Peg3 (paternally expressed gene 3) is an imprinted gene encoding a DNA-binding protein. This gene plays important roles in controlling fetal growth rates and nurturing behaviors. In the current study, a new mutant mouse model has been generated to further characterize the functions of this DNA-binding protein. Besides known phenotypes, this new mutant model also revealed potential roles of Peg3 in mammalian reproduction. Female heterozygotes produce a much smaller number of mature oocytes than the wild-type littermates, resulting in reduced litter sizes. According to genome-wide expression analyses, several placenta-specific gene families are de-repressed in the brain of Peg3 heterozygous embryos, including prolactin, cathepsin and carcinoembryonic antigen cell adhesion molecule (Ceacam) families. The observed de-repression is more pronounced in females than in males. The de-repression of several members of these gene families is observed even in the adult brain, suggesting potential defects in epigenetic setting of the placenta-specific gene families in the Peg3 mutants. Overall, these results indicate that Peg3 likely controls the transcription of several placenta-specific gene families, and further suggest that this predicted transcriptional control by Peg3 might be mediated through unknown epigenetic mechanisms.

We have constructed mathematical models of the electrical activity of two hypothalamic supraoptic neuro-secretory cell-types, and we support our models with new calcium imaging and in vitro electrophysiological data. These cells are neurones that project to the pituitary gland and secrete either of two hormones, oxytocin or vasopressin, into the blood from their axonal terminals. Oxytocin-secreting and vasopressin-secreting cells are closely related and physically they differ only subtly, however when physiologically stressed their discharge patterns are dramatically distinct. We first show how each potassium current contributes to the action-potentials and after-potentials observed in these cells, and we show how these after-potentials are correlated to intra-cellular calcium elevations. We then show how these currents regulate the excitability of these cells and consequently shape their discharge pattern.

To better understand the plasticity of intrinsic membrane properties of supraoptic magnocellular neuroendocrine cells associated with reproductive function, intracellular recordings were performed in oxytocin (OT) and vasopressin (VP) neurones from virgin, late pregnant (E19-22), and lactating (8-12 days of lactation) rats in vitro, using hypothalamic explants. OT neurones from virgin rats displayed a narrower spike width than neurones from pregnant and lactating rats, characterized by faster rise and decay times. Spike width changes in VP neurones were not as prominent as those observed in OT neurones. In OT neurones, the amplitude and the decay of the afterhyperpolarization following spike trains was significantly larger and faster, respectively, in pregnant and lactating rats compared to virgin rats. These properties did not change during pregnancy and lactation in VP neurones. The incidence of the depolarizing afterpotential following spikes significantly increased from approximately 20% in virgin rats to 40-50% during pregnancy and lactation in OT neurones, but was stable (80-90%) across states in VP neurones. Repetitive firing properties (frequency adaptation, the first interspike interval frequency and frequency-current (F-I) relationship) were altered during pregnancy and lactation in OT neurones, but not VP neurones. The increased incidence of depolarizing afterpotentials in OT neurones enhances excitability, while the increased afterhyperpolarization results in suppression of firing rate. Thus, the changes may favour the short bursting activity seen in OT neurones during lactation. These results confirmed reproductive state-dependent changes in intrinsic membrane properties of OT neurones during lactation, and suggest these changes are in place during late pregnancy. This argues that the plasticity in the electrical properties in OT neurones associated with lactation is not instigated by suckling.

