Association of Higher Ocrelizumab Exposure With Reduced Disability Progression in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Ocrelizumab improved clinical and MRI measures of disease activity and progression in three phase 3 multiple sclerosis (MS) studies. Post hoc analyses demonstrated a correlation between the ocrelizumab serum concentration and the degree of blood B-cell depletion, and body weight was identified as the most influential covariate on ocrelizumab pharmacokinetics. The magnitude of ocrelizumab treatment benefit on disability progression was greater in lighter vs heavier patients. These observations suggest that higher ocrelizumab serum levels provide more complete B-cell depletion and a greater delay in disability progression. The current post hoc analyses assessed population exposure-efficacy/safety relationships of ocrelizumab in patients with relapsing and primary progressive MS. METHODS: Patients in OPERA I/II and ORATORIO were grouped in exposure quartiles based on their observed individual serum ocrelizumab level over the treatment period. Exposure-response relationships were analyzed for clinical efficacy (24-week confirmed disability progression (CDP), annualized relapse rate [ARR], and MRI outcomes) and adverse events. RESULTS: Ocrelizumab reduced new MRI lesion counts to nearly undetectable levels in patients with relapsing or primary progressive MS across all exposure subgroups, and reduced ARR in patients with relapsing MS to very low levels (0.13-0.18). A consistent trend of higher ocrelizumab exposure leading to lower rates of CDP was seen (0%-25% [lowest] to 75%-100% [highest] quartile hazard ratios and 95% confidence intervals; relapsing MS: 0.70 [0.41-1.19], 0.85 [0.52-1.39], 0.47 [0.25-0.87], and 0.34 [0.17-0.70] vs interferon ß-1a; primary progressive MS: 0.88 [0.59-1.30], 0.86 [0.60-1.25], 0.77 [0.52-1.14], and 0.55 [0.36-0.83] vs placebo). Infusion-related reactions, serious adverse events, and serious infections were similar across exposure subgroups. DISCUSSION: The almost complete reduction of ARR and MRI activity already evident in the lowest quartile, and across all ocrelizumab-exposure groups, suggests a ceiling effect. A consistent trend of higher ocrelizumab exposure leading to greater reduction in risk of CDP was observed, particularly in the relapsing MS trials, and was not associated with a higher rate of adverse events. Higher ocrelizumab exposure may provide improved control of disability progression by reducing disease activity below that detectable by ARR and MRI, and/or by attenuating other B-cell-related pathologies responsible for tissue damage. CLASSIFICATION OF EVIDENCE: This analysis provides Class III evidence that higher ocrelizumab serum levels are related to greater reduction in risk of disability progression in patients with multiple sclerosis. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in the open-label extension. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov Identifier: NCT01247324 (OPERA I), NCT01412333 (OPERA II), and NCT01194570 (ORATORIO).

Activity within specific enteric neurochemical subtypes is correlated with distinct patterns of gastrointestinal motility in the murine colon.

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.

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels.

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.

A model of the enteric neural circuitry underlying the generation of rhythmic motor patterns in the colon: the role of serotonin.

We discuss the role of multiple cell types involved in rhythmic motor patterns in the large intestine that include tonic inhibition of the muscle layers interrupted by rhythmic colonic migrating motor complexes (CMMCs) and secretomotor activity. We propose a model that assumes these motor patterns are dependent on myenteric descending 5-hydroxytryptamine (5-HT, serotonin) interneurons. Asynchronous firing in 5-HT neurons excite inhibitory motor neurons (IMNs) to generate tonic inhibition occurring between CMMCs. IMNs release mainly nitric oxide (NO) to inhibit the muscle, intrinsic primary afferent neurons (IPANs), glial cells, and pacemaker myenteric pacemaker interstitial cells of Cajal (ICC-MY). Mucosal release of 5-HT from enterochromaffin (EC) cells excites the mucosal endings of IPANs that synapse with 5-HT descending interneurons and perhaps ascending interneurons, thereby coupling EC cell 5-HT to myenteric 5-HT neurons, synchronizing their activity. Synchronized 5-HT neurons generate a slow excitatory postsynaptic potential in IPANs via 5-HT7 receptors and excite glial cells and ascending excitatory nerve pathways that are normally inhibited by NO. Excited glial cells release prostaglandins to inhibit IMNs (disinhibition) to allow full excitation of ICC-MY and muscle by excitatory motor neurons (EMNs). EMNs release ACh and tachykinins to excite pacemaker ICC-MY and muscle, leading to the simultaneous contraction of both the longitudinal and circular muscle layers. Myenteric 5-HT neurons also project to the submucous plexus to couple motility with secretion, especially during a CMMC. Glial cells are necessary for switching between different colonic motor behaviors. This model emphasizes the importance of myenteric 5-HT neurons and the likely consequence of their coupling and uncoupling to mucosal 5-HT by IPANs during colonic motor behaviors.

Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity.

Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8-fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (σmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype.

Inclusion of brain volume loss in a revised measure of 'no evidence of disease activity' (NEDA-4) in relapsing-remitting multiple sclerosis.

BACKGROUND: 'No evidence of disease activity' (NEDA), defined as absence of magnetic resonance imaging activity (T2 and/or gadolinium-enhanced T1 lesions), relapses and disability progression ('NEDA-3'), is used as a comprehensive measure of treatment response in relapsing multiple sclerosis (RMS), but is weighted towards inflammatory activity. Accelerated brain volume loss (BVL) occurs in RMS and is an objective measure of disease worsening and progression. OBJECTIVE: To assess the contribution of individual components of NEDA-3 and the impact of adding BVL to NEDA-3 ('NEDA-4') METHODS: We analysed data pooled from two placebo-controlled phase 3 fingolimod trials in RMS and assessed NEDA-4 using different annual BVL mean rate thresholds (0.2%-1.2%). RESULTS: At 2 years, 31.0% (217/700) of patients receiving fingolimod 0.5 mg achieved NEDA-3 versus 9.9% (71/715) on placebo (odds ratio (OR) 4.07; p < 0.0001). Adding BVL (threshold of 0.4%), the respective proportions of patients achieving NEDA-4 were 19.7% (139/706) and 5.3% (38/721; OR 4.41; p < 0.0001). NEDA-4 status favoured fingolimod across all BVL thresholds tested (OR 4.01-4.41; p < 0.0001). CONCLUSION: NEDA-4 has the potential to capture the impact of therapies on both inflammation and neurodegeneration, and deserves further evaluation across different compounds and in long-term studies.

Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon.

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.

Colonic migrating motor complexes, high amplitude propagating contractions, neural reflexes and the importance of neuronal and mucosal serotonin.

The colonic migrating motor complex (CMMC) is a critical neurally mediated rhythmic propulsive contraction observed in the large intestine of many mammals. It seems to be equivalent to the high amplitude propagating contractions (HAPCs) in humans. This review focuses on the probable neural mechanisms involved in producing the CMMC or HAPC, their likely de-pendence on mucosal and neuronal serotonin and pacemaker insterstitial cells of Cajal networks and how intrinsic neural re-flexes affect them. Discussed is the possibility that myenteric 5-hydroxytryptamine (5-HT) neurons are not only involved in tonic inhibition of the colon, but are also involved in generating the CMMC and modulation of the entire enteric nervous system, including coupling motility to secretion and blood flow. Mucosal 5-HT appears to be important for the initiation and effective propagation of CMMCs, although this mechanism is a longstanding controversy since the 1950s, which we will address. We argue that the slow apparent propagation of the CMMC/HAPC down the colon is unlikely to result from a slowly conducting wave front of neural activity, but more likely because of an interaction between ascending excitatory and descending (serotonergic) inhibitory neural pathways interacting both within the myenteric plexus and at the level of the muscle. That is, CMMC/HAPC propagation appears to be similar to esophageal peristalsis. The suppression of inhibitory (neuronal nitric oxide synthase) motor neurons and mucosal 5-HT release by an upregulation of prostaglandins has important implications in a num-ber of gastrointestinal disorders, especially slow transit constipation.(J Neurogastroenterol Motil 2014;20:423-446).

Neural crest cell-specific inactivation of Nipbl or Mau2 during mouse development results in a late onset of craniofacial defects.

Nipbl (Scc2) and Mau2 (Scc4) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell-specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl;Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner.

Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms.

Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.

Important role of mucosal serotonin in colonic propulsion and peristaltic reflexes: in vitro analyses in mice lacking tryptophan hydroxylase 1.

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.

Insights from a novel model of slow-transit constipation generated by partial outlet obstruction in the murine large intestine.

The mechanisms underlying slow-transit constipation (STC) are unclear. In 50% of patients with STC, some form of outlet obstruction has been reported; also an elongated colon has been linked to patients with STC. Our aims were 1) to develop a murine model of STC induced by partial outlet obstruction and 2) to determine whether this leads to colonic elongation and, consequently, activation of the inhibitory "occult reflex," which may contribute to STC in humans. Using a purse-string suture, we physically reduced the maximal anal sphincter opening in C57BL/6 mice. After 4 days, the mice were euthanized (acutely obstructed), the suture was removed (relieved), or the suture was removed and replaced repeatedly (chronically obstructed, over 24-31 days). In partially obstructed mice, we observed increased cyclooxygenase (COX)-2 levels in muscularis and mucosa, an elongated impacted large bowel, slowed transit, nonpropagating colonic migrating motor complexes (CMMCs), a lack of mucosal reflexes, a depolarized circular muscle with slow-wave activity due to a lack of spontaneous inhibitory junction potentials, muscle hypertrophy, and CMMCs in mucosa-free preparations. Elongation of the empty obstructed colon produced a pronounced occult reflex. Removal of the obstruction or addition of a COX-2 antagonist (in vitro and in vivo) restored membrane potential, spontaneous inhibitory junction potentials, CMMC propagation, and mucosal reflexes. We conclude that partial outlet obstruction increases COX-2 leading to a hyperexcitable colon. This hyperexcitability is largely due to suppression of only descending inhibitory nerve pathways by prostaglandins. The upregulation of motility is suppressed by the occult reflex activated by colonic elongation.

Analysis of spontaneous and nerve-evoked calcium transients in intact extraocular muscles in vitro.

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.

Ca2+ transients in myenteric glial cells during the colonic migrating motor complex in the isolated murine large intestine.

Enteric glia cells (EGCs) form a dense network around myenteric neurons in a ganglia and are likely to have not only a supportive role but may also regulate or be regulated by neural activity. Our aims were to determine if EGCs are activated during the colonic migrating motor complex (CMMC) in the isolated murine colon. Strips of longitudinal muscle were removed and Ca(2+) imaging (Fluo-4) used to study activity in EGCs within myenteric ganglia during CMMCs, followed by post hoc S100 staining to reveal EGCs. The cell bodies of EGCs and their processes formed caps and halos, respectively, around some neighbouring myenteric neurons. Some EGCs (36%), which were largely quiescent between CMMCs, exhibited prolonged tetrodotoxin (TTX; 1 µm)-sensitive Ca(2+) transients that peaked ∼39 s following a mucosal stimulus that generated the CMMC, and often outlasted the CMMC (duration ∼23 s). Ca(2+) transients in EGCs often varied in duration within a ganglion; however, the duration of these transients was closely matched by activity in closely apposed nerve varicosities, suggesting EGCs were not only innervated but the effective innervation was localized. Furthermore, all EGCs, even those that were quiescent, responded with robust Ca(2+) transients to KCl, caffeine, nicotine, substance P and GR 64349 (an NK2 agonist), suggesting they were adequately loaded with indicator and that some EGCs may be inhibited by substances released by neighbouring neurons. Intracellular Ca(2+) waves were visualised propagating between closely apposed glia and from glial cell processes to the soma (velocity 12 µm s(-1)) where they produced an accumulative rise in Ca(2+), suggesting that the soma acts as an integrator of Ca(2+) activity. In conclusion, Ca(2+) transients in EGCs occur secondary to nerve activity; their activation is driven by intrinsic excitatory nerve pathways that generate the CMMC.

Identification and characterization of an inhibitory fibroblast growth factor receptor 2 (FGFR2) molecule, up-regulated in an Apert Syndrome mouse model.

AS (Apert syndrome) is a congenital disease composed of skeletal, visceral and neural abnormalities, caused by dominant-acting mutations in FGFR2 [FGF (fibroblast growth factor) receptor 2]. Multiple FGFR2 splice variants are generated through alternative splicing, including PTC (premature termination codon)-containing transcripts that are normally eliminated via the NMD (nonsense-mediated decay) pathway. We have discovered that a soluble truncated FGFR2 molecule encoded by a PTC-containing transcript is up-regulated and persists in tissues of an AS mouse model. We have termed this IIIa-TM as it arises from aberrant splicing of FGFR2 exon 7 (IIIa) into exon 10 [TM (transmembrane domain)]. IIIa-TM is glycosylated and can modulate the binding of FGF1 to FGFR2 molecules in BIAcore-binding assays. We also show that IIIa-TM can negatively regulate FGF signalling in vitro and in vivo. AS phenotypes are thought to result from gain-of-FGFR2 signalling, but our findings suggest that IIIa-TM can contribute to these through a loss-of-FGFR2 function mechanism. Moreover, our findings raise the interesting possibility that FGFR2 signalling may be a regulator of the NMD pathway.

Ca2+ imaging of activity in ICC-MY during local mucosal reflexes and the colonic migrating motor complex in the murine large intestine.

Colonic migrating motor complexes (CMMCs) are neurally mediated, cyclical contractile and electrical events, which typically propagate along the colon every 2-3 min in the mouse. We examined the interactions between myenteric neurons, interstitial cells of Cajal in the myenteric region (ICC-MY) and smooth muscle cells during CMMCs using Ca(2+) imaging. CMMCs occurred spontaneously or were evoked by stimulating the mucosa locally, or by brushing it at either end of the colon. Between CMMCs, most ICC-MY were often quiescent; their lack of activity was correlated with ongoing Ca(2+) transients in varicosities on the axons of presumably inhibitory motor neurons that were on or surrounded ICC-MY. Ca(2+) transients in other varicosities initiated intracellular Ca(2+) waves in adjacent ICC-MY, which were blocked by atropine, suggesting they were on the axons of excitatory motor neurons. Following TTX (1 µM), or blockade of inhibitory neurotransmission with N(ω)-nitro-L-arginine (L-NA, a NO synthesis inhibitor, 10 µM) and MRS 2500 (a P2Y(1) antagonist, 1 µM), ongoing spark/puff like activity and rhythmic intracellular Ca(2+) waves (38.1 ± 2.9 cycles min(-1)) were observed, yet this activity was uncoupled, even between ICC-MY in close apposition. During spontaneous or evoked CMMCs there was an increase in the frequency (62.9 ± 1.4 cycles min(-1)) and amplitude of Ca(2+) transients in ICC-MY and muscle, which often had synchronized activity. At the same time, activity in varicosites along excitatory and inhibitory motor nerve fibres increased and decreased respectively, leading to an overall excitation of ICC-MY. Atropine (1 µM) reduced the evoked responses in ICC-MY, and subsequent addition of an NK1 antagonist (RP 67580, 500 nM) completely blocked the responses to stimulation, as did applying these drugs in reverse order. An NKII antagonist (MEN 10,376, 500 nM) had no effect on the evoked responses in ICC-MY. Following TTX application, carbachol (1 µM), substance P (1 µM) and an NKI agonist (GR73632, 100 nM) produced the fast oscillations superimposed on a slow increase in Ca(2+) in ICC-MY, whereas SNP (an NO donor, 10 µM) abolished all activity in ICC-MY. In conclusion, ICC-MY, which are under tonic inhibition, are pacemakers whose activity can be synchronized by excitatory nerves to couple the longitudinal and circular muscles during the CMMC. ICC-MY receive excitatory input from motor neurons that release acetylcholine and tachykinins acting on muscarinic and NK1 receptors, respectively.

Colonic elongation inhibits pellet propulsion and migrating motor complexes in the murine large bowel.

The colonic migrating motor complex (CMMC) is a rhythmically occurring neurally mediated motor pattern. Although the CMMC spontaneously propagates along an empty colon it is responsible for faecal pellet propulsion in the murine large bowel. Unlike the peristaltic reflex, the CMMC is an 'all or none' event that appears to be dependent upon Dogiel Type II/AH neurons for its regenerative slow propagation down the colon. A reduction in the amplitude of CMMCs or an elongated colon have both been thought to underlie slow transit constipation, although whether these phenomena are related has not been considered. In this study we examined the mechanisms by which colonic elongation might affect the CMMC using video imaging of the colon, tension and electrophysiological recordings from the muscle and Ca(2+) imaging of myenteric neurons. As faecal pellets were expelled from the murine colon, it shortened by up to 29%. Elongation of the colon resulted in a linear reduction in the velocity of a faecal pellet and the amplitude of spontaneous CMMCs. Elongation of the oral end of a colonic segment reduced the amplitude of CMMCs, whereas elongation of the anal end of the colon evoked a premature CMMC, and caused the majority of CMMCs to propagate in an anal to oral direction. Dogiel Type II/AH sensory neurons and most other myenteric neurons responded to oral elongation with reduced amplitude and frequency of spontaneous Ca(2+) transients, whereas anal elongation increased their amplitude and frequency in most neurons. The inhibitory effects of colonic elongation were reduced by blocking nitric oxide (NO) production with l-NA (100 mum) and soluble guanylate cyclase with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 mum); whereas, l-arginine (1-2 mm) enhanced the inhibitory effects of colonic elongation. In conclusion, polarized neural reflexes can be triggered by longitudinal stretch. The dominant effect of elongation is to reduce CMMCs primarily by inhibiting Dogiel Type II/AH neurons, thus facilitating colonic accommodation and slow transit.

Critical role of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex.

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT(3) receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT(1A), 5-HT(3), and 5-HT(7) receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 microM; 5-HT(3) antagonist) plus hexamethonium (100 microM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 microM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1-5 microM; 5-HT(7) antagonists). Both NAN-190 and (S)-WAY100135 (1-5 microM; 5-HT(1A) antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 microM), and 5-carboxamidotryptamine (5-CT) (5 microM; 5-HT(1/5/7) agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT(1A), 5-HT(3), and 5-HT(7) receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT(7) receptors on AH neurons these interneurons also contribute to the generation of the CMMC.