Search

The enteric nervous system in the large intestine generates two important patterns relating to motility: 1) propagating rhythmic peristaltic smooth muscle contractions referred to as colonic migrating motor complexes (CMMCs) and 2) tonic inhibition, during which colonic smooth muscle contractions are suppressed. The precise neurobiological substrates underlying each of these patterns are unclear. Using transgenic animals expressing the genetically encoded calcium indicator GCaMP3 to monitor activity or the optogenetic actuator channelrhodopsin (ChR2) to drive activity in defined enteric neuronal subpopulations, we provide evidence that cholinergic and nitrergic neurons play significant roles in mediating CMMCs and tonic inhibition, respectively. Nitrergic neurons [neuronal nitric oxide synthase (nNOS)-positive neurons] expressing GCaMP3 exhibited higher levels of activity during periods of tonic inhibition than during CMMCs. Consistent with these findings, optogenetic activation of ChR2 in nitrergic neurons depressed ongoing CMMCs. Conversely, cholinergic neurons [choline acetyltransferase (ChAT)-positive neurons] expressing GCaMP3 markedly increased their activity during the CMMC. Treatment with the NO synthesis inhibitor Nω-nitro-l-arginine also augmented the activity of ChAT-GCaMP3 neurons, suggesting that the reciprocal patterns of activity exhibited by nitrergic and cholinergic enteric neurons during distinct phases of colonic motility may be related. NEW & NOTEWORTHY Correlating the activity of neuronal populations in the myenteric plexus to distinct periods of gastrointestinal motility is complicated by the difficulty of measuring the activity of specific neuronal subtypes. Here, using mice expressing genetically encoded calcium indicators or the optical actuator channelrhodopsin-2, we provide compelling evidence that cholinergic and nitrergic neurons play important roles in mediating coordinated propagating peristaltic contractions or tonic inhibition, respectively, in the murine colon.

Volume accommodation occurs via a novel mechanism involving interstitial cells in detrusor muscles. The interstitial cells in the bladder are PDGFRα+ , and they restrain the excitability of smooth muscle at low levels and prevent the development of transient contractions (TCs). A common clinical manifestation of spinal cord injury (SCI)-induced bladder dysfunction is detrusor overactivity (DO). Although a myogenic origin of DO after SCI has been suggested, a mechanism for development of SCI-induced DO has not been determined. In this study we hypothesized that SCI-induced DO is related to loss of function in the regulatory mechanism provided by PDGFRα+ cells. Our results showed that transcriptional expression of Pdgfra and Kcnn3 was decreased after SCI. Proteins encoded by these genes also decreased after SCI, and a reduction in PDGFRα+ cell density was also documented. Loss of PDGFRα + cells was due to apoptosis. TCs in ex vivo bladders during filling increased dramatically after SCI, and this was related to the loss of regulation provided by SK channels, as we observed decreased sensitivity to apamin. These findings show that damage to the mechanism restraining muscle contraction during bladder filling that is provided by PDGFRα + cells is causative in the development of DO after SCI.