Neuronal excitability in the adult brain is controlled by a balance between synaptic excitation and inhibition mediated by glutamate and GABA, respectively. While generally inhibitory in the adult brain, GABAA receptor activation is excitatory under certain conditions in which the GABA reversal potential is shifted positive due to intracellular Cl− accumulation, such as during early postnatal development and brain injury. However, the conditions under which GABA is excitatory are generally either transitory or pathological. Here, we reveal GABAergic synaptic inputs to be uniformly excitatory in vasopressin (VP)-secreting magnocellular neurons in the adult hypothalamus under normal conditions. The GABA reversal potential (EGABA) was positive to resting potential and spike threshold in VP neurons, but not in oxytocin (OT)-secreting neurons. The VP neurons lacked expression of the K+-Cl− co-transporter 2 (KCC2), the predominant Cl− exporter in the adult brain. The EGABA was unaffected by inhibition of KCC2 in VP neurons, but was shifted positive in OT neurons, which express KCC2. Alternatively, inhibition of the Na+-K+-Cl− co-transporter 1 (NKCC1), a Cl− importer expressed in most cell types mainly during postnatal development, caused a negative shift in EGABA in VP neurons, but had no effect on GABA currents in OT neurons. GABAA receptor blockade caused a decrease in the firing rate of VP neurons, but an increase in firing in OT neurons. Our findings demonstrate that GABA is excitatory in adult VP neurons, suggesting that the classical excitation/inhibition paradigm of synaptic glutamate and GABA control of neuronal excitability does not apply to VP neurons.

The firing pattern of oxytocin (OT) hormone synthesizing neurons changes dramatically immediately before each milk ejection, when a brief burst of action potentials is discharged. OT neurons possess intrinsic currents that would modulate this burst. Our previous studies showed the amplitude of the Ca 2+ -dependent afterhyperpolarization (AHP) following spike trains is significantly larger during lactation. In the present study we sought to determine which component of the AHP is enhanced, and whether the enhancement could be related to changes in whole-cell Ca 2+ current or the Ca 2+ transient in identified OT or vasopressin (VP) neurons during lactation. We confirmed, with whole-cell current-clamp recordings, our previous finding from sharp electrodes that the size of the AHP following spike trains increased in OT, but not VP neurons during lactation. We then determined that an apamin-sensitive medium-duration AHP (mAHP) and an apamin-insensitive slow AHP (sAHP) were specifically increased in OT neurons. Simultaneous Ca 2+ imaging revealed that the peak change in somatic [Ca 2+ ] i was not altered in either cell type, but the slow decay of the Ca 2+ transient was faster in both cell types during lactation. In voltage clamp, the whole-cell, Ca 2+ current was slightly larger during lactation in OT cells only, but current density was unchanged when corrected for somatic hypertrophy. The currents, I mAHP and I sAHP , also were increased in OT neurons only, but only the apamin-sensitive I mAHP showed an increase in current density after adjusting for somatic hypertrophy. These findings suggest a specific modulation (e.g. increased number) of the small-conductance Ca 2+ -dependent K + (SK) channels, or their interaction with Ca 2+ , underlies the increased mAHP/I mAHP during lactation. This larger mAHP may be necessary to limit the explosive bursts during milk ejection.

BackgroundDespite the availability of several antihypertensive medications, the morbidity and mortality caused by hypertension is on the rise, suggesting the need for investigation of novel signaling pathways involved in its pathogenesis. Recent evidence suggests the role of toll-like receptor (TLR) 4 in various inflammatory diseases, including hypertension. The role of the brain in the initiation and progression of all forms of hypertension is well established, but the role of brain TLR4 in progression of hypertension has never been explored. Therefore, we investigated the role of TLR4 within the paraventricular nucleus (PVN; an important cardioregulatory center in the brain) in an animal model of human essential hypertension. We hypothesized that a TLR4 blockade within the PVN causes a reduction in mean arterial blood pressure (MAP), inflammatory cytokines and sympathetic drive in hypertensive animals.MethodsSpontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were administered either a specific TLR4 blocker, viral inhibitory peptide (VIPER), or control peptide in their PVN for 14 days. MAP was recorded continuously by radiotelemetry. PVN and blood were collected for the measurement of pro-inflammatory cytokines (Tumor Necrosis Factor (TNF)-α, interleukin (IL)-1β), anti-inflammatory cytokine IL-10, inducible nitric oxide synthase (iNOS), TLR4, nuclear factor (NF) κB activity and plasma norepinephrine (NE) and high mobility group box (HMGB)1 expression, respectively.ResultsHypertensive rats exhibited significantly higher levels of TLR4 in the PVN. TLR4 inhibition within the PVN attenuated MAP, improved cardiac hypertrophy, reduced TNF-α, IL-1β, iNOS levels, and NFκB activity in SHR but not in WKY rats. These results were associated with a reduction in plasma NE and HMGB1 levels and an increase in IL-10 levels in SHR.ConclusionsThis study demonstrates that TLR4 upregulation in PVN plays an important role in hypertensive response. Our results provide mechanistic evidence that hypertensive response in SHR are mediated, at least in part, by TLR4 in the PVN and that inhibition of TLR4 within the PVN attenuates blood pressure and improves inflammation, possibly via reduction in sympathetic activity.

Oxytocin (OT) and vasopressin (VP) synthesizing magnocellular cells (MNCs) in the supraoptic nucleus (SON) display distinct firing patterns during the physiological demands for these hormones. Depolarizing afterpotentials (DAPs) in these neurons are involved in controlling phasic bursting in VP neurons. Our whole cell recordings demonstrated a Cs(+)-resistant fast DAP (fDAP; decay tau = approximately 200 ms), which has not been previously reported, in addition to the well-known Cs(+)-sensitive slower DAP (sDAP; decay tau = approximately 2 s). Immunoidentification of recorded neurons revealed that all VP neurons, but only 20% of OT neurons, expressed the fDAP. The activation of the fDAP required influx of Ca(2+) through voltage-gated Ca(2+) channels as it was strongly suppressed in Ca(2+)-free extracellular solution or by bath application of Cd(2+). Additionally, the current underlying the fDAP (I(fDAP)) is a Ca(2+)-activated current rather than a Ca(2+) current per se as it was abolished by strongly buffering intracellular Ca(2+) with BAPTA. The I-V relationship of the I(fDAP) was linear at potentials less than -60 mV but showed pronounced outward rectification near -50 mV. I(fDAP) is sensitive to changes in extracellular Na(+) and K(+) but not Cl(-). A blocker of Ca(2+)-activated nonselective cation (CAN) currents, flufenamic acid, blocked the fDAP, suggesting the involvement of a CAN current in the generation of fDAP in VP neurons. We speculate that the two DAPs have different roles in generating after burst discharges and could play important roles in determining the distinct firing properties of VP neurons in the SON neurons.

Teruyama R, Sakuraba M, Wilson LL, Wandrey NE, Armstrong WE. Epithelial Na ϩ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei. Am J Physiol Endocrinol Metab 302: E273-E285, 2012. First published November 1, 2011; doi:10.1152/ajpendo.00407.2011.-The epithelial Na ϩ channels (ENaCs) are present in kidney and contribute to Na ϩ and water homeostasis. All three ENaC subunits (␣, ␤, and ␥) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the ␣-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for ␣-ENaC. In contrast, immunoreactivity to ␤-and ␥-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT. aldodsterone; vasopressin; oxytocin THE NEUROHYPOPHYSIAL HORMONES vasopressin (VP) and oxytocin (OT) are synthesized in the magnocellular neurons (MNCs) located within the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) of the hypothalamus and released from the neurohypophysis into the general circulation in response to physiological demands. The secretion of VP increases in response to hyperosmolality, hypovolemia, and hypotension and produces antidiuretic and pressor effects (59). In addition to the well-known effects of OT during parturition and lactation, plasma OT also increases with hyperosmolality and hypernatremia (32) and induces natriuresis (13, 31).The non-voltage-dependent, amiloride-sensitive epithelial Na ϩ channels (ENaCs) are present in the apical membrane of epithelial cells in a variety of tissues, such as urinary bladder, renal collecting duct, distal colon, sweat and salivary glands, lung, and taste buds, and are known to mediate the transport of Na ϩ across epithelia (7, 21). Thus, together with the Na ϩ /K ϩ -ATPase present in the basal membrane of epithelial cells, ENaCs regulate transe...