Effects of in utero exposure to fluoxetine on the gastrointestinal tract of rat offspring.

Perinatal exposure to selective serotonin reuptake inhibitors (SSRIs) has been shown to disrupt the development of serotonergic signaling pathways in the brain and enteric nervous system. Serotonin (5-hydroxytryptamine; 5-HT) signaling is critical for gastrointestinal homeostasis; changes in 5-HT expression and regulation have been associated with gastrointestinal diseases of motility and inflammation. We tested the hypothesis that perinatal exposure to the SSRI fluoxetine can influence the development of the gastrointestinal tract in exposed offspring. Female nulliparous Wistar rats were given fluoxetine (10 mg/kg) or vehicle control from 2 wk before mating until weaning; small and large intestines of female and male offspring were collected at postnatal days 1, 21 (P1, P21, respectively), and 6 mo of age. In histological preparations, the proportion of serotonergic neurons significantly increased in the colons of both female and male fluoxetine-exposed compared with control offspring at P21, a time point that signifies maximal exposure to fluoxetine. At 6 mo of age, male but not female fluoxetine-exposed offspring had a significant increase in circulating 5-HT, with a significant decrease in transcripts encoding the 5-HT2A receptor and monoamine oxidase as compared with control offspring. Measurement of spatiotemporal mapping of contractile activity of the small and large intestine at 6 mo of age revealed no changes in motility in the small bowel of fluoxetine-exposed offspring but revealed a significant increase in the frequency of colonic contractions in the female fluoxetine-exposed compared with control animals. Susceptibility to inflammation was examined at 6 mo using the dextran sulfate sodium model of acute colitis. In utero exposure to fluoxetine was not found to exacerbate colitis severity. These findings suggest that fluoxetine exposure during fetal and early postnatal development can lead to changes in serotonergic neurons at the peak of exposure with sex-specific changes in 5-HT signaling and colonic motility in adulthood.NEW & NOTEWORTHY There is increasing recognition of the relevance of in utero and early postnatal exposures in the developmental programming of the gastrointestinal tract. Perinatal exposure to selective serotonin reuptake inhibitors and antidepressant medications is of particular relevance as they are commonly prescribed during pregnancy, and serotonergic pathways play key roles during gastrointestinal development and in postnatal homeostasis. Here, we provide a comprehensive evaluation of clinically relevant outcomes of gastrointestinal motility and susceptibility to colitis in fluoxetine-exposed offspring and highlight changes in colonic serotonergic neurons at the peak of perinatal fluoxetine exposure with sex-dependent changes in serotonin signaling and colonic motility in adulthood.

Jet-Cooled Phosphorescence Excitation Spectrum of the T1(n,π*) ← S0 Transition of 4H-Pyran-4-one.

The 4H-pyran-4-one (4PN) molecule is a cyclic conjugated enone with spectroscopically accessible singlet and triplet (n,π*)excited states. Vibronic spectra of 4PN provide a stringent test of electronic-structure calculations, through comparison of predicted vs measured vibrational frequencies in the excited state. We report here the T1(n,π*) ← S0 phosphorescence excitation spectrum of 4PN, recorded under the cooling conditions of a supersonic free-jet expansion. The jet cooling has eliminated congestion appearing in previous room-temperature measurements of the T1 ← S0 band system and has enabled us to determine precise fundamental frequencies for seven vibrational modes of the molecule in its T1(n,π*) state. We have also analyzed the rotational contour of the 000 band, obtaining experimental values for spin-spin and spin-rotation constants of the T1(n,π*) state. We used the experimental results to test predictions from two commonly used computational methods, equation-of-motion excitation energies with dynamical correlation incorporated at the level of coupled cluster singles doubles (EOM-EE-CCSD) and time-dependent density functional theory (TDDFT). We find that each method predicts harmonic frequencies within a few percent of observed fundamentals, for in-plane vibrational modes. However, for out-of-plane modes, each method has specific liabilities that result in frequency errors on the order of 20-30%. The calculations have helped to identify a perturbation from the T2(π,π*) state that leads to unexpected features observed in the T1(n,π*) ← S0 origin band rotational contour.