Key points Studies of urothelial cells, bladder sheets or lumens of filled bladders have suggested that mediators released from urothelium into suburothelium (SubU)/lamina propria (LP) activate mechanisms controlling detrusor excitability. None of these approaches, however, has enabled direct assessment of availability of mediators at SubU/LP during filling. We developed an ex vivo mouse bladder preparation with intact urothelium and SubU/LP but no detrusor, which allows direct access to the SubU/LP surface of urothelium during filling. Pressure-volume measurements during filling demonstrated that bladder compliance is governed primarily by the urothelium. Measurements of purine mediators in this preparation demonstrated asymmetrical availability of purines in lumen and SubU/LP, suggesting that interpretations based solely on intraluminal measurements of mediators may be inaccurate. The preparations are suitable for assessments of release, degradation and transport of mediators in SubU/LP during bladder filling, and are superior to experimental approaches previously used for urothelium research. Abstract The purpose of this study was to develop a decentralized (ex vivo) detrusor smooth muscle (DSM)-denuded mouse bladder preparation, a novel model that enables studies on availability of urothelium-derived mediators at the luminal and anti-luminal aspects of the urothelium during filling. Urinary bladders were excised from C57BL6/J mice and the DSM was removed by fine-scissor dissection without touching the mucosa. Morphology and cell composition of the preparation wall, pressure-volume relationships during filling, and fluorescent dye permeability of control, protamine sulfate- and lipopolysaccharide-treated denuded bladders were characterized. The preparation wall contained intact urothelium and suburothelium (SubU)/lamina propria (LP) and lacked the DSM and the serosa. The utility of the model for physiological research was validated by measuring release, metabolism and transport of purine mediators at SubU/LP and in bladder lumen during filling. We determined asymmetrical availability of purines (e.g. ATP, ADP, AMP and adenosine) in lumen and at SubU/LP during filling, suggesting differential mechanisms of release, degradation and bilateral transurothelial transport of purines during filling. Some observations were validated in DSM-denuded bladder of the cynomolgus monkey (Macaca fascicularis). The novel model was superior to current models utilized to study properties of the urothelium (e.g. cultured urothelial cells, bladder mucosa sheets mounted in Ussing chambers or isolated bladder strips in organ baths) in that it enabled direct access to the vicinity of SubU/LP during authentic bladder filling. The model is particularly suitable for understanding local mechanisms of urothelium-DSM connectivity and for broad understanding of the role of urothelium in regulating continence and voiding.

Previous studies have established the release of chemical substances from flat bladder mucosa sheets affixed in Ussing chambers and exposed to changes in hydrostatic pressure or mechanical stretch and from cultured urothelial cells upon hydrostatic pressure changes, stretch, cell swelling, or drag forces, and in bladder lumen at end of filling. Such findings led to the assumption that these mediators are also released in suburothelium (SubU)/lamina propria (LP) during bladder filling, where they affect cells deep in the bladder wall to ultimately regulate bladder excitability. There are at least two obvious limitations in such studies: 1) none of these approaches provide direct information about the presence of mediators in SubU/LP, and 2) the stimuli used are not physiological and do not recapitulate authentic filling of the bladder. Here, we discuss a procedure that enables direct access to the suburothelial surface of the bladder mucosa in the course of bladder filling. The murine detrusor-free preparation we created closely resembles filling of the intact bladder and allows pressure-volume studies to be performed on the bladder in the absence of confounding signaling from spinal reflexes and detrusor smooth muscle. Using the novel detrusor-free bladder model, we recently demonstrated that intravesical measurements of mediators cannot be used as a proxy to what has been released or present in the SubU/LP during bladder filling. The model enables examination of urothelium-derived signaling molecules that are released, generated by metabolism and/or transported into the SubU/LP during the course of bladder filling to transmit information to neurons and smooth muscle of the bladder and regulate its excitability during continence and micturition.

During urinary bladder filling the bladder urothelium releases chemical mediators that in turn transmit information to the nervous and muscular systems to regulate sensory sensation and detrusor muscle activity. Defects in release of urothelial mediators may cause bladder dysfunctions that are characterized with aberrant bladder sensation during bladder filling. Previous studies have demonstrated release of ATP from the bladder urothelium during bladder filling, and ATP remains the most studied purine mediator that is released from the urothelium. However, the micturition cycle is likely regulated by multiple purine mediators, since various purine receptors are found present in many cell types in the bladder wall, including urothelial cells, afferent nerves, interstitial cells in lamina propria, and detrusor smooth muscle cells. Information about the release of other biologically active purines during bladder filling is still lacking. Decentralized bladders from C57BL/6 mice and Cynomolgus monkeys ( Macaca fascicularis) were filled with physiological solution at different rates. Intraluminal fluid was analyzed by high-performance liquid chromatography with fluorescence detection for simultaneous evaluation of ATP, ADP, AMP, adenosine, nicotinamide adenine dinucleotide (NAD+), ADP-ribose, and cADP-ribose content. We also measured ex vivo bladder filling pressures and performed cystometry in conscious unrestrained mice at different filling rates. ATP, ADP, AMP, NAD+, ADPR, cADPR, and adenosine were detected released intravesically at different ratios during bladder filling. Purine release increased with increased volumes and rates of filling. Our results support the concept that multiple urothelium-derived purines likely contribute to the complex regulation of bladder sensation during bladder filling.

KEY POINTS: Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine‐sensitive but nicardipine‐insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca(2+)‐independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI‐17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C–CPI‐17 Ca(2+) sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI‐17 phosphorylation. ABSTRACT: Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C‐kinase potentiated protein phosphatase‐1 inhibitor of 17 kDa (CPI‐17) and myosin phosphatase targeting subunit of myosin light‐chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of N (ω)‐nitro‐l‐arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF‐431396 inhibited the contractions and the increase in pY397. PF‐431396 also inhibited the electrical field stimulation‐induced increase in CPI‐17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca(2+) influx was unaffected by PF‐431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13‐dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca(2+) independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI‐17 phosphorylation.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4−/− mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4−/− bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.

Key points • Previous studies have indicated that neither neuronal nor mucosal 5-hydroxytryptamine (5-HT) are important for colonic migrating motor complexes (CMMCs) or faecal pellet propulsion. Therefore, tryptophan hydroxylase 1 knockout (TPH1KO) mice were used to examine the role of mucosal 5-HT in generating CMMCs and faecal pellet propulsion, as TPH1 is the regulatory enzyme necessary for the synthesis of 5-HT in enterochromaffin cells in the mucosa. • Control mice generated a robust CMMC when the mucosa was mechanically stimulated, which was blocked by ondansetron (5-HT3 antagonist), and could propagate faecal pellets that did not significantly distend the bowel, suggesting that they were propelled by mucosal reflexes in the absence of stretch reflexes. • TPH1KO mice exhibited no mucosal reflexes, reduced responses to intraluminal distension and propelled only larger faecal pellets, suggesting that they relied upon stretch reflexes alone. • In control mice, CMMCs, which can propel a faecal pellet, propagated in an oral to anal direction, whereas, in TPH1KO mice, they rarely propagated. • Both the propagation and amplitude of CMMCs were reduced by ondansetron in control mice, whereas this drug did not affect CMMCs in TPH1KO mice. • This suggests that 5-HT release from the mucosa and stretch reflexes are important for normal colonic propulsion. Abstract Although there is general agreement that mucosal 5-hydroxytryptamine (5-HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5-HT is critical. We therefore tested the hypothesis that the secretion of 5-HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1−/− mice), which is the rate-limiting enzyme in the biosynthesis of mucosal but not neuronal 5-HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1+/+) mice, Tph1−/− animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1+/+ colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1+/+); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1−/− colon; and (9) the 5-HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1+/+ mice, failed to alter CMMCs in Tph1−/− animals. Our observations suggest that mucosal 5-HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.

Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2(max) duration of 2-12 s, velocity of 25-50 μm/s) and two fast "flash-like" types (Type 1, 30-90 ms; Type 2, 90-150 ms 1/2(max) duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs.

Objective Morphology and functional role of PDGFRα+ cells have been recently characterized in the detrusor muscle layer. Detrusor relaxation is caused by activation of small conductance Ca2+ activated-K+ (SK) channels and purinergic inhibitory responses in detrusor PDGFRα+ cells. Loss of PDGFRα+ cells or alteration of P2Y receptors and SK channels will affect detrusor excitability. Cyclophosphamide (CYP)-treated animals exhibited overactive bladder (OAB). We hypothesized that the downregulation of P2Y receptors and/or SK channels in PDGFRα+ cells will display the phenotype of CYP-induced OAB. Methods CYP was injected intraperitoneally in PDGFRα+/eGFP and SMC/eGFP mice. We harvested the detrusor muscle without urothelium and disperse the cells for the fluorescence activated cell sorting (FACS). Sorted PDGFRα+ cells and smooth muscle cells (SMCs) were used for molecular study to compare the changes in transcripts between CYP-injected and control group. Transcripts were examined included; Pdgfrα, P2ry1, P2ry2, P2ry4, Kcnn1, Kcnn2, Kcnn3 and inflammation marker (Il-6). Immunohistochemistry, mechanical contractility and ex vivo cystometry were also performed. Results Quantitative analysis of PCR revealed that CYP-injected detrusor muscle increased transcriptional expression of Il-6, but decreased the expression of Pdgfrα. Transcriptional changes in CYP-injected sorted PDGFRα+ cells from PDGFRα+/eGFP mice showed Pdgfα, Kccn3 (SK3), P2ry1, P2ry2 and P2ry4 genes were decreased compared with saline-injected control. Sorted SMCs from SMC/eGFP mice did not show significant expression of those genes and no detectable changes. Immunohistochemistry showed SK3 in PDGFRα immunoreactivity was downregulated in CYP-injected detrusor muscle. Apamin (a SK blocker) sensitivity on spontaneous contractile activity was decreased in CYP-injected mice compared to saline-injected mice. In ex vivo cystometry, increased spontaneous non-voiding contractions and less apamin sensitivity were observed in CYP-injected mice. Conclusions These findings are the first report to investigate the role of PDGFRα+ cells in relation to OAB mechanisms. In conclusion, we found that CYP-induced OAB is resulted from down regulation of PDGFRα, P2Y receptors and SK channels in CYP-injected bladder. These results provide novel mechanisms of functional role of PDGFRα+ cells on OAB. Funding Source(s) NIDDK, RO1 DK098388 and Urology Care Foundation Research Scholar Award (Interstitial Cystitis Association)

Objective Neurogenic bladder dysfunction due to spinal cord injury (SCI) poses a significant threat to the well-being of patients. The complications of this condition include but not are limited to incontinence, renal impairment, urinary tract infection, stones, and poor quality of life. Clinical manifestations of SCI involve combination of storage and voiding bladder problems. Although a number of clinical studies have reported overactive bladder (OAB) after SCI, the pathophysiological mechanisms remain unclear. SCI is widely used to induce neurogenic bladder in rodent models. These animals exhibited dysfunctional condition results in different symptoms, ranging from acute urinary retention to an OAB or a combination of both. There is an abundance of PDGFRα+ cells in detrusor muscles. This cell involves the membrane stabilization via activation of SK channels in detrusor PDGFRα+ cells during filling. Thus we investigate the molecular and protein expression of PDGFRα+ cells from SCI mice to characterize the role of these cells that contributes to development of OAB in SCI. Methods SCI was induced by complete compression of T12-L1 spinal cord. Experiments were performed on 24, 48 and 72 hr after surgery. We employed molecular approaches and ex vivo cystometry. Pdgfrα and Kccn1−3 transcripts were analyzed for molecular expression. Ex vivo compliance was used for testing SK channel sensitivity in control and SCI mice. Results In quantitative analysis of transcripts, Pdgfrα and Kcnn3 transcripts in SCI detrusor were significantly decreased in a time-dependent manner after SCI surgery compared with control detrusor. In ex vivo cystometry, SCI bladder revealed an increase in the amplitude and frequency of non−voiding pressure responses during filling. Effects of a SK blocker (apamin) and a SK channel activator (SKA-31) were reduced in non-voiding contractions in SCI mice compared to control. Conclusions These findings support that downregulation of PDGFRα+ cells and SK channels in SCI detrusors might involve the development of OAB in SCI. Funding Source(s) NIDDK, RO1 DK098388

Genetically encoded Ca(2+) indicators (GECIs) have been used extensively in many body systems to detect Ca(2+) transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca(2+) indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca(2+) imaging. In this preliminary study, we observed neuronal and glial cell Ca(2+) transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100-1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca(2+) transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca(2+) transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca(2+) transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Although there is general agreement that mucosal 5-hydroxytryptamine (5-HT) can initiate peristaltic reflexes in the colon, recent studies have differed as to whether or not the role of mucosal 5-HT is critical. We therefore tested the hypothesis that the secretion of 5-HT from mucosal enterochromaffin (EC) cells is essential for the manifestation of murine colonic peristaltic reflexes. To do so, we analysed the mechanisms underlying faecal pellet propulsion in isolated colons of mice lacking tryptophan hydroxylase 1 (Tph1(-/-) mice), which is the rate-limiting enzyme in the biosynthesis of mucosal but not neuronal 5-HT. We used video analysis of faecal pellet propulsion, tension transducers to record colonic migrating motor complexes (CMMCs) and intracellular microelectrodes to record circular muscle activity occurring spontaneously or following intraluminal distension. When compared with control (Tph1(+/+)) mice, Tph1(-/-) animals exhibited: (1) an elongated colon; (2) larger faecal pellets; (3) orthograde propulsion followed by retropulsion (not observed in Tph1(+/+) colon); (4) slower in vitro propulsion of larger faecal pellets (28% of Tph1(+/+)); (5) CMMCs that infrequently propagated in an oral to anal direction because of impaired descending inhibition; (6) reduced CMMCs and inhibitory responses to intraluminal balloon distension; (7) an absence of reflex activity in response to mucosal stimulation. In addition, (8) thin pellets that propagated along the control colon failed to do so in Tph1(-/-) colon; and (9) the 5-HT3 receptor antagonist ondansetron, which reduced CMMCs and blocked their propagation in Tph1(+/+) mice, failed to alter CMMCs in Tph1(-/-) animals. Our observations suggest that mucosal 5-HT is essential for reflexes driven by mucosal stimulation and is also important for normal propagation of CMMCs and propulsion of pellets in the isolated colon.

The Sola Concentrator Advanced Development Project, which has achieved the successful design, fabrication, and testing of a full-scale prototypical solar dynamic concentrator is discussed. The design and fabrication process are summarized, and the test results for the reflective facet optical performance and the concentrator structural repeatability are reported. Initial testing of structural repeatability of a seven panel portion of the concentrator was followed by assembly and testing of the full nineteen-panel structure. The testing, which consisted of theodolite and optical measurements over an assembly-disassembly-reassembly cycle, demonstrated that the concentrator maintained the as-built contour and optical characteristics. The facet development effort, which entailed developing a vapor-deposited reflective facet, produced a viable design with demonstrated optical characteristics that are within the project goals. >

The Solar Concentrator Advanced Development Project has successfully designed, fabricated, and tested a full scale prototypical solar dynamic concentrator for space station applications. A Truss Hexagonal Panel reflector was selected as a viable solar concentrator concept to be used for space station applications. This concentrator utilizes a modular design approach and is flexible in attainable flux profiles and assembly techniques. The detailed design of the concentrator, which included structural, thermal and optical analysis, identified the feasibility of the design and specific technologies that were required to fabricate it. The needed surface accuracy of the reflectors surface was found to be very tight, within 5 mrad RMS slope error, and results in very close tolerances for fabrication. To meet the design requirements, a modular structure composed of hexagonal panels was used. The panels, made up of graphite epoxy box beams provided the strength, stiffness and dimensional stability needed. All initial project requirements were met or exceeded by hardware demonstration. Initial testing of structural repeatability of a seven panel portion of the concentrator was followed by assembly and testing of the full nineteen panel structure. The testing, which consisted of theodolite and optical measurements over an assembly-disassembly-reassembly cycle, demonstrated that the concentrator maintained the as-built contour and optical characteristics. The facet development effort within the project, which included developing the vapor deposited reflective facet, produced a viable design with demonstrated optical characteristics that are within the project goals.