Long-lasting intrinsic optical changes observed in the neurointermediate lobe of the mouse pituitary reflect volume changes in cells of the pars intermedia.

BACKGROUND/AIMS: Complex intrinsic optical changes (light scattering) are readily observed in the neurointermediate lobe of the mouse pituitary gland following electrical stimulation of the infundibular stalk. Our laboratory has previously identified three distinct phases within the light scattering signal: two rapid responses to action potential stimulation and a long duration recovery. The rapid light scattering signals, restricted to the neurohypophysial portion (posterior pituitary) of the neurointermediate lobe, consist of an E-wave and an S-wave that reflect excitation and secretion, respectively. The E-wave has the approximate shape of the action potential and includes voltage- and current-related components and is independent of Ca(2+) entry. The S-wave is related to Ca(2+) entry and exocytosis. The slow recovery phase of the light scattering signal, which we designated the R-wave, is less well characterized. METHODS: Using high temporal resolution light scattering measurements, we monitored intrinsic optical changes in the neurointermediate lobe of the mouse pituitary gland. Pharmacological interventions during the measurements were employed. RESULTS: The data presented here provide optical and pharmacological evidence suggesting that the R-wave, which comprises signals from the posterior pituitary as well as from the pars intermedia, mirrors volume changes in pars intermedia cells following a train of stimuli applied to the infundibular stalk. These volume changes were blocked by the GABA-receptor antagonists bicuculline and picrotoxin, and were mimicked by direct application of GABA in the absence of electrical stimulation. CONCLUSIONS: These results emphasize the importance of central GABAergic projections into the neurointermediate lobe, and the potential role of GABA in effecting hormone release from the pars intermedia.

Optical recording of electrical activity in guinea-pig enteric networks using voltage-sensitive dyes.

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 microm in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall (1); the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes (2,7,8,9). We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) (3) that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of approximately 2.5 microm at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

A mechanical spike accompanies the action potential in Mammalian nerve terminals.

Large and rapid changes in light scattering accompany secretion from nerve terminals of the mammalian neurohypophysis (posterior pituitary). In the mouse, these intrinsic optical signals are intimately related to the arrival of the action potential E-wave and the release of arginine vasopressin and oxytocin (S-wave). Here we have used a high bandwidth atomic force microscope to demonstrate that these light-scattering signals are associated with changes in terminal volume that are detected as nanometer-scale movements of a cantilever positioned on top of the neurohypophysis. The most rapid mechanical response ("spike"), having a duration shorter than the action potential but comparable to that of the E-wave, represents a transient increase in terminal volume due to water movement associated with Na(+)-influx. The slower mechanical event ("dip"), on the other hand, depends upon Ca(2+)-entry as well as on intraterminal Ca(2+)-transients and, analogously to the S-wave, seems to monitor events associated with secretion.

Daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), for the treatment of moderately to severely active ulcerative colitis: a randomised, double blind, placebo controlled, dose ranging trial.

BACKGROUND: An uncontrolled pilot study demonstrated that daclizumab, a humanised monoclonal antibody to the interleukin 2 receptor (CD25), might be effective for the treatment of active ulcerative colitis. METHODS: A randomised, double blind, placebo controlled trial was conducted to evaluate the efficacy of daclizumab induction therapy in patients with active ulcerative colitis. A total of 159 patients with moderate ulcerative colitis were randomised to receive induction therapy with daclizumab 1 mg/kg intravenously at weeks 0 and 4, or 2 mg/kg intravenously at weeks 0, 2, 4, and 6, or placebo. The primary end point was induction of remission at week 8. Remission was defined as a Mayo score of 0 on both endoscopy and rectal bleeding components and a score of 0 or 1 on stool frequency and physician's global assessment components. Response was defined as a decrease from baseline in the Mayo score of at least 3 points. RESULTS: Two per cent of patients receiving daclizumab 1 mg/kg (p = 0.11 v placebo) and 7% of patients receiving 2 mg/kg (p = 0.73) were in remission at week 8, compared with 10% of those who received placebo. Response occurred at week 8 in 25% of patients receiving daclizumab 1 mg/kg (p = 0.04) and in 33% of patients receiving 2 mg/kg (p = 0.30) versus 44% of those receiving placebo. Daclizumab was well tolerated. The most frequently reported adverse events in daclizumab treated patients compared with placebo treated patients were nasopharyngitis (14.6%) and pyrexia (10.7%). CONCLUSION: Patients with moderate ulcerative colitis who are treated with daclizumab are not more likely to be in remission or response at eight weeks than patients treated with placebo.

Optical studies of nicotinic acetylcholine receptor subtypes in the guinea-pig enteric nervous system.

Nicotinic transmission in the enteric nervous system (ENS) is extensive, but the role of individual nicotinic acetylcholine receptor (nAChR) subtypes in the functional connectivity of its plexuses has been elusive. Using monoclonal antibodies (mAbs) against neuronal alpha3-, alpha4-, alpha3/alpha5-, beta2-, beta4- and alpha7-subunits, combined with radioimmunoassays and immunocytochemistry, we demonstrate that guinea-pig enteric ganglia contain all of these nAChR-subunits with the exception of alpha4, and so, differ from mammalian brain. This information alone, however, is insufficient to establish the functional role of the identified nAChR-subtypes within the enteric networks and, ultimately, their specific contributions to gastrointestinal physiology. We have used voltage-sensitive dyes and a high-speed CCD camera, in conjunction with specific antagonists to various nAChRs, to elucidate some of the distinct contributions of the individual subtypes to the behaviour of enteric networks. In the guinea-pig, the submucous plexus has the extraordinary advantage that it is virtually two-dimensional, permitting optical recording, with single cell resolution, of the electrical activity of all of its neurones. In this plexus, the block of alpha3beta2-, alpha3beta4- and/or alpha7-nAChRs always results in a decrease in the magnitude of the synaptic response. However, the magnitude of the fast excitatory post-synaptic potentials (epsps) evoked by electrical stimulation of a neighbouring ganglion varies from cell to cell, reflecting the differential expression of subunits already observed using mAbs, as well as the strengths of the activated synaptic inputs. At the same time, we observe that submucous neurones have a substantial mecamylamine (Mec)-insensitive (non-nicotinic) component to their fast epsps, which may point to the presence of purinergic or serotonergic fast epsps in this system. In the myenteric plexus, on the other hand, the antagonist-induced changes in the evoked synaptic response vary depending upon the location of the stimulating electrode with respect to the ganglion under study. The range of activity patterns that follows sequential pharmacological elimination of individual subtypes suggests that nAChRs may be capable of regulating the activity of both excitatory and inhibitory pathways, in a manner similar to that described in the central nervous system.

A randomized, double-blind, placebo-controlled trial of CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, in patients with corticosteroid-dependent Crohn's disease.

AIM: To evaluate CDP571, a humanized monoclonal antibody to tumour necrosis factor-alpha, for the treatment of corticosteroid-dependent Crohn's disease. METHODS: Patients with corticosteroid-dependent Crohn's disease (use of prednisolone 15-40 mg/day or budesonide 9 mg/day for at least 8 weeks, a previous failed attempt to discontinue corticosteroids within 8 weeks, and Crohn's Disease Activity Index score 150 points or less) were enrolled in a 16-week, randomized, double-blind, placebo-controlled trial. The patients received intravenous CDP571 (20 mg/kg at week 0 and 10 mg/kg at week 8) or placebo. Corticosteroid therapy was decreased following a predefined schedule. The primary efficacy end-point was the percentage of patients with corticosteroid-sparing [i.e. no disease flare (Crohn's Disease Activity Index score > or =220 points) and no longer requiring corticosteroid therapy] at week 10. The major secondary efficacy end-point was corticosteroid-sparing at week 16. RESULTS: Seventy-one patients received treatment. Corticosteroid-sparing was achieved by 19 of 39 (48.7%) CDP571 patients and 13 of 42 (40.6%) placebo patients (P = 0.452) at week 10, and by 18 of 39 (46.2%) CDP571 patients and seven of 32 (21.9%) placebo patients (P = 0.032) at week 16. CDP571 therapy was well-tolerated and the incidence of serious adverse events was similar to placebo. CONCLUSIONS: The CDP571 was effective for corticosteroid-sparing at week 16 but not week 10, and was well-tolerated in patients with corticosteroid-dependent Crohn's disease.

Changes in FAD and NADH fluorescence in neurosecretory terminals are triggered by calcium entry and by ADP production.

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.

Properties of new, long-wavelength, voltage-sensitive dyes in the heart.

Membrane potential measurements using voltage-sensitive dyes (VSDs) have made important contributions to our understanding of electrophysiological properties of multi-cellular systems. Here, we report the development of long wavelength VSDs designed to record cardiac action potentials (APs) from deeper layers in the heart. The emission spectrum of styryl VSDs was red-shifted by incorporating a thienyl group in the polymethine bridge to lengthen and retain the rigidity of the chromophore. Seven dyes, Pittsburgh I to IV and VI to VIII (PGH I-VIII) were synthesized and characterized with respect to their spectral properties in organic solvents and heart muscles. PGH VSDs exhibited 2 absorption, 2 excitation and 2 voltage-sensitive emission peaks, with large Stokes shifts (> 100 nm). Hearts (rabbit, guinea pig and Rana pipiens) and neurohypophyses (CD-1 mice) were effectively stained by injecting a bolus (10-50 microl) of stock solution of VSD (2-5 mM) dissolved in in dimethylsulfoxide plus low molecular weight Pluronic (16% of L64). Other preparations were better stained with a bolus of VSD (2-5 mM) Tyrode's solution at pH 6.0. Action spectra measured with a fast CCD camera showed that PGH I exhibited an increase in fractional fluorescence, DeltaF/F = 17.5 % per AP at 720 nm with 550 nm excitation and DeltaF/F = - 6% per AP at 830 nm with 670 nm excitation. In frog hearts, PGH1 was stable with approximately 30% decrease in fluorescence and AP amplitude during 3 h of intermittent excitation or 1 h of continuous high intensity excitation (300 W Xe-Hg Arc lamp), which was attributed to a combination of dye wash out > photobleaching > dynamic damage > run down of the preparation. The long wavelengths, large Stokes shifts, high DeltaF/F and low baseline fluorescence make PGH dyes a valuable tool in optical mapping and for simultaneous mapping of APs and intracellular Ca(2+).

An ultra-stable non-coherent light source for optical measurements in neuroscience and cell physiology.

We demonstrate that high power light-emitting diodes (LED's) exhibit low-frequency noise characteristics that are clearly superior to those of quartz tungsten halogen lamps, the non-coherent light source most commonly employed when freedom from intensity variation is critical. Their extreme stability over tens of seconds (combined with readily selectable wavelength) makes high power LED's ideal light sources for DC recording of optical changes, from living cells and tissues, that last more than a few hundred milliseconds. These optical signals (DeltaI/I(0)) may be intrinsic (light scattering, absorbance or fluorescence) or extrinsic (absorbance or fluorescence from probe molecules) and we show that changes as small as approximately 8 x 10(-5) can be recorded without signal averaging when LED's are used as monochromatic light sources. Here, rapid and slow changes in the intrinsic optical properties of mammalian peptidergic nerve terminals are used to illustrate the advantages of high power LED's compared to filament bulbs.

Novel naphthylstyryl-pyridium potentiometric dyes offer advantages for neural network analysis.

The submucous plexus of the guinea pig intestine is a quasi-two-dimensional mammalian neural network that is particularly amenable to study using multiple site optical recording of transmembrane voltage (MSORTV) [Biol. Bull. 183 (1992) 344; J. Neurosci. 19 (1999) 3073]. For several years the potentiometric dye of choice for monitoring the electrical activity of its individual neurons has been di-8-ANEPPS [Neuron 9 (1992) 393], a naphthylstyryl-pyridinium dye with a propylsulfonate headgroup that provides relatively large fluorescence changes during action potentials and synaptic potentials. Limitations to the use of this dye, however, have been its phototoxicity and its low water solubility which requires the presence of DMSO and Pluronic F-127 in the staining solution. In searching for less toxic and more soluble dyes exhibiting larger fluorescence signals, we first tried the dienylstyryl-pyridinium dye RH795 [J. Neurosci. 14 (1994) 2545] which is highly soluble in water. This dye yielded relatively large signals, but it was internalized quickly by the submucosal neurons resulting in rapid degradation of the signal-to-noise ratio. We decided to synthesize a series of naphthylstyryl-pyridinium dyes (di-n-ANEPPDHQ) having the same chromophore as di-8-ANEPPS and the quaternary ammonium headgroup (DHQ) of RH795 (resulting in two positive charges versus the neutral propylsulfonate-ring nitrogen combination), and we tested the di-methyl (JPW3039), di-ethyl (JPW2081), di-propyl (JPW3031), di-butyl (JPW5029), and di-octyl (JPW5037) analogues, all of them soluble in ethanol. We found that the di-propyl (di-3-ANEPPDHQ) and the di-butyl (di-4-ANEPPDHQ) forms yielded the best combination of signal-to-noise ratio, moderate phototoxicity and absence of dye internalization.

Activity-dependent depression of excitability and calcium transients in the neurohypophysis suggests a model of "stuttering conduction".

Using millisecond time-resolved optical recordings of transmembrane voltage and intraterminal calcium, we have determined how activity-dependent changes in the population action potential are related to a concurrent modulation of calcium transients in the neurohypophysis. We find that repetitive stimulation dramatically alters the amplitude of the population action potential and significantly increases its temporal dispersion. The population action potentials and the calcium transients exhibit well correlated frequency-dependent amplitude depression, with broadening of the action potential playing only a limited role. High-speed camera recordings indicate that the magnitude of the spike modulation is uniform throughout the neurohypophysis, thereby excluding propagation failure as the underlying mechanism. In contrast, temporal dispersion and latency of the population spike do increase with distance from the stimulation site. This increase is enhanced during repeated stimulation and by raising the stimulation frequency. Changes in Ca influx directly affect the decline in population spike amplitude, consistent with electrophysiological measurements of the local loss of excitability in nerve terminals and varicosities, mediated by a Ca-activated K conductance. Our observations suggest a model of "stuttering conduction": repeated action potential stimulation causes excitability failures limited to nerve terminals and varicosities, which account for the rapid decline in the population spike amplitude. These failures, however, do not block action potential propagation but generate the cumulative increases in spike latency.

Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn's disease.

BACKGROUND AND AIMS: To evaluate the safety and efficacy of the intercellular adhesion molecule 1 (ICAM-1) antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) in Crohn's disease. METHODS: Active (Crohn's disease activity index (CDAI) 200-350), steroid dependent (prednisone 10-40 mg) Crohn's patients were randomised into three treatment groups: placebo versus ISIS 2302 (2 mg/kg intravenously three times a week) for two or four weeks. Patients were treated in months 1 and 3, with steroid withdrawal attempted by week 10. The primary end point (steroid free remission) was a CDAI <150 off steroids at the end of week 14. RESULTS: A total of 299 patients were enrolled, with a mean baseline CDAI of 276 and steroid dose of 23 mg/day. Rates of steroid free remission were equivalent for the two and four week ISIS 2302 groups (20.2% and 21.2%) and the placebo group (18.8%). At week 14, steroid withdrawal was successful in more ISIS 2302 patients compared with placebo treated patients (78% v 64%; p=0.032). Steroid free remission was highly correlated with exposure (p=0.0064). Other clinical responses were correlated with exposure, with significant results versus placebo being observed in the highest area under the curve subgroup. CDAI scores decreased by 136 (112) at week 14 versus 52 (107) for placebo (p=0.027) and inflammatory bowel disease score questionnaire improved by 43 (31) versus 15 (36) for placebo (p=0.027). CONCLUSIONS: Although the primary outcomes failed to demonstrate efficacy, pharmacodynamic modelling suggests that alicaforsen (ISIS 2302) may be an effective therapy for steroid dependent Crohn's disease.

Randomized, controlled trial of recombinant human interleukin-11 in patients with active Crohn's disease.

BACKGROUND: Interleukin-11 is a mesenchymally derived cytokine with pleiotropic activities. A pilot study suggested therapeutic benefit of recombinant human interleukin-11 (rhIL-11) in patients with Crohn's disease. AIM: To determine the safety and preliminary estimate of efficacy of rhIL-11 in treating active Crohn's disease. METHODS: Patients with mild to moderately active Crohn's disease, defined as a Crohn's disease activity index (CDAI) > or = 220 and < or = 450, were enrolled in a multicentre trial. Stable doses of 5-aminosalicylates, antibiotics, 6-mercaptopurine or azathioprine were permitted with appropriate wash-in periods. Oral, intravenous or rectally administered corticosteroids were not allowed. Patients were randomized to 6 weeks of subcutaneous injection with rhIL-11 15 microg/kg or placebo weekly, or rhIL-11 7.5 microg/kg or placebo twice weekly. The primary end-point was per cent change in CDAI at week 6; the major secondary end-point was the proportion of patients in remission, defined as a 100 point decrease in CDAI and absolute CDAI < or = 150. RESULTS: Baseline characteristics were similar among the 148 evaluated patients (49 placebo, 49 rhIL-11 15 microg/kg once weekly, 50 rhIL-11 7.5 microg/kg twice weekly). Treatment was well-tolerated, with mild injection site reactions occurring more frequently among patients treated with rhIL-11. Headache, oedema, and increased platelet count occurred significantly more often in the rhIL-11 7.5 microg/kg twice weekly group, but not the 15 microg/kg once weekly group. There was a trend toward decreased mean per cent change in CDAI in the rhIL-11 15 micro/kg once weekly group vs. placebo (-31.5% vs. -18.5%, 95% confidence interval for the difference -27.9-1.6%). A significantly greater proportion of patients receiving rhIL-11 15 microg/kg once weekly achieved remission compared to placebo (36.7% vs. 16.3%, 95% confidence interval for the difference 3.4-37.4%; 16.4% for rhIL-11 7.5 microg/kg, N.S.). CONCLUSIONS: Weekly subcutaneous injection with rhIL-11 15 microg/kg is safe and effective in inducing remission in a subset of patients with active Crohn's disease.

Dependence of transient and residual calcium dynamics on action-potential patterning during neuropeptide secretion.

Secretion of the neuropeptide arginine vasopressin (AVP) from the neurohypophysis is optimized by short phasic bursts of action potentials with a mean intraburst frequency around 10 Hz. Several hypotheses, most prominently action-potential broadening and buildup of residual calcium, have been proposed to explain this frequency dependence of AVP release. However, how either of these mechanisms would optimize release at any given frequency remains an open question. We have addressed this issue by correlating the frequency-dependence of intraterminal calcium dynamics and AVP release during action-potential stimulation. By monitoring the intraterminal calcium changes with low-affinity indicator dyes and millisecond time resolution, the signal could be dissected into three separate components: rapid Ca(2+) rises (Delta[Ca(2+)](tr)) related to action-potential depolarization, Ca(2+) extrusion and/or uptake, and a gradual increase in residual calcium (Delta[Ca(2+)](res)) throughout the stimulus train. Action-potential stimulation modulated all three components in a manner dependent on both the stimulation frequency and number of stimuli. Overall, the cumulative Delta[Ca(2+)](tr) amplitude initially increased with f(Stim) and then rapidly deteriorated, with a maximum around f(Stim)

Caffeine interaction with fluorescent calcium indicator dyes.

We report that caffeine, in millimolar concentrations, interacts strongly with four common calcium indicator dyes: mag-fura-2, magnesium green, fura-2, and fluo-3. Fluorescence intensities are either noticeably enhanced (mag-fura-2, fura-2) or diminished (magnesium green, fluo-3). The caffeine-induced changes in the fluorescence spectra are clearly distinct from those of metal ion binding at the indicator chelation sites. Binding affinities for calcium of either mag-fura-2 or magnesium green increased only slightly in the presence of caffeine. Caffeine also alters the fluorescence intensities of two other fluorescent dyes lacking a chelation site, fluorescein and sulforhodamine 101, implicating the fluorophore itself as the interaction site for caffeine. In the absence of caffeine, variation of solution hydrophobicity by means of water/dioxane mixtures yielded results similar to those for caffeine. These observations suggest that hydrophobic substances, in general, can alter dye fluorescence in a dye-specific manner. For the particular case of caffeine, and perhaps other commonly used pharmacological agents, the dye interactions can seriously distort fluorescence measurements of intracellular ion concentrations with metal indicator dyes.

Preliminary evaluation of safety and activity of recombinant human interleukin 11 in patients with active Crohn's disease.

BACKGROUND & AIMS: Recombinant human interleukin 11 (rhIL-11) is a cytokine with thrombocytopoietic activity and anti-inflammatory and mucosal protective effects. The objectives of this study were to investigate the safety and tolerability of rhIL-11 in patients with Crohn's disease and to explore the effects of dose and schedule on platelet count and Crohn's disease activity. METHODS: A multicenter, double-masked, placebo-controlled, dose-escalation study of 76 patients with active Crohn's disease was performed. Patients were randomized to receive subcutaneous placebo or rhIL-11 at doses of 5, 16, or 40 microgram. kg-1. wk-1 given 2 or 5 times weekly for 3 weeks. Clinical and laboratory safety data were recorded, and disease activity was measured at each visit. RESULTS: Subcutaneous injection of rhIL-11 generally was well tolerated. Significantly greater increases in platelet counts were found among patients receiving rhIL-11 40 microgram. kg-1. wk-1 as 2 or 5 weekly doses and 16 microgram. kg-1. week-1 as 5 weekly doses compared with patients receiving placebo (P < 0.05). Patients receiving 16 microgram. kg-1. wk-1 had the highest clinical response rates, with a response seen in 42% of patients (5/12) receiving 5 weekly doses and 33% of patients (4/12) receiving 2 weekly doses, compared with 7% of patients (1/15) receiving placebo. CONCLUSIONