The application of human pluripotent stem cells to model the neuronal and glial components of neurodevelopmental disorders.

Cellular models of neurodevelopmental disorders provide a valuable experimental system to uncover disease mechanisms and novel therapeutic strategies. The ability of induced pluripotent stem cells (iPSCs) to generate diverse brain cell types offers great potential to model several neurodevelopmental disorders. Further patient-derived iPSCs have the unique genetic and molecular signature of the affected individuals, which allows researchers to address limitations of transgenic behavioural models, as well as generate hypothesis-driven models to study disorder-relevant phenotypes at a cellular level. In this article, we review the extant literature that has used iPSC-based modelling to understand the neuronal and glial contributions to neurodevelopmental disorders including autism spectrum disorder (ASD), Rett syndrome, bipolar disorder (BP), and schizophrenia. For instance, several molecular candidates have been shown to influence cellular phenotypes in three-dimensional iPSC-based models of ASD patients. Delays in differentiation of astrocytes and morphological changes of neurons are associated with Rett syndrome. In the case of bipolar disorders and schizophrenia, patient-derived models helped to identify cellular phenotypes associated with neuronal deficits (e.g., excitability) and mutation-specific abnormalities in oligodendrocytes (e.g., CSPG4). Further we provide a critical review of the current limitations of this field and provide methodological suggestions to enhance future modelling efforts of neurodevelopmental disorders. Future developments in experimental design and methodology of disease modelling represent an exciting new avenue relevant to neurodevelopmental disorders.

Adiponectin increases insulin content and cell proliferation in MIN6 cells via PPARγ-dependent and PPARγ-independent mechanisms.

AIMS: Adiponectin is an important adipokine whose levels are decreased in obesity despite increases in adipocyte mass. Studies in animal models implicate adiponectin as an insulin sensitizer in skeletal muscle and liver. Thiazolidinediones (TZDs) are insulin sensitizers and ligands for peroxisome proliferator-activated γ receptors (PPARγ) and these receptors are expressed in ß cells where their activation promotes cell survival. We hypothesize that adiponectin promotes ß cell survival by activating PPARγ. METHODS: We used MIN6 cells to investigate the effect of adiponectin on PPARγ expression, ß-cell proliferation, insulin synthesis and insulin secretion. RESULTS: We demonstrate that MIN6 cells contain adiponectin receptors and that adiponectin activates PPARγ mRNA and protein expression. This increase in PPARγ expression is blocked by the PPARγ antagonist, GW9662, indicating a transcriptional feedback loop involving PPARγ activation of itself. Adiponectin causes a significant increase in insulin content and secretion and this occurs also via PPARγ activation due to the inhibitory effect of GW9662. Adiponectin also promotes MIN6 cell proliferation, however, this effect is independent of PPARγ activation. CONCLUSIONS: Our results identify novel roles for the adipokine, adiponectin, in ß-cells function. Adiponectin upregulates PPARγ expression, insulin content and insulin secretion through PPARγ-dependent mechanisms. Reductions in circulating adiponectin levels in obese individuals could therefore result in negative effects on ß-cell function and this may have direct relevance to ß-cell dysfunction in type 2 diabetes.

Glucocorticoid treatment does not alter early cardiac adaptations to growth restriction in preterm sheep fetuses.

OBJECTIVE: To study the consequences of glucocorticoid treatment in fetal growth restriction (FGR) on cardiac function. SETTING: Laboratory. SAMPLE: Sheep. METHODS: Growth restriction was induced in sheep fetuses using single umbilical artery ligation (SUAL) on days 105-110 of gestation (term 147). Control fetuses were not ligated. Betamethasone (BM) (11.4 mg intramuscularly) or saline was administered to ewes on days 5 and 6 after surgery. Ewes were anaesthetised on day 7, the fetuses were removed, and their hearts were mounted on a Langendorff apparatus. Balloon catheters were inserted into the right and left ventricles. OUTCOME MEASURES: Ventricular contractile function and infarct area following ischaemia/reperfusion. RESULTS: The SUAL resulted in FGR (body weight 77% of control). The FGR was associated with increases in basal left ventricular pressure development and rates of contraction and relaxation. Right ventricular contraction was unaffected. Following brief ischaemia/reperfusion, the infarct area in FGR hearts was increased four-fold compared with controls. Antenatal BM resulted in a proportional increase in heart size and coronary flow, especially in FGR fetuses, and left ventricular pressure and heart rate responses to ß-adrenoceptor activation were increased. CONCLUSIONS: Fetal hearts rapidly adapt to FGR to maintain substrate delivery to the brain and heart. The FGR greatly enhanced the area of ischaemia, with implications for susceptibility in postnatal life. Antenatal BM treatment does not interfere with these cardiac changes but appears to increase left ventricle ß-adrenoceptor responsiveness, which may render the offspring vulnerable to subsequent cardiac dysfunction.

Oxytocin depolarizes mitochondria in isolated myometrial cells.

Oxytocin is known to play important roles in uterine contractions, mediated at least in part by increasing intracellular Ca(2+) concentration ([Ca(2+)](i)), through enhancing extracellular Ca(2+) entry and Ca(2+) release from the sarcoplasmic reticulum, processes that are intimately linked with mitochondria. This study examined the effects of oxytocin on mitochondrial function. This was achieved by measuring the ratiometric JC-1 fluorescence signal in isolated myometrial cells, which provides a relative measure of the mitochondrial membrane potential (ψ(m)), and also by loading the cells with Oregon Green BAPTA-AM to examine changes in [Ca(2+)](i). Oxytocin (1 nm) depolarized the ψ(m) to 73.8 ± 3.7% of the control value (P < 0.05; perfused for 11 min) and also caused a transient increase in [Ca(2+)](i). The depolarization of mitochondrial membrane potential was effectively reversed by 2-aminoethoxydiphenyl borate, nifedipine, Ca(2+)-free solution or oligomycin, with the ratiometric JC-1 fluorescence signal becoming no different from the control value in all cases (i.e. P > 0.05). The reduction in ψ(m) is likely to occur at least in part through the oxytocin-induced increase in [Ca(2+)](i), causing enhanced mitochondrial uptake of Ca(2+) and resultant dissipation of the mitochondrial electrochemical gradient. ATP synthase is also stimulated, which would further contribute to a decrease in ψ(m).

Role of Ca2+ entry and Ca2+ stores in atypical smooth muscle cell autorhythmicity in the mouse renal pelvis.

BACKGROUND AND PURPOSE: Electrically active atypical smooth muscle cells (ASMCs) within the renal pelvis have long been considered to act as pacemaker cells driving pelviureteric peristalsis. We have investigated the role of Ca2+ entry and uptake into and release from internal stores in the generation of Ca2+ transients and spontaneous transient depolarizations (STDs) in ASMCs. EXPERIMENTAL APPROACH: The electrical activity and separately visualized changes in intracellular Ca2+ concentration in typical smooth muscle cells (TSMCs), ASMCs and interstitial cells of Cajal-like cells (ICC-LCs) were recorded using intracellular microelectrodes and a fluorescent Ca2+ indicator, fluo-4. RESULTS: In 1 microM nifedipine, high frequency (10-30 min(-1)) Ca2+ transients and STDs were recorded in ASMCs, while ICC-LCs displayed low frequency (1-3 min(-1)) Ca2+ transients. All spontaneous electrical activity and Ca2+ transients were blocked upon removal of Ca2+ from the bathing solution, blockade of Ca2+ store uptake with cyclopiazonic acid (CPA) and with 2-aminoethoxy-diphenylborate (2-APB). STD amplitudes were reduced upon removal of the extracellular Na+ or blockade of IP3 dependent Ca2+ store release with neomycin or U73122. Blockade of ryanodine-sensitive Ca2+ release blocked ICC-LC Ca2+ transients but only reduced Ca2+ transient discharge in ASMCs. STDs in ASMCS were also little affected by DIDS, La3+, Gd3+ or by the replacement of extracellular Cl(-) with isethionate. CONCLUSIONS: ASMCs generated Ca2+ transients and cation-selective STDs via mechanisms involving Ca2+ release from IP3-dependent Ca2+ stores, STD stimulation of TSMCs was supported by Ca2+ entry through L type Ca2+ channels and Ca2+ release from ryanodine-sensitive stores.

Nonlinear effects of potassium channel blockers on endothelium-dependent hyperpolarization.

In a number of published studies on endothelium-dependent hyperpolarization and relaxation, the results of the effects of K+ blockers have been difficult to interpret. When the effects of two blockers have been studied, often either blocker by itself had little effect, whereas the two blockers combined tended to abolish the responses. Explanations suggested in the literature include an unusual pharmacology of the K+ channels, and possible blocker binding interactions. In contrast, when we applied the same blockers to segments of small blood vessels under voltage clamp, the blockers reduced the endothelium-dependent K+ current in a linearly additive manner. Resolution of these contrasting results is important as endothelium-derived hyperpolarization (EDH) makes its greatest contribution to vasorelaxation in arterioles and small resistance arteries, where it can exert a significant role in tissue perfusion and blood pressure regulation. Furthermore, EDH is impaired in various diseases. Here, we consider why the voltage-clamp results differ from earlier free-running membrane potential and contractility studies. We fitted voltage-clamp-derived current-voltage relationships with mathematical functions and considered theoretically the effects of partial and total block of endothelium-derived K+ -currents on the membrane potential of small blood vessels. When the K+ -conductance was partially reduced, equivalent to applying a single blocker, the effect on EDH was small. Total block of the endothelium-dependent K+ conductance abolished the hyperpolarization, in agreement with various published studies. We conclude that nonlinear summation of the hyperpolarizing response evoked by endothelial stimulation can explain the variable effectiveness of individual K+ channel blockers on endothelium-dependent hyperpolarization and resulting relaxation.

Response of the rat myometrium to phenylephrine in early pregnancy and the effects of 6-hydroxydopamine.

1. The contractile responses of the longitudinal and circular muscle layers of the rat uterus to the alpha 1-adrenoceptor agonist phenylephrine were measured on days 3-6 of gestation. There was a progressive increase in sensitivity to phenylephrine in both muscle layers between days 3 and 6 of gestation. Overall, this amounted to a 13 and 9 fold increase in sensitivity in longitudinal and circular muscles, respectively. In longitudinal muscle the slope of the Hill plot was 2 on day 3 of pregnancy and was decreased to 1 thereafter. 2. The sympathetic nerve terminals innervating the smooth muscle of the uterus were destroyed by administration of 6-hydroxydopamine (2 x 50 mg kg-1) 4-7 days before testing with phenylephrine. Following this treatment there was a significant increase in sensitivity to phenylephrine on day 3 in both muscle layers. After day 4, the longitudinal muscle was less sensitive to phenylephrine. 3. In the longitudinal muscle there was a progressive increase in the contractile response to maximal concentrations of phenylephrine and to high potassium (100 mM) between days 3 and 6 of pregnancy. In the circular muscle the responsiveness to both phenylephrine and potassium remained unchanged between days 3 and 6 of gestation. 6-Hydroxydopamine had no effect on the maximal responses to phenylephrine or high potassium in either muscle layer. 4. In conclusion, denervation supersensitivity of uterine smooth muscle following injection of 6-hydroxydopamine is observed only on day 3 of pregnancy and appears to be replaced by subsensitivity by day 6. The decrease in the slope of the Hill plot in longitudinal muscle after day 3 may be explained by changes in events between activation of alpha 1-adrenoceptors and contraction.

EDHF, NO and a prostanoid: hyperpolarization-dependent and -independent relaxation in guinea-pig arteries.

The contribution of endothelium-derived hyperpolarizing factor (EDHF), nitric oxide (NO) and a prostanoid (PG) to endothelium-dependent hyperpolarization and relaxation were assessed in coronary and mammary arteries of guinea-pigs by integration of the responses evoked during discrete applications of acetylcholine (ACh). The results of this integration approach were compared with those using traditional peak analysis methods. N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) and indomethacin (1 microM), alone or in combination, were without effect on peak hyperpolarizations or relaxations while they markedly reduced the integrated responses in both arteries. Integrated responses attributed to NO and PG were larger than those attributed to EDHF in the coronary artery (at 2 microM ACh, hyperpolarization (mV s): NO, 4200+/-91; PG, 5046+/-157; EDHF, 1532+/-94; relaxation (mN s mm(-1)): NO, 2488+/-122; PG, 2234+/-96; EDHF, 802+/-54). Integrated responses attributed to NO, PG and EDHF were similar in the mammary artery (at 2 microM ACh, hyperpolarization: NO, 347+/-69; PG, 217+/-49; EDHF, 310+/-63; relaxation: NO, 462+/-94; PG, 456+/-144; EDHF, 458+/-40). Gilbenclamide (1 microM) all but abolished the hyperpolarization attributable to NO and PG but not EDHF in both arteries allowing assessment of the role of the hyperpolarization in relaxation. Gilbenclamide was without effect on the integrated relaxation due to NO but significantly reduced the relaxation associated with PG in the two arteries. In conclusion, integration of the responses enabled a more complete assessment of the contribution of EDHF, NO and PG to endothelium-dependent responses, which were strikingly different in the two arteries. There is commonality in the role of hyperpolarization in relaxation in both arteries: EDHF-dependent relaxation is strongly dependent on hyperpolarization; hyperpolarization plays an important role in PG relaxation, whereas it has a small facilitatory role in NO-dependent relaxation.

Pilocarpine-induced relaxation of rat tail artery by a non-cholinergic mechanism and in the absence of an intact endothelium.

1. The partial muscarinic agonist, pilocarpine, evoked concentration-dependent relaxation with an EC50 of 2.4 x 10(-3) M in isolated segments of rat tail artery that were constricted with phenylephrine (10(-8) to 2 x 10(-7) M). Acetylcholine also evoked concentration-dependent relaxation but was more potent than pilocarpine (EC50, 6.5 x 10(-7) M). 2. The concentration-relaxation curves for pilocarpine were not affected by the muscarinic antagonists, atropine (10(-9) M) or pirenzepine (5 x 10(-7) M), while the concentration-relaxation curves for acetylcholine-evoked relaxation of the same tissues were shifted some 10 fold to the right by these concentrations of atropine and pirenzepine. 3. Acetylcholine failed to evoke relaxation following removal of the endothelium. The smooth muscle of the rat tail artery was some 10 fold more sensitive to the relaxing action of pilocarpine following denudation of the endothelium. 4. The effects of pilocarpine and acetylcholine on membrane potential were studied in tissues that were depolarized to -39 +/- 1 mV with phenylephrine (5 x 10(-8) to 2 x 10(-7) M). In intact tissues, pilocarpine caused hyperpolarization, an effect that persisted in the presence of muscarinic antagonists. Acetylcholine also evoked hyperpolarization. 5. Following removal of the endothelium, pilocarpine (10(-5) to 10(-3) M) evoked hyperpolarization in 6 of 15 preparations and a decrease in the frequency of action potentials in the remainder. Both of these responses were associated with relaxation. 6. The effects of pilocarpine were compared with other agents that evoke endothelium-independent relaxation. The concentration-relaxation curves in response to pilocarpine and nitroprusside were shifted to the right by ferricyanide (10-5 M) and methylene blue (10-5 M). Glibenclamide (10-6 M) was without effect on the hyperpolarization and relaxation evoked by pilocarpine (10' to 10- M).7. Thus, pilocarpine evokes relaxation of rat tail artery independently of the cholinergic system and it is suggested that this is achieved by decreasing the frequency of action potentials in the smooth muscle.

Release of endothelium-derived hyperpolarizing factor (EDHF) by M3 receptor stimulation in guinea-pig coronary artery.

1. The muscarinic receptor subtype(s) involved in the release of endothelium-derived hyperpolarizing factor (EDHF) were studied in the guinea-pig coronary artery by recording the membrane potential in the smooth muscle cells with intracellular microelectrodes. 2. Acetylcholine (ACh, pD2 6.68) was 10 times more potent than the M2 agonist, oxotremorine (pD2 5.65) and 500 fold more potent than the M1 agonist, McN-A-343 (pD2 3.95) in evoking the EDHF hyperpolarization. 3. The M3 muscarinic antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was the most potent (pA2 9.5) in inhibiting the release of EDHF evoked by ACh, being more potent than pirenzepine (pA2 6.7), and AFDX-116 (pA2 6.1) which preferentially block M1 and M2 receptors, respectively. 4. These results suggest that EDHF is released from the endothelium of the guinea-pig coronary artery upon the activation of the muscarinic M3 receptor subtype.

Endothelium-dependent hyperpolarization in resting and depolarized mammary and coronary arteries of guinea-pigs.

1. The membrane potential responses in guinea-pig coronary and mammary arteries attributable to endothelium-derived nitric oxide (NO), prostaglandin (PG) and hyperpolarizing factor (EDHF), and to exogenous NO and the prostacyclin analogue, iloprost, were compared at rest and when depolarized with the thromboxane analogue, U46619. 2. In the coronary artery, stimulation of the endothelium with acetylcholine (ACh) evoked hyperpolarization attributable to NO and a PG with similar pD2s at rest and in the presence of U46619. However, in depolarized tissues, the pD2 of the response attributed to EDHF required a 10 fold lower concentration of ACh compared with at rest. 3. In the mammary artery, lower concentrations of ACh were required to evoke NO- and EDHF-dependent hyperpolarizations in depolarized mammary artery compared with at rest, while PG-dependent hyperpolarization did not occur until the concentration of ACh was increased some 10 fold both at rest and in U46619. 4. The smooth muscle of the coronary artery of guinea-pigs was some 4 fold more sensitive to exogenous NO and iloprost than was the mammary artery. 5. In conclusion, the membrane potential response in arteries at rest, that is, in the absence of constrictor, may be extrapolated to events in the presence of constrictor when NO and PG are under study. However, the sensitivity to ACh and the magnitude of the hyperpolarization attributed to EDHF obtained in tissues at rest may underestimate these parameters in depolarized tissues.

Sustained effects on synthetic ovarian steroids of rat myometrial contractility.

Ovariectomized rats were given a single dose of synthetic oestrogen or progestogen, or both combined. On selected days after treatment one rat from each treatment group was killed and myometrical contractility in response to spasmogens was measured isometrically in isolated tissue baths. Contractility persisted at a low level after ovariectomy without steroid replacement (controls) for the 60 days of the experiment. The powerful potentiating action of oestrogen reached a maximum in about 10 days and sustained a high level thereafter. Progestogen treatment did not influence contractility to a significant degree when compared with saline, except that the contractions were sometimes of higher frequency and more irregular in size. There was a less powerful potentiating action on contractility with combined oestrogen and progestogen treatment than with oestrogen alone. It reached a maximum in 4 days and declined more rapidly than with oestrogen-treated preparation but was still vigorous for up to 30 days.

PIP: Virgin female Wistar rats were ovariectomized and 2 days later were given a single .2 ml dose of steroid im. Medroxyprogesterone acetate, 100 mg/kg of body weight or estradiol valerate 100 mcg were used. Group 1 (controls) received only solvents, Group 2 received progestogen in saline, Group 3 received the estrogen in oil, and Group 4 received both steroids. On selected days after treatment a rat from each group was killed, the uterus removed, and 2 myometrial strips were suspended in isolated organ baths on Statha G10B strain gauges for isometric recording. After 30 minutes, a dose of either 10 mcg/ml of acetylcholine or .25 mcg/ml of oxytocin was added to each bath. Tension was recorded for 10 minutes followed by a 15-minute wash. These doses were supramaximal. Responses began with a brief contraction and then became a series of fast powerful contrations which continued for 30-60 minutes. Each strip was later removed, prepared for sectioning, and length and cross-section were measured. Controls responded similarly to saline and to oil injections. There was a significnt initial increase in the response to both acetylcholine (p less than .05) and oxytocin (p less than .01). From Days 6 to 20 the responses did not change significantly. After Day 20 the response to acetylcholine declined significantly (p less than .01) but the response to oxytocin was unchanged for the 60 days of the test. With estrogen treatment, increase in response to both spasmogens was highly significant (p less than .001) in the first 18 days at which time it reached a plateau and remained the same for 60 days. With the progestogen treatment the mean response to acetylcholine increased significantly for the first 2 days (p less than .01) then maintained a plateau for the next 3 days equal to the control group and subsequently declined slowly (p less than .001). The contractile response to oxytocin remained constant for 60 days. With estrogen plus progestogen treatment there was a significant increase in contractility up to Day 5 (p less than .001). From Days 5 to 9 plateau levels were maintained with acetycholine and at a lower level with oxytocin. After Day 9 there was a significant decline in both spasmogens effect (p less than .001). With controls and all steroid treatments there was a highly significant difference in responses to acetylcholine and to oxytocin, with acetylcholine consistently giving stronger responses (p less than .001) than oxytocin. Results indicated that medroxyprogesterone acetate required the presence of estrogen for its myometrial action. There was also evidence that estrogen actions were modified by progestogen. It is concluded that single doses of estrogen and progestogen had depot actions of adequate intensity and duration for prolonged studies in myometrial contractility and that there was significant interaction between them when given together. Rat myometrium retained a significant response to spasmogens for at least 60 days after ovariectomy without steroid replacement, perhaps from adrenocortical steroid secretion.

Effects of ovarian steroids and prolactin on the contractility and sodium pump site density of guinea-pig myometrium.

Ovariectomized guinea-pigs were given a single dose of oestrogen or progestogen or the two steroids combined. Controls were given 0.9% saline. Four days after the injection, myometrial contractility in response to spasmogens was measured isometrically in isolated tissue baths, both before and after incubation in Kreb's solution with or without added prolactin. Further myometrial strips were also incubated with or without prolactin and the density of Na+/K+ ATPase 'pump' sites on the surface of the myometrial cell was estimated by labelling with tritiated ouabain. Incubation with prolactin significantly reduced the contractility of myometrial tissue from guinea-pigs given saline alone, progestogen alone or progestogen plus oestrogen, but not in tissue from animals pretreated with oestrogen. When myometrial strips from animals pretreated with oestrogen or progestogen were incubated without prolactin in the medium, there was a significant increase in the density of pump sites compared with the number in saline-pretreated animals; incubation with prolactin did not further modify this effect. When both steroids were administered together there was a significant increase only when prolactin was present in the incubation medium. There was also a significant increase in the density of pump sites after incubation of myometrium from the control (saline-pretreated) animals in the presence of prolactin.

Force, membrane potential and cytoplasmic Ca2+ responses to cyclic nucleotides in rat anococcygeus muscle.

Simultaneous recordings of membrane potential and force, and cytoplasmic calcium ([Ca2+]i) and force were made in rat anococcygeus to determine whether membrane hyperpolarisation plays a role in cyclic nucleotide-induced relaxation. In the presence of phenylephrine (0.2 microM), which evoked sustained contraction, an elevation in [Ca2+]i, and depolarisation, nitroprusside (5 microM) caused 96+/-3% relaxation, 77+/-3% decrease in suprabasal [Ca2+]i, and 16+/-2 mV hyperpolarisation. Forskolin (1 microM) caused 98+/-1% relaxation, 92+/-2% decrease in suprabasal [Ca2+]i, and 18+/-1 mV hyperpolarisation. These responses persisted in the presence of a variety of K+ channel blockers or in ouabain. The decrease in [Ca2+]i preceded the commencement of relaxation whereas the onset of hyperpolarisation lagged behind. Thus, cyclic nucleotide-mediated relaxation in rat anococcygeus is not dependent on hyperpolarisation mediated by the opening of K+ channels. Rather, it is suggested that the decrease in [Ca2+]i gives rise to hyperpolarisation, which reflects a decline in the Ca2+ dependent conductance(s) activated by phenylephrine.

Currents caused by the spontaneous release of quanta of acetylcholine onto chromaffin cells in guinea-pig adrenal medulla.

Membrane potentials were recorded from chromaffin cells in isolated bisected adrenal glands from guinea-pigs. Spontaneous excitatory synaptic potentials (SESPs) were recorded whose frequency was increased following brief (up to 10 s) periods of presynaptic nerve stimulation at 10-30 Hz. The single electrode voltage-clamp method was used to record the currents underlying all but the largest SESPs. Spontaneous excitatory synaptic currents (SESCs) decayed according to a single exponential with a time constant of about 8 ms at 30 degrees C. Thus the neuronal nicotinic receptor-channels giving rise to SESCs in chromaffin cells are probably very similar to those that are opened by quanta of acetylcholine in sympathetic ganglion cells.

Paradoxical effect of gonadotrophin-inhibiting hormone to negatively regulate neuropeptide Y neurones in mouse arcuate nucleus.

Regulation of reproduction and energy homeostasis are linked, although our understanding of the central neural mechanisms subserving this connection is incomplete. Gonadotrophin-inhibiting hormone (GnIH) is a neuropeptide that negatively regulates reproduction and stimulates food intake. Neuropeptide Y (NPY) and products of the pro-opiomelanocortin (POMC) precursor (ß-endorphin melanocortins) are appetite regulating peptides produced in the neurones of the arcuate nucleus; these peptides also regulate reproduction. In the present study, we determined the effects of GnIH on NPY and POMC neurones. Using brain slices from mice with transgenes for fluorescent tags in the two types of neurone and patch clamp electrophysiology, a predominant inhibitory effect of GnIH was observed. GnIH (100 nM) inhibited the firing rate in POMC cells, confirming the results of previous studies and consistent with the stimulatory effect of GnIH on food intake. Paradoxically (i.e. because both GnIH and NPY stimulate food intake), GnIH also had a predominantly inhibitory effect on action potential activity in NPY cells. GnIH also inhibited the secretion of NPY and α-melanocyte-stimulating hormone secretion in incubated hypothalamic blocks. GnIH (100 ng) injected into the cerebral ventricles of mice did not increase the number of NPY cells that were positively immunostained for c-Fos. Finally, dual label immunocytochemistry showed that 20% of NPY neurones had close contacts from GnIH fibres/varicosities. In conclusion, we confirm a negative effect of GnIH on POMC cells and demonstrate a paradoxical reduction of electrophysiological and functional activity in NPY cells.

Alcohol exposure during late gestation: multiple developmental outcomes in sheep.

Alcohol consumption during pregnancy remains common in many countries. Exposure to even low amounts of alcohol (i.e. ethanol) in pregnancy can lead to the heterogeneous fetal alcohol spectrum disorders (FASD), while heavy alcohol consumption can result in the fetal alcohol syndrome (FAS). FAS is characterized by cerebral dysfunction, growth restriction and craniofacial malformations. However, the effects of lower doses of alcohol during pregnancy, such as those that lead to FASD, are less well understood. In this article, we discuss the findings of recent studies performed in our laboratories on the effects of fetal alcohol exposure using sheep, in which we investigated the effects of late gestational alcohol exposure on the developing brain, arteries, kidneys, heart and lungs. Our studies indicate that alcohol exposure in late gestation can (1) affect cerebral white matter development and increase the risk of hemorrhage in the fetal brain, (2) cause left ventricular hypertrophy with evidence of altered cardiomyocyte maturation, (3) lead to a decrease in nephron number in the kidney, (4) cause altered arterial wall stiffness and endothelial and smooth muscle function and (5) result in altered surfactant protein mRNA expression, surfactant phospholipid composition and pro-inflammatory cytokine mRNA expression in the lung. These findings suggest that fetal alcohol exposure in late gestation can affect multiple organs, potentially increasing the risk of disease and organ dysfunction in later life.

Effects of birth asphyxia on neonatal hippocampal structure and function in the spiny mouse.

Studies of human neonates, and in animal experiments, suggest that birth asphyxia results in functional compromise of the hippocampus, even when structural damage is not observable or resolves in early postnatal life. The aim of this study was to determine if changes in hippocampal function occur in a model of birth asphyxia in the precocial spiny mouse where it is reported there is no major lesion or infarct. Further, to assess if, as in human infants, this functional deficit has a sex-dependent component. At 37 days gestation (term=39 days) spiny mice fetuses were either delivered immediately by caesarean section (control group) or exposed to 7.5min of in utero asphyxia causing systemic acidosis and hypoxia. At 5 days of age hippocampal function was assessed ex vivo in brain slices, or brains were collected for examination of structure or protein expression. This model of birth asphyxia did not cause infarct or cystic lesion in the postnatal day 5 (P5) hippocampus, and the number of proliferating or pyknotic cells in the hippocampus was unchanged, although neuronal density in the CA1 and CA3 was increased. Protein expression of synaptophysin, brain-derived neurotrophic factor (BDNF), and the inositol trisphosphate receptor 1 (IP(3)R1) were all significantly increased after birth asphyxia, while long-term potentiation (LTP), paired pulse facilitation (PPF), and post-tetanic potentiation (PTP) were all reduced at P5 by birth asphyxia. In control P5 pups, PPF and synaptic fatigue were greater in female compared to male pups, and after birth asphyxia PPF and synaptic fatigue were reduced to a greater extent in female vs. male pups. In contrast, the asphyxia-induced increase in synaptophysin expression and neuronal density were greater in male pups. Thus, birth asphyxia in this precocial species causes functional deficits without major structural damage, and there is a sex-dependent effect on the hippocampus. This may be a clinically relevant model for assessing treatments delivered either before or after birth to protect this vulnerable region of the developing brain.

Role of mitochondria in contraction and pacemaking in the mouse uterus.

BACKGROUND AND PURPOSE: Uterine spontaneous contraction and pacemaking are poorly understood. This study investigates the role of the mitochondrial Ca(2+) store in uterine activity. EXPERIMENTAL APPROACH: We investigated the effects of mitochondrial and sarco-endoplasmic reticulum (SER) inhibitors on contraction, membrane potential (Vm) and cytosolic Ca(2+) concentration ([Ca(2+) ](c) ) in longitudinal smooth muscle of the mouse uterus. KEY RESULTS: The mitochondrial agents rotenone, carbonylcyanide-3-chlorophenylhydrazone (CCCP), 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157) and kaempferol decreased the force of contractions. The ATP synthase inhibitor oligomycin had no significant effect. The effects of these agents were compared with those of SER inhibitors cyclopiazonic acid (CPA), 2-amino ethoxyphenylborate (2-APB) and caffeine. All agents, except CPA and oligomycin, decreased contractile force. CPA and CCCP transiently increased contraction frequency, which returned to control levels, whereas rotenone, CGP37157, kaempferol and 2-APB decreased frequency and caffeine had no significant effect. Application of the mitochondrial agents when CPA functionally inhibited stores did not change contraction frequency but, with the exception of kaempferol, decreased force. CCCP caused depolarization and maintained increase in [Ca(2+) ](c) or depolarization/transient hyperpolarization and transient increase in [Ca(2+) ](c) for oestrus and di-oestrus tissues respectively. Rotenone caused hyperpolarization and maintained increase in [Ca(2+) ](c) . CGP37157 and kaempferol caused hyperpolarization but no measurable change in [Ca(2+) ](c) . Application of a range of K(+) channel blockers indicated a role of Ca(2+) -activated K(+) (K(Ca) ) channels in the CCCP- and CGP37157-induced actions. CONCLUSIONS AND IMPLICATIONS: Mitochondria have a modulatory role on uterine contractions, with mitochondrial inhibition reducing contraction amplitude and pacemaker frequency by changes in Vm, [Ca(2+) ](c) and/or Ca(2+) influx.