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Mammalian oviducts play an essential role in female fertility by picking up ovulated oocytes and transporting and nurturing gametes (sperm/oocytes) and early embryos. However, the relative contributions to these functions from various cell types within the oviduct remain controversial. The oviduct in mice deficient in two microRNA (miRNA) clusters (miR-34b/c and miR-449) lacks cilia, thus allowing us to define the physiological role of oviductal motile cilia. Here, we report that the infundibulum without functional motile cilia failed to pick up the ovulated oocytes. In the absence of functional motile cilia, sperm could still reach the ampulla region, and early embryos managed to migrate to the uterus, but the efficiency was reduced. Further transcriptomic analyses revealed that the five messenger ribonucleic acids (mRNAs) encoded by miR-34b/c and miR-449 function to stabilize a large number of mRNAs involved in cilium organization and assembly and that Tubb4b was one of their target genes. Our data demonstrate that motile cilia in the infundibulum are essential for oocyte pickup and thus, female fertility, whereas motile cilia in other parts of the oviduct facilitate gamete and embryo transport but are not absolutely required for female fertility.

The deficit in cerebral blood flow (CBF) seen in patients with hypertension-induced vascular dementia is increasingly viewed as a therapeutic target for disease-modifying therapy. Progress is limited, however, due to uncertainty surrounding the mechanisms through which elevated blood pressure reduces CBF. To investigate this, we used the BPH/2 mouse, a polygenic model of hypertension. At 8 mo of age, hypertensive mice exhibited reduced CBF and cognitive impairment, mimicking the human presentation of vascular dementia. Small cerebral resistance arteries that run across the surface of the brain (pial arteries) showed enhanced pressure-induced constriction due to diminished activity of large-conductance Ca2+-activated K+ (BK) channels--key vasodilatory ion channels of cerebral vascular smooth muscle cells. Activation of BK channels by transient intracellular Ca2+ signals from the sarcoplasmic reticulum (SR), termed Ca2+ sparks, leads to hyperpolarization and vasodilation. Combining patch-clamp electrophysiology, high-speed confocal imaging, and proximity ligation assays, we demonstrated that this vasodilatory mechanism is uncoupled in hypertensive mice, an effect attributable to physical separation of the plasma membrane from the SR rather than altered properties of BK channels or Ca2+ sparks, which remained intact. This pathogenic mechanism is responsible for the observed increase in constriction and can now be targeted as a possible avenue for restoring healthy CBF in vascular dementia. [ABSTRACT FROM AUTHOR]

Dickson E J, Heredia D J, McCann C J, Hennig GW, Smith TK. The mechanisms underlying the generation of the colonic migrating motor complex in both wild-type and nNOS knockout mice. Am J Physiol Gastrointest Liver Physiol 298: G222-G232, 2010. First published December 3, 2009; doi:1O.1152/ajpgi.00399.2009.--Colonic migrating motor complexes (CMMCs) propel fecal contents and are altered in diseased states, including slow-transit constipation. However, the mechanisms underlying the CMMCs are controversial because it has been proposed that disinhibition (turning off of inhibitory neurotransmission) or excitatory nerve activity generate the CMMC. Therefore, our aims were to reexamine the mechanisms underlying the CMMC in the colon of wild-type and neuronal nitric oxide synthase [(nNOS).sup.-/-] mice. CMMCs were recorded from the isolated murine large bowel using intracellular recordings of electrical activity from circular muscle (CM) combined with tension recording. Spontaneous CMMCs occurred in both wild-type (frequency: 0.3 cycles/min) and [nNOS.sup.-/-] mice (frequency: 0.4 cycles/min). CMMCs consisted of a hyperpolarization, followed by fast oscillations (slow waves) with action potentials superimposed on a slow depolarization (wild-type: 14.0 [+ or -] 0.6 mV; [nNOS.sup.-/-]: 11.2 [+ or -] 1.5 mV). Both atropine (1 [micro]M) and MEN 10,376 [neurokinin 2 (NK2) antagonist; 0.5 [micro]M] added successively reduced the slow depolarization and the number of action potentials but did not abolish the fast oscillations. The further addition of RP 67580 (NK1 antagonist; 0.5 [micro]M) blocked the fast oscillations and the CMMC. Importantly, none of the antagonists affected the resting membrane potential, suggesting that ongoing tonic inhibition of the CM was maintained. Fecal pellet propulsion, which was blocked by the NK2 or the NK1 antagonist, was slower down the longer, more constricted [nNOS.sup.-/-] mouse colon (wild-type: 47.9 [+ or -] 2.4 mm; [nNOS.sup.-/-] : 57.8 [+ or -] 1.4 mm). These observations suggest that excitatory neurotransmission enhances pacemaker activity during the CMMC. Therefore, the CMMC is likely generated by a synergistic interaction between neural and interstitial cells of Cajal networks. cholinergic transmission; NK2 receptors; NK1 receptors; tachykinins; neuronal nitric oxide synthase; circular muscle; longitudinal muscle; smooth muscle; interstitial cells of Cajal; slow waves doi: 10.1152/ajpgi.00399.2009

Gastric emptying depends on functional coupling of slow waves between the corpus and antrum, to allow slow waves initiated in the gastric corpus to propagate to the pyloric sphincter and generate gastric peristalsis. Functional coupling depends on a frequency gradient where slow waves are generated at higher frequency in the corpus and drive the activity of distal pacemakers. Simultaneous intracellular recording from corpus and antrum was used to characterize the effects of PGEa on slow waves in the murine stomach. [PGE.sub.2] increased slow-wave frequency, and this effect was mimicked by [EP.sub.3], but not by [EP.sub.2], receptor agonists. Chronotropic effects were due to [EP.sub.3] receptors expressed by intramuscular interstitial cells of Cajal because these effects were not observed in W/[W.sup.v] mice. Although the integrated chronotropic effects of [EP.sub.3] receptor agonists were deduced from electrophysiological experiments, no clear evidence of functional uncoupling was observed with two-point electrical recording. Gastric peristalsis was also monitored by video imaging and spatiotemporal maps to study the impact of chronotropic agonists on propagating contractions. [EP.sub.3] receptor agonists increased the frequency of peristaltic contractions and caused ectopic sites of origin and collisions of peristaltic waves. The impact of selective regional application of chronotropic agonists was investigated by use of a partitioned bath. Antral slow waves followed enhanced frequencies induced by stimulation of the corpus, and corpus slow waves followed when slow-wave frequency was elevated in the antrum. This demonstrated reversal of slow-wave propagation with selective antral chronotropic stimulation. These studies demonstrate the impact of chronotropic agonists on regional intrinsic pacemaker frequency and integrated gastric peristalsis. interstitial cells of Cajal; EP receptor; gastroparesis; pacemaker; tachygastria

The mechanosensitive endings of low-threshold, slowly adapting pelvic afferents that innervate the rectum have been previously identified as rectal intraganglionic laminar endings (rlGLEs) that lie within myenteric ganglia. We tested whether the aganglionic rectum of piebald-lethal ([s.sup.I]/[s.sup.I]) mice lacks rIGLEs and whether this could explain impaired distension-evoked reflexes from this region. Extracellular recordings were made from fine rectal nerves in C57BL/6 wild-type and [s.sup.I]/[s.sup.I] mice, combined with anterograde labeling. In C57BL/6 mice, graded circumferential stretch applied to the rectum activated graded increases in firing of slowly adapting rectal mechanoreceptors. In [s.sup.I]/[s.sup.I] mice, graded stretch of the aganglionic rectum activated similar graded increases in rectal afferent firing. Stretch-sensitive afferents responded at low mechanical thresholds and fired more intensely at noxious levels of stretch. They could also be activated by probing their receptive fields with von Frey hairs and by muscle contraction. Anterograde labeling from recorded rectal nerves identified the mechanoreceptors of muscular afferents in the aganglionic rectal smooth muscle. A population of afferents were also recorded in both C57BL/6 and [s.sup.I]/[s.sup.I] mice that were activated by von Frey hair probing, but not stretch. In summary, the aganglionic rectum is innervated by a population of stretch-sensitive rectal afferent mechanoreceptor which develops and functions in the absence of any enteric ganglia. These results suggest that in patients with Hirschsprung's disease the inability to activate extrinsic distension reflexes from the aganglinnic rectum is unlikely to be due to the absence of stretch-sensitive extrinsic mechanoreceptors. mechanoreceptor; rectal nerve; afferent; aganglionic; Hirschsprung's

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR) [Ca.sup.2+]-ATPase (SERCA), and this inhibition is relieved by [Ca.sup.2+] calmodulin-dependent protein kinase II (CaM kinase II) phosphorylation. We previously reported significant differences in contractility, SR [Ca.sup.2+] release, and CaM kinase II activity in gastric fundus smooth muscles as a result of PLB phosphorylation by CaM kinase II. In this study, we used PLB-knockout (PLB-KO) mice to directly examine the effect of PLB absence on contractility, CaM kinase II activity, and intracellular [Ca.sup.2+] waves in gastric antrum smooth muscles. The frequencies and amplitudes of spontaneous phasic contractions were elevated in antrum smooth muscle strips from PLB-KO mice. Bethanecol increased the amplitudes of phasic contractions in antrum smooth muscles from both control and PLB-KO mice. Caffeine decreased and cyclopiazonic acid (CPA) increased the basal tone of antrum smooth muscle strips from PLB-KO mice, hut the effects were less pronounced compared with control strips. The CaM kinase II inhibitor KN-93 was less effective at inhibiting caffeine-induced relaxation in antrum smooth muscle strips from PLB-KO mice. CaM kinase II autonomous activity was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. Similarly, the intracellular [Ca.sup.2+] wave frequency was elevated, and not further increased by caffeine, in antrum smooth muscles from PLB-KO mice. These findings suggest that PLB is an important modulator of gastric antrum smooth muscle contractility by modulation of SR [Ca.sup.2+] release and CaM kinase II activity. gastrointestinal smooth muscle; [Ca.sup.2+] calmodulin-dependent protein kinase II

In mammals that develop rectal aganglionosis, the aganglionic segment still exhibits spontaneous phasic contractions that contribute to dysmotility and pseudoobstruction in this region. However, almost nothing is known about the mechanisms that generate these myogenic contractions or the effects of aganglionosis on the generation of [Ca.sup.2+] waves that underlie contractions of the longitudinal muscle (LM) and circular muscle (CM). In a mouse model of Hirschsprung's disease [endothelin type B receptor-deficient ([Ednrb.sup.1-l]/[Ednrb.sup.s-l]) mice], the [Ca.sup.2+] indicator fluo-4 was used to simultaneously monitor the temporal activation and spread of intercellular [Ca.sup.2+] waves in the LM and CM during spontaneous colonic motor activities. During the intervals between colonic migrating motor complexes (CMMCs) in control mice, [Ca.sup.2+] waves discharged asynchronously between the LM and CM. However, in these same mice, during CMMCs, a burst of discreet [Ca.sup.2+] waves fired simultaneously in both muscle layers, where the propagation velocity of [Ca.sup.2+] waves significantly increased, as did the rate of initiation and number of collisions between [Ca.sup.2+] waves. Hexamethonium (300 [micro]M) or atropine (1 [micro]M) prevented synchronized firing of [Ca.sup.2+] waves. In the aganglionic distal colon of [Ednrb.sup.s-l]/[Ednrb.sup.s-l] mice, not only were CMMCs absent, but [Ca.sup.2+] waves between the two muscle layers fired asynchronously, despite increased propagation velocity. The generation of CMMCs in control mice involves synchronized firing of enteric motor nerves to both the LM and CM, explaining the synchronized firing of discreet [Ca.sup.2+] waves between the two muscle layers. Aganglionosis results in a sporadic and sustained asynchrony in [Ca.sup.2+] wave firing between the LM and CM and an absence of CMMCs. colon; myenteric; peristalsis; colonic migrating motor complexes

Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used [Ca.sup.2+] imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular [Ca.sup.2+] transients that occurred after electrical slow waves. ICC-MY formed networks, and [Ca.sup.2+] transient wave fronts propagated through the ICC-MY networks at ~2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked [Ca.sup.2+] transients in LM but had no visible effect on the transients in ICC-MY. [beta]-Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant [Ca.sup.2+] entry ([Ni.sup.2+] and mibefradil) and elevated external [K.sup.+] reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic [Ca.sup.2+] transients in ICC-MY. These data show that global [Ca.sup.2+] transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular [Ca.sup.2+] handling mechanisms and entrained by voltage-dependent [Ca.sup.2+] entry and coupling of cells via gap junctions. [Ca.sup.2+] signaling; slow waves; gastrointestinal motility

Vanden Berghe, Pieter, Grant W. Hennig, and Terence K. Smith. Characteristics of intermittent mitochondrial transport in guinea pig enteric nerve fibers. Am J Physiol Gastrointest Liver Physiol 286: G671-G682, 2004. First published October 30, 2003; 10.1152/ajpgi.00283.2003.--Enteric neurons controlling various gut functions are prone to oxidative insults that might damage mitochondria (e.g., intestinal inflammation). To resume local energy supply, mitochondria need to be transported. We used MitoTracker dyes and confocal microscopy to investigate basic characteristics of mitochondrial transport in guinea pig myenteric neurites. During a 10-s observation of 1 mm nerve fiber, on average, three mitochondria were transported at an average speed of 0.41 [+ or -] 0.02 [micro]m/s. Movement patterns were clearly erratic, and velocities were independent of mitochondrial size. The velocity oscillated periodically (~6 s) but was not consistently affected by structures such as en route boutons, bifurcations, or stationary mitochondria. Also, mitochondria transported in opposite directions did not necessarily affect each others' mobility. Transport was blocked by microtubule disruption (100 [micro]M colchicine), and destabilization (1 [micro]M cytochalasin-D) or stabilization (10 [micro]M phalloidin) of actin filaments, respectively, decreased (0.22 [+ or -] 0.02 [micro]m/s, P < 0.05) or increased (0.53 [+ or -] 0.02 [micro]m/s, P < 0.05) transport speed. Transport was inhibited by TTX (1 [micro]M), and removal of extracellular [Ca.sup.2+] (plus 2 mM EGTA) had no effect. However, depletion of intracellular stores (thapsigargin) reduced (to 33%) and slowed the transport significantly (0.18 [+ or -] 0.02 [micro]m/s, P < 0.05), suggesting an important role for stored [Ca.sup.2+] in mitochondrial transport. Transport was also reduced (to 21%) by the mitochondrial uncoupler FCCP (1 [micro]M) in a time-dependent fashion and slowed by oligomycin (10 [micro]M). We conclude that mitochondrial transport is remarkably independent of structural nerve fiber properties. We also show that mitochondrial transport is TTX sensitive and speeds up by stabilizing actin and that functional [Ca.sup.2+] stores are required for efficient transport. myenteric neuron; axonal transport; intracellular calcium stores; MitoTracker

The role of the longitudinal muscle (LM) layer during the peristaltic reflex in the small and large intestine is unclear. In this study, we have made double and quadruple simultaneous intracellular recordings from LM and circular muscle (CM) cells of guinea pig distal colon to correlate the electrical activities in the two different muscle layers during circumferential stretch. Simultaneous recordings from LM and CM cells ( circular muscle; inhibitory junction potential; excitatory junction potential; myenteric neuron; peristaltic reflex; peristalsis

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 [micro]m in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 [micro]M) or tetrodotoxin (1 [micro]M) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 [micro]M) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually interstitial cells of Cajal; slow wave; pacemaker cell