A myogenic motor pattern in mice lacking myenteric interstitial cells of Cajal explained by a second coupled oscillator network.

The interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are a network of coupled oscillators in the small intestine that generate rhythmic electrical phase waves leading to corresponding waves of contraction, yet rhythmic action potentials and intercellular calcium waves have been recorded from c-kit-mutant mice that lack the ICC-MP, suggesting that there may be a second pacemaker network. The gap junction blocker carbenoxolone induced a "pinstripe" motor pattern consisting of rhythmic "stripes" of contraction that appeared simultaneously across the intestine with a period of ~4 s. The infinite velocity of these stripes suggested they were generated by a coupled oscillator network, which we call X. In c-kit mutants rhythmic contraction waves with the period of X traveled the length of the intestine, before the induction of the pinstripe pattern by carbenoxolone. Thus X is not the ICC-MP and appears to operate under physiological conditions, a fact that could explain the viability of these mice. Individual stripes consisted of a complex pattern of bands of contraction and distension, and between stripes there could be slide waves and v waves of contraction. We hypothesized that these phenomena result from an interaction between X and the circular muscle that acts as a damped oscillator. A mathematical model of two chains of coupled Fitzhugh-Nagumo systems, representing X and circular muscle, supported this hypothesis. The presence of a second coupled oscillator network in the small intestine underlines the complexity of motor pattern generation in the gut.NEW & NOTEWORTHY Physiological experiments and a mathematical model indicate a coupled oscillator network in the small intestine in addition to the c-kit-expressing myenteric interstitial cells of Cajal. This network interacts with the circular muscle, which itself acts as a system of damped oscillators, to generate physiological contraction waves in c-kit (W) mutant mice.

On the nature of high-amplitude propagating pressure waves in the human colon.

Characterization of high-amplitude propagating pressure waves (HAPWs or HAPCs) plays a key role in diagnosis of colon dysmotility using any type of colonic manometry. With the introduction of high-resolution manometry, more insight is gained into this most prominent propulsive motor pattern. Here, we use a water-perfused catheter with 84 sensors with intervals between measuring points of 1 cm throughout the colon, for 6-8 h, in 19 healthy subjects. The catheter contained a balloon to evoke distention. We explored as stimuli a meal, balloon distention, oral prucalopride, and bisacodyl injection, with a goal to optimally evoke HAPWs. We developed a quantitative measure of HAPW activity, the "HAPW Index." Our protocol elicited 290 HAPWs. 21% of HAPWs were confined to the proximal colon with an average amplitude of 75.3 ± 3.3 mmHg and an average HAPW Index of 440 ± 58 mmHg·m·s. 29% of HAPWs started in the proximal colon and ended in the transverse or descending colon, with an average amplitude of 87.9 ± 3.1 mmHg and an average HAPW Index of 3,344 ± 356 mmHg·m·s. Forty-nine percent of HAPWs started and ended in the transverse or descending colon with an average amplitude of 109.3 ± 3.3 mmHg and an average HAPW Index of 2,071 ± 195 mmHg·m·s. HAPWs with and without simultaneous pressure waves (SPWs) initiated the colo-anal reflex, often abolishing 100% of anal sphincter pressure. Rectal bisacodyl and proximal balloon distention were the most optimal stimuli to evoke HAPWs. These measures now allow for a confident diagnosis of abnormal motility in patients with colonic motor dysfunction.NEW & NOTEWORTHY High-amplitude propagating pressure waves (HAPWs) were characterized using 84 sensors throughout the entire colon in healthy subjects, taking note of site of origin, site of termination, amplitude, and velocity, and to identify optimal stimuli to evoke HAPWs. Three categories of HAPWs were identified, including the associated colo-anal reflex. Proximal balloon distention and rectal bisacodyl were recognized as reliable stimuli for evoking HAPWs, and a HAPW Index was devised to quantify this essential colonic motor pattern.

Phase waves and trigger waves: emergent properties of oscillating and excitable networks in the gut.

The gut is enmeshed by a number of cellular networks, but there is only a limited understanding of how these networks generate the complex patterns of activity that drive gut contractile functions. Here we review two fundamental types of cell behaviour, excitable and oscillating, and the patterns that networks of such cells generate, trigger waves and phase waves, respectively. We use both the language of biophysics and the theory of nonlinear dynamics to define these behaviours and understand how they generate patterns. Based on this we look for evidence of trigger and phase waves in the gut, including some of our recent work on the small intestine.

Abnormal absorptive colonic motor activity in germ-free mice is rectified by butyrate, an effect possibly mediated by mucosal serotonin.

The role of short-chain fatty acids (SCFAs) in the control of colonic motility is controversial. Germ-free (GF) mice are unable to produce these metabolites and serve as a model to study how their absence affects colonic motility. GF transit is slower than controls, and colonization of these mice improves transit and serotonin [5-hydroxytryptamine (5-HT)] levels. Our aim was to determine the role SCFAs play in improving transit and whether this is dependent on mucosal 5-HT signaling. Motility was assessed in GF mice via spatiotemporal mapping. First, motor patterns in the whole colon were measured ex vivo with or without luminal SCFA, and outflow from the colon was recorded to quantify outflow caused by individual propulsive contractions. Second, artificial fecal pellet propulsion was measured. Motility was then assessed in tryptophan hydroxylase-1 (TPH1) knockout (KO) mice, devoid of mucosal 5-HT, with phosphate buffer, butyrate, or propionate intraluminal perfusion. GF mice exhibited a lower proportion of propulsive contractions, lower volume of outflow/contraction, slower velocity of contractions, and slower propulsion of fecal pellets compared with controls. SCFAs changed motility patterns to that of controls in all parameters. Butyrate administration increased the proportion of propulsive contractions in controls yet failed to in TPH1 KO mice. Propionate inhibited propulsive contractions in all mice. Our results reveal significant abnormalities in the propulsive nature of colonic motor patterns in GF mice, explaining the decreased transit time in in vivo studies. We show that butyrate but not propionate activates propulsive motility and that this may require mucosal 5-HT. NEW & NOTEWORTHY Understanding the role that the microbiota play in governing the physiology of colonic motility is lacking. Here, we offer for the first time, to our knowledge, a detailed analysis of colonic motor patterns and pellet propulsion using spatiotemporal mapping in the absence of microbiota. We show a striking difference in germ-free and control phenotypes and attribute this to a lack of fermentation-produced short-chain fatty acid. We then show that butyrate but not propionate can restore motility and that the butyrate effect likely requires mucosal 5-hydroxytryptamine.

Slow wave contraction frequency plateaux in the small intestine are composed of discrete waves of interval increase associated with dislocations.

NEW FINDINGS: What is the central question of this study? What is the nature of slow wave-driven contraction frequency gradients in the small intestine? What is the main finding and its importance? Frequency plateaux are composed of discrete waves of increased interval, each wave associated with a contraction dislocation. Smooth frequency gradients are generated by localized neural modulation of wave frequency, leading to functionally important wave turbulence. Both patterns are emergent properties of a network of coupled oscillators, the interstitial cells of Cajal. ABSTRACT: A gut-wide network of interstitial cells of Cajal generates electrical oscillations (slow waves) that orchestrate waves of muscle contraction. In the small intestine there is a gradient in slow wave frequency from high at the duodenum to low at the terminal ileum. Time-averaged measurements of frequency have suggested either a smooth or a stepped (plateaued) gradient. We measured individual contraction intervals from diameter maps of the mouse small intestine to create interval maps (IMaps). The IMaps showed that each frequency plateau was composed of discrete waves of increased interval. Each interval wave originated at a terminating contraction wave, a 'dislocation', at the proximal boundary of the plateau. In a model chain of coupled phase oscillators, interval wave frequency increased as coupling decreased or as the natural frequency gradient or noise increased. Injuring the intestine at a proximal point, to destroy coupling, suppressed distal steps, which then reappeared with gap junction block by carbenoxolone. This lent further support to our previous hypothesis that lines of dislocations were fixed by points of low coupling strength. Dislocations, induced by electrical field pulses in the intestine and by equivalent phase shift in the model, were associated with interval waves. When the enteric nervous system was active, IMaps showed a chaotic, turbulent pattern of interval change, with no frequency steps or plateaux. This probably resulted from local, stochastic release of neurotransmitters. Plateaux, dislocations, interval waves and wave turbulence arise from a dynamic interplay between natural frequency and coupling in the network of interstitial cells of Cajal.

The phase response and state space of slow wave contractions in the small intestine.

NEW FINDINGS: What is the central question of this study? What are the dynamical rules governing interstitial cell of Cajal (ICC)-generated slow wave contractions in the small intestine, as reflected in their phase response curve and state space? What is the main finding and its importance? The phase response curve has a region of phase advance surrounding a phase delay peak. This pattern is important in generating a stable synchrony within the ICC network and is related to the state space of the ICC; in particular, the phase delay peak corresponds to the unstable equilibrium point that threads the ICC's limit cycle. Interstitial cells of Cajal (ICCs) generate electrical oscillations in the gut. Synchronization of the ICC population is required for generation of coherent electrical waves ('slow waves') that cause muscular contraction and thereby move gut content. The phase response curve (PRC) is an experimental measure of the dynamical rules governing a population of oscillators that determine their synchrony and gives an experimental window onto the state space of the oscillator, its dynamical landscape. We measured the PRC of slow wave contractions in the mouse small intestine by the novel combination of diameter mapping and single pulse electrical field stimulation. Phase change (τ) was measured as a function of old phase (ϕ) and distance from the stimulation electrode (d). Plots of τ(ϕ, d) showed an arrowhead-shaped region of phase advance enclosing at its base a phase delay peak. The phase change mirrored the perturbed pattern of contraction waves in response to a pulse. The (ϕ, d) plane is the surface of a displacement tube extending from the limit cycle through state space. To visualize the state space vector field on this tube, latent phase (ϕlat ) was calculated from τ. At the transition from advance to delay, isochrons made boomerang turns before tightening and winding around the phase delay peak corresponding to the unstable equilibrium point that threads the limit cycle. This isochron foliation had previously been observed in oscillator models such as the Fitzhugh-Nagumo but has not been demonstrated experimentally. The spatial extension of the PRC afforded by diameter mapping allows a better understanding of the dynamical properties of ICCs and how they synchronize as a population.

Network properties of interstitial cells of Cajal affect intestinal pacemaker activity and motor patterns, according to a mathematical model of weakly coupled oscillators.

NEW FINDINGS: What is the central question of this study? What are the effects of interstitial cells of Cajal (ICC) network perturbations on intestinal pacemaker activity and motor patterns? What is the main finding and its importance? Two-dimensional modelling of the ICC pacemaker activity according to a phase model of weakly coupled oscillators showed that network properties (coupling strength between oscillators, frequency gradient and frequency noise) strongly influence pacemaker network activity and subsequent motor patterns. The model explains motor patterns observed in physiological conditions and provides predictions and testable hypotheses for effects of ICC loss and frequency modulation on the motor patterns. Interstitial cells of Cajal (ICC) are the pacemaker cells of gut motility and are associated with motility disorders. Interstitial cells of Cajal form a network, but the contributions of its network properties to gut physiology and dysfunction are poorly understood. We modelled an ICC network as a two-dimensional network of weakly coupled oscillators with a frequency gradient and showed changes over time in video and graphical formats. Model parameters were obtained from slow-wave-driven contraction patterns in the mouse intestine and pacemaker slow-wave activities from the cat intestine. Marked changes in propagating oscillation patterns (including changes from propagation to non-propagating) were observed by changing network parameters (coupling strength between oscillators, the frequency gradient and frequency noise), which affected synchronization, propagation velocity and occurrence of dislocations (termination of an oscillation). Complete uncoupling of a circumferential ring of oscillators caused the proximal and distal section to desynchronize, but complete synchronization was maintained with only a single oscillator connecting the sections with high enough coupling. The network of oscillators could withstand loss; even with 40% of oscillators lost randomly within the network, significant synchronization and anterograde propagation remained. A local increase in pacemaker frequency diminished anterograde propagation; the effects were strongly dependent on location, frequency gradient and coupling strength. In summary, the model puts forth the hypothesis that fundamental changes in oscillation patterns (ICC slow-wave activity or circular muscle contractions) can occur through physiological modulation of network properties. Strong evidence is provided to accept the ICC network as a system of coupled oscillators.

Motor patterns of the small intestine explained by phase-amplitude coupling of two pacemaker activities: the critical importance of propagation velocity.

Phase-amplitude coupling of two pacemaker activities of the small intestine, the omnipresent slow wave activity generated by interstitial cells of Cajal of the myenteric plexus (ICC-MP) and the stimulus-dependent rhythmic transient depolarizations generated by ICC of the deep muscular plexus (ICC-DMP), was recently hypothesized to underlie the orchestration of the segmentation motor pattern. The aim of the present study was to increase our understanding of phase-amplitude coupling through modeling. In particular the importance of propagation velocity of the ICC-DMP component was investigated. The outcome of the modeling was compared with motor patterns recorded from the rat or mouse intestine from which propagation velocities within the different patterns were measured. The results show that the classical segmentation motor pattern occurs when the ICC-DMP component has a low propagation velocity (<0.05 cm/s). When the ICC-DMP component has a propagation velocity in the same order of magnitude as that of the slow wave activity (∼1 cm/s), cluster type propulsive activity occurs which is in fact the dominant propulsive activity of the intestine. Hence, the only difference between the generation of propagating cluster contractions and the Cannon-type segmentation motor pattern is the propagation velocity of the low-frequency component, the rhythmic transient depolarizations originating from the ICC-DMP. Importantly, the proposed mechanism explains why both motor patterns have distinct rhythmic waxing and waning of the amplitude of contractions. The hypothesis is brought forward that the velocity is modulated by neural regulation of gap junction conductance within the ICC-DMP network.

Effects of gap junction inhibition on contraction waves in the murine small intestine in relation to coupled oscillator theory.

Waves of contraction in the small intestine correlate with slow waves generated by the myenteric network of interstitial cells of Cajal. Coupled oscillator theory has been used to explain steplike gradients in the frequency (frequency plateaux) of contraction waves along the length of the small intestine. Inhibition of gap junction coupling between oscillators should lead to predictable effects on these plateaux and the wave dislocation (wave drop) phenomena associated with their boundaries. It is these predictions that we wished to test. We used a novel multicamera diameter-mapping system to measure contraction along 25- to 30-cm lengths of murine small intestine. There were typically two to three plateaux per length of intestine. Dislocations could be limited to the wavefronts immediately about the terminated wave, giving the appearance of a three-pronged fork, i.e., a fork dislocation; additionally, localized decreases in velocity developed across a number of wavefronts, ending with the terminated wave, which could appear as a fork, i.e., slip dislocations. The gap junction inhibitor carbenoxolone increased the number of plateaux and dislocations and decreased contraction wave velocity. In some cases, the usual frequency gradient was reversed, with a plateau at a higher frequency than its proximal neighbor; thus fork dislocations were inverted, and the direction of propagation was reversed. Heptanol had no effect on the frequency or velocity of contractions but did reduce their amplitude. To understand intestinal motor patterns, the pacemaker network of the interstitial cells of Cajal is best evaluated as a system of coupled oscillators.

Enteric sensory neurons communicate with interstitial cells of Cajal to affect pacemaker activity in the small intestine.

Enteric sensory neurons (the AH neurons) play a role in control of gastrointestinal motor activity; AH neuron activation has been proposed to change propulsion into segmentation. We sought to find a mechanism underlying this phenomenon. We formulated the hypothesis that AH neurons increase local ICC-MP (interstitial cells of Cajal associated with the myenteric plexus) pacemaker frequency to disrupt peristalsis and promote absorption. To that end, we sought structural and physiological evidence for communication between ICC-MP and AH neurons. We designed experiments that allowed us to simultaneously activate AH neurons and observe changes in ICC calcium transients that underlie its pacemaker activity. Neurobiotin injection in AH neurons together with ICC immunohistochemistry proved the presence of multiple contacts between AH neuron varicosities and the cell bodies and processes of ICC-MP. Generating action potential activity in AH neurons led to increase in the frequency and amplitude of calcium transients underlying pacemaker activity in ICC. When no rhythmicity was seen, rhythmic calcium transients were evoked in ICC. As a control, we stimulated nitrergic S neurons, which led to reduction in ICC calcium transients. Hence, we report here the first demonstration of communication between AH neurons and ICC. The following hypothesis can now be formulated: AH neuron activation can disrupt peristalsis directed by ICC-MP slow wave activity, through initiation of a local pacemaker by increasing ICC pacemaker frequency through increasing the frequency of ICC calcium transients. Evoking new pacemakers distal to the proximal lead pacemaker will initiate both retrograde and antegrade propulsion causing back and forth movements that may disrupt peristalsis.

Cholinergic signalling-regulated KV7.5 currents are expressed in colonic ICC-IM but not ICC-MP.

Interstitial cells of Cajal (ICC) and the enteric nervous system orchestrate the various rhythmic motor patterns of the colon. Excitation of ICC may evoke stimulus-dependent pacemaker activity and will therefore have a profound effect on colonic motility. The objective of the present study was to evaluate the potential role of K(+) channels in the regulation of ICC excitability. We employed the cell-attached patch clamp technique to assess single channel activity from mouse colon ICC, immunohistochemistry to determine ICC K(+) channel expression and single cell RT-PCR to identify K(+) channel RNA. Single channel activity revealed voltage-sensitive K(+) channels, which were blocked by the KV7 blocker XE991 (n = 8), which also evoked inward maxi channel activity. Muscarinic acetylcholine receptor stimulation with carbachol inhibited K(+) channel activity (n = 8). The single channel conductance was 3.4 ± 0.1 pS (n = 8), but with high extracellular K(+), it was 18.1 ± 0.6 pS (n = 22). Single cell RT-PCR revealed Ano1-positive ICC that were positive for KV7.5. Double immunohistochemical staining of colons for c-Kit and KV7.5 in situ revealed that intramuscular ICC (ICC-IM), but not ICC associated with the myenteric plexus (ICC-MP), were positive for KV7.5. It also revealed dense cholinergic innervation of ICC-IM. ICC-IM and ICC-MP networks were found to be connected. We propose that the pacemaker network in the colon consists of both ICC-MP and ICC-IM and that one way of exciting this network is via cholinergic KV7.5 channel inhibition in ICC-IM.

Visualizing partial solvation at the air-water interface.

Despite significant research, the mechanistic nuances of unusual reactivity at the air-water interface, especially in microdroplets, remain elusive. The likely contributors include electric fields and partial solvation at the interface. To reveal these intricacies, we measure the frequency shift of a well-defined azide vibrational probe at the air-water interface, while independently controlling the surface charge density by introducing surfactants. First, we establish the response of the probe in the bulk and demonstrate that it is sensitive to both electrostatics and hydrogen bonding. From interfacial spectroscopy we infer that the azide is neither fully hydrated nor in a completely aprotic dielectric environment; instead, it experiences an intermediate environment. In the presence of hydrogen bond-accepting sulphate surfactants, competition arises for interfacial water with the azide. However, the dominant influence stems from the electrostatic effect of their negative heads, resulting in a significant blue-shift. Conversely, for the positive ammonium surfactants, our data indicate a balanced interplay between electrostatics and hydrogen bonding, leading to a minimal shift in the probe. Our results demonstrate partial solvation at the interface and highlights that both hydrogen bonding and electrostatics may assist or oppose each other in polarizing a reactant, intermediate, or product at the interface, which is important for understanding and tuning interfacial reactivity.

Gating of maxi channels observed from pseudo-phase portraits.

Phase space has been used to visualize and analyze the dynamic behavior of stochastic and chaotic systems. We applied this concept to maxi channels recorded from excised inside-out patches of in situ interstitial cells of Cajal. Pseudo-phase portraits of channel current were fairly homogeneous from patch to patch. They showed three main peaks, α, ß, and γ, in increasing conductance. These represented single or near aggregated states. The α-peak was the closed state. The ß-peak was small, consisting of a single conductance state, or in some cases two (a doublet). The ß-peak state(s) had a long lifetime and displayed a characteristic behavior of frequent short transitions to γ but not to α. It was always preceded by a short series of α/γ-transitions. The γ-peak was the largest and consisted of a large number of conductance states with fast state transitions, sometimes to the extent of causing a diffusive-type behavior. Phase portraits allowed us to construct a provisional gating scheme for the maxi channel and suggest that further analysis of recordings in higher dimensional phase space and with related techniques may be promising.

Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins ß1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

Statistical assessment of change point detectors for single molecule kinetic analysis.

Change point detectors (CPDs) are used to segment recordings of single molecules for the purpose of kinetic analysis. The assessment of the accuracy of CPD algorithms has usually been based on testing them with simulated data. However, there have not been methods to assess the output of CPDs from real data independent of simulation. Here we present one method to do this based on the assumption that the elementary kinetic unit is a stationary period (SP) with a normal distribution of samples, separated from other SPs by change points (CPs). Statistical metrics of normality can then be used to assess SPs detected by a CPD algorithm (detected SPs, DSPs). Two statistics in particular were found to be useful, the z-transformed skew (S(Z)) and z-transformed kurtosis (K(Z)). K(Z)(S(Z)) plots of DSP from noise, simulated data and single ion channel recordings showed that DSPs with false negative CP could be distinguished. Also they showed that filtering had a significant effect on the normality of data and so filtering should be taken into account when calculating statistics. This method should be useful for analyzing single molecule recordings where there is no simple model for the data.

Endothelial LSP1 is involved in endothelial dome formation, minimizing vascular permeability changes during neutrophil transmigration in vivo.

The endothelium actively participates in neutrophil migration out of the vasculature via dynamic, cytoskeleton-dependent rearrangements leading to the formation of transmigratory cups in vitro, and to domes that completely surround the leukocyte in vivo. Leukocyte-specific protein 1 (LSP1), an F-actin-binding protein recently shown to be in the endothelium, is critical for effective transmigration, although the mechanism has remained elusive. Herein we show that endothelial LSP1 is expressed in the nucleus and cytosol of resting endothelial cells and associates with the cytoskeleton upon endothelial activation. Two-photon microscopy revealed that endothelial LSP1 was crucial for the formation of endothelial domes in vivo in response to neutrophil chemokine keratinocyte-derived chemokine (KC) as well as in response to endogenously produced chemokines stimulated by cytokines (tumor necrosis factor α [TNFα] or interleukin-1ß [IL-1ß]). Endothelial domes were significantly reduced in Lsp1(-/-) compared with wild-type (WT) mice. Lsp1(-/-) animals not only showed impaired neutrophil emigration after KC and TNFα stimulation, but also had disproportionate increases in vascular permeability. We demonstrate that endothelial LSP1 is recruited to the cytoskeleton in inflammation and plays an important role in forming endothelial domes thereby regulating neutrophil transendothelial migration. The permeability data may underscore the physiologic relevance of domes and the role for LSP1 in endothelial barrier integrity.

The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia.

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.