Anandamide (ANA) alters sleep by increasing the amount of time spent in slow wave sleep 2 (SWS2) and rapid eye movement sleep (REMS) at the expense of wakefulness (W) in rats. In this report, we describe a similar effect of ANA when injected itracerebroventricularly (i.c.v.) or into the peduriculopontine tegmental nucleus (PPTg) and the lack of an effect when ANA is administered into the medial preoptic area (MPOA). Furthermore, the i.c.v. or PPTg administration of SR141716A, a CB1 antagonist, or U73122, a PLC inhibitor, 15 min prior to ANA, readily prevents the ANA induced changes in sleep. The present results suggest that a cannabinoid system in the PPTg may be involved in sleep regulation and that the cannabinoid effect is mediated by the CB1 receptor coupled to a PLC second messenger system.

Uterine contractions are tightly regulated by the electrical activity of myometrial smooth muscle cells (MSMCs). These cells require a depolarizing current to initiate Ca(2+) influx and induce contraction. Cationic leak channels, which permit a steady flow of cations into a cell, are known to cause membrane depolarization in many tissue types. Previously, a Gd(3+)-sensitive, Na(+)-dependent leak current was identified in the rat myometrium, but the presence of such a current in human MSMCs and the specific ion channel conducting this current was unknown. Here, we report the presence of a Na(+)-dependent leak current in human myometrium and demonstrate that the Na(+)-leak channel, NALCN, contributes to this current. We performed whole-cell voltage-clamp on fresh and cultured MSMCs from uterine biopsies of term, non-laboring women and isolated the leak currents by using Ca(2+) and K(+) channel blockers in the bath solution. Ohmic leak currents were identified in freshly isolated and cultured MSMCs with normalized conductances of 14.6 pS/pF and 10.0 pS/pF, respectively. The myometrial leak current was significantly reduced (P < 0.01) by treating cells with 10 μM Gd(3+) or by superfusing the cells with a Na(+)-free extracellular solution. Reverse transcriptase PCR and immunoblot analysis of uterine biopsies from term, non-laboring women revealed NALCN messenger RNA and protein expression in the myometrium. Notably, ∼90% knockdown of NALCN protein expression with lentivirus-delivered shRNA reduced the Gd(3+)-sensitive leak current density by 42% (P < 0.05). Our results reveal that NALCN, in part, generates the leak current in MSMCs and provide the basis for future research assessing NALCN as a potential molecular target for modulating uterine excitability.

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.

Pierce SL, Kutschke W, Cabeza R, England SK. In vivo measurement of intrauterine pressure by telemetry: a new approach for studying parturition in mouse models. Physiol Genomics 42: 310 -316, 2010. First published May 11, 2010 doi:10.1152/physiolgenomics.00058.2010.-Transgenic and knockout mouse models have proven useful in the study of genes necessary for parturition-including genes that affect the timing and/or progression of labor contractions. However, taking full advantage of these models will require a detailed characterization of the contractile patterns in the mouse uterus. Currently the best methodology for this has been measurement of isometric tension in isolated muscle strips in vitro. However, this methodology does not provide a real-time measure of changes in uterine pressure over the course of pregnancy. Recent advances have opened the possibility of using radiotelemetric devices to more accurately and comprehensively study intrauterine pressure in vivo. We tested the effectiveness of this technology in the mouse, in both wild-type (WT) mice and a mouse model of defective parturition (SK3 channel-overexpressing mice), after surgical implant of telemetry transmitters into the uterine horn. Continuous recordings from day 18 of pregnancy through delivery revealed that WT mice typically deliver during the 12-h dark cycle after 19.5 days postcoitum. In these mice, intrauterine pressure gradually increases during this cycle, to threefold greater than that measured during the 12-h cycle before delivery. SK3-overexpressing mice, by contrast, exhibited lower intrauterine pressure over the same period. These results are consistent with the outcome of previous in vitro studies, and they indicate that telemetry is an accurate method for measuring uterine contraction, and hence parturition, in mice. The use of this technology will lead to important novel insights into changes in intrauterine pressure during the course of pregnancy. potassium channel; SK3 channel-overexpressing mice; preterm labor; uterine contraction; myometrium PRETERM DELIVERIES alone account for over 12.6% of all births in the U.S. and are associated with a high percentage (ϳ85%) of perinatal morbidity and mortality (2). Despite increased medical intervention over the past 30 years, preterm birth rates have risen by 28%-with most of these births occurring in women who do not have any of the known risk factors. Risk factors that have been identified are related to four distinct processes, each of which activates multiple signaling pathways and leads to preterm birth: precocious fetal endocrine activation, uterine overdistension, decidual bleeding, and intrauterine inflammation/infection (14).Genetically modified mouse models have served as effective systems in which to investigate the signaling pathways essential to labor-both preterm and term. Despite certain differences in parturition between human and mouse, these species are similar with respect to many of the pathways that are known to modify pregnancy outcome (8). Mouse models of alte...