterine lumen occurs as a part of the DCR and that both an appropriate endocrine balance and physical stimulation are essential for maximal IgG accumulation. Further, they suggest that IgG may play a central role in early pregnancy, which results in the successful implantation of the blastocysts.

Nitric oxide inhibits the firing activity of hypothalamic paraventricular neurons that innervate the medulla oblongata: role of GABA.

Nitric oxide (NO) has been shown to modulate autonomic function by acting both peripherally and centrally. A growing body of evidence indicates that the paraventricular nucleus of the hypothalamus (PVN), an important site for autonomic and endocrine homeostasis, constitutes an important locus mediating central NO actions. However, the cellular targets and mechanisms mediating NO actions within the PVN are not completely understood. Here, we examined whether NO influences the firing activity of identified PVN neurons that innervate two functionally different autonomic centers, the dorsal vagal complex (DVC) and the rostral ventrolateral medulla (RVLM). Perforated patch-clamp recordings were performed in hypothalamic slices containing retrogradely labeled PVN neurons innervating the DVC or the RVLM. Application of the NO donors dyethylamine- or 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) NONOate inhibited the firing activity of both DVC- and RVLM-projecting PVN neurons. Furthermore, application of 2-(4-carboxypheny)-4,4,5,5,-tetramethilimidazoline-1-oxyl-3-oxide (carboxy-PTIO), or the relatively selective neuronal nitric oxide synthase (nNOS) inhibitor 7-nitroindazole alone, increased their basal firing activity, suggesting the presence of an endogenous NO inhibitory tone. GABAergic synaptic activity in PVN neurons was potentiated by NO donors, an action that involved a presynaptic mechanism. Furthermore, the NO-mediated inhibition of firing activity was blocked by the GABA(A) receptor antagonist bicuculline, suggesting that NO-inhibitory actions involved potentiation of local GABAergic synaptic activity. Immunohistochemical studies showed that approximately 25% of DVC- and RVLM-projecting PVN neurons express nNOS, suggesting that a proportion of these medullary-projecting PVN neurons contribute to the cellular source of NO within the PVN. In summary, NO has been identified as an important molecule controlling autonomic function under physiological and pathological conditions. Here, we provide information on the cellular mechanisms mediating central NO actions. Our results demonstrate for the first time that NO modulates the activity of identified populations of PVN neurons that innervate the medulla oblongata, an action that is likely mediated by enhancing synaptic GABAergic function. This work suggests that NO-GABA interaction in PVN neurons that innervate the medulla constitutes an efficient cellular mechanism mediating NO central regulation of autonomic function.

Phenotypic and state-dependent expression of the electrical and morphological properties of oxytocin and vasopressin neurones.

Oxytocin and vasopressin secreting neurones of the hypothalamic supraoptic nucleus share many membrane characteristics and a roughly similar morphology. However, these two neurone types differ in the relative expression of some intrinsic and synaptic currents, and in the extent of their respective dendritic arbors. Spike depolarizing afterpotentials are present in both types, but more frequently give rise to prolonged burst discharges in vasopressin neurones. Oxytocin, but not vasopressin neurones, are characterized by a depolarization-activated, sustained outward rectifier which turns on near spike threshold, and which can produce prolonged spike frequency adaptation. When this sustained current is deactivated by small hyperpolarizing pulses, a rebound depolarization sufficient to evoke short spike trains follows the offset of these pulses. Both oxytocin and vasopressin neurones exhibit a transient outward rectification underlain by an Ia-type current. This transient rectifier delays spiking to depolarizing stimuli from a relatively hyperpolarized baseline, and is more prominent in vasopressin neurones. As a result, oxytocin neurones may be more reactive to depolarizing inputs. Both cell types receive glutamatergic, excitatory synaptic inputs and both possess R,S- alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subtypes. The AMPA receptor channel on both cell types is characterized by a relatively high calcium permeability and voltage-dependent rectification, characteristic of a diminished presence of the GluR2 AMPA subunit. However, AMPA-mediated synaptic transients are larger, and decay faster, in oxytocin compared with vasopressin neurones, suggesting a potential difference for synaptic integration. The characteristics of NMDA-mediated synaptic transients are similar in oxytocin and vasopressin neurones, but some data suggest NMDA receptors may be less involved in the glutamatergic activation of oxytocin neurones. In both cell types, synaptic release of glutamate often coactivates AMPA and NMDA receptors. The dendritic morphology of oxytocin and vasopressin neurones in female rats differs from one another and exhibits considerable plasticity as a function of endocrine state. In virgin rats, oxytocin neurones have more dendritic branches and a greater total dendritic length compared with lactation, when the arbor is much less extensive. A complementary change occurs in vasopressin dendrites, which are more extensive during lactation. This reorganization suggests that oxytocin neurones may be more electronically compact during lactation. In addition, such dramatic shifts in overall dendritic length imply that significant gains and losses in either the total number of synapses, or in synaptic density, are incurred by both cell types as a function of reproductive state.

Activation of postsynaptic GABAB receptors modulate the firing activity of supraoptic oxytocin and vasopressin neurones: role of calcium channels.

Oxytocin and vasopressin release from neurohypophysial terminals is closely related to the firing activity of magnocellular neurones in the supraoptic (SON) and paraventricular nuclei. It is well established that activation of GABAA receptors potently inhibits the activity of SON neurones and, thus, hormone release. However, whether postsynaptic GABAB receptors are expressed in magnocellular neurones, and the role they play in controlling their firing activity, is still controversial. In the present work, we combined immunohistochemical and electrophysiological techniques to determine whether activation of GABAB receptors in identified oxytocin and vasopressin neurones modulates their firing activity. Patch-clamp recordings from SON neurones were obtained either in the slice preparation or from acutely dissociated neurones. Activation of GABAB receptors with the selective agonist baclofen (10 micro m) inhibited voltage-gated Ca2+ currents, reduced the duration of individual action potentials, as well as the magnitude of the hyperpolarizing after-potential. SON firing activity was reduced by baclofen, and effect that was accompanied by a small membrane hyperpolarization. The inhibition of firing discharge persisted in the presence of synaptic blockade media, and was also observed in acutely dissociated SON neurones. Finally, GABAB-mediated modulation of firing activity was largely blocked by the Ca2+ channel blocker Co2+ (2 mm). In general, baclofen modulatory actions were significantly larger, or observed more predominantly, in vasopressin neurones. In summary, these results support the expression of functional postsynaptic GABAB receptors in SON neurones, activation of which efficiently modulates neuronal excitability, in a Ca2+- and cell-type dependent manner.

Effect of parathyroid hormone and calcitonin on cholinergic markers in rat parathyroid gland.

The effect of parathyroid hormone (PTH) and calcitonin on 3H-choline uptake and on 3H-acetylcholine synthesis by rat parathyroid glands were examined. Incubation of tissue for 120 min with PTH or calcitonin resulted in an inverted bell-shaped, dose-dependent inhibition of 3H-choline uptake, with a maximal effect at 10(-9) M concentration. The effect of PTH on parathyroid choline uptake was blunted by preincubation with the PTH antagonist NLe(8-18)-PTH (3-34) amide. PTH brought about a dose-dependent inhibition of 3H-choline conversion to 3H-acetylcholine, with significant effects at 10(-8) M concentration or higher. This inhibitory effect of PTH on 3H-acetylcholine synthesis was blocked by co-incubation with the PTH antagonist NLe(8-18)-PTH (3-34) amide. Only at a 10(-7) M concentration calcitonin was effective to impair the in vitro conversion of radioactive choline into 3H-acetylcholine by parathyroid fragments. The results indicate that, in vitro, PTH, and to a less extent, calcitonin, inhibit cholinergic activity in rat parathyroid glands.

The secretory immune system in the uterus of the pregnant rat: production of secretory component by uterine tissues.

Secretory component (SC) was measured in amniotic fluid, fetal serum, and maternal serum and compared with SC production during in vitro culture of uterine tissue segments from pregnant rats. The concentrations of SC in amniotic fluid did not change between days 14 and 20 of pregnancy. Similarly, there was no change in maternal or fetal serum during pregnancy, although, the levels of SC in sera were consistently higher than those in amniotic fluid. When uterine segments were incubated in vitro, release of SC was greater in the absence of cycloheximide than in the presence of cycloheximide at all stages of pregnancy. In contrast to SC values in amniotic fluid, however, SC production by uterine tissue changed markedly during pregnancy. SC levels were low during early pregnancy (day 7 post coitus) and increased to levels found in non-pregnant diestrous rats just prior to parturition (day 20). The findings suggest that the endocrine balance during pregnancy may play a central role in regulation of the uterus immune system. The pattern of SC release may reflect a need both to ensure protection of the fetus from the IgA immune system in early pregnancy and to prevent maternal infection during parturition by reactivation of this system.

Secretory immune system of the male reproductive tract: effects of dihydrotestosterone and estradiol on IgA and secretory component levels.

Immunoglobulin A (IgA) is present at mucosal sites of the body which are exposed to the external environment. In this study we evaluated the levels of IgA and its transport protein secretory component (SC) in organs of the male reproductive tract of both intact and castrate-hormone-treated rats. Our goals were to determine whether these proteins are present in the male reproductive tract and whether sex hormones can influence the amounts of IgA and SC in selected organs. We found that in intact animals, IgA was present in the prostate, epididymis, vas deferens and testis and that SC levels in the prostate were 22-fold greater than in these same organs. Dihydrotestosterone (DHT) and/or estradiol, when administered to castrate animals, dramatically increased the levels of prostatic SC. In contrast, the levels of IgA were only minimally affected. DHT administration also resulted in a significant increase in SC found in the seminal vesicles. These studies demonstrate that IgA and SC are present in the male reproductive tract of the rat. Further, they show that androgens and estrogens act at selected sites in the male reproductive tract to play an important role in maintaining SC levels and thereby suggest that these hormones influence the movement of IgA from tissues into secretions.

Regulation by human uterine cells of PBMC proliferation: influence of the phase of the menstrual cycle and menopause.

To determine the influence of human uterine cells recovered at different stages of the menstrual cycle and following menopause on the proliferation of peripheral blood mononuclear cells (PBMC), whole cell suspensions of uterine tissues were co-cultured with autologous and heterologous PBMC. PBMC proliferation in response to tetanus toxoid (TT) or Con A was inhibited by uterine endometrial cells and was dependent on the phase of the menstrual cycle. Inhibition by cells from the proliferative phase was significantly greater than by cells from the secretory phase. Uterine cells isolated from post-menopausal women also inhibited proliferation of PBMC. Cell fractionation studies indicated that epithelial cells are the primary source of uterine inhibitory activity. When epithelial cells and PBMC were cultured in separate compartments, epithelial cells released a soluble factor(s) that inhibited the PBMC proliferation. These results suggest that uterine epithelial cells produce cytokines that down-regulate the proliferation of PBMC in response to antigens and mitogens. This may be important for the control of uterine immune responses, as well as the growth of the reproductive tract in preparation for implantation during the secretory phase of the menstrual cycle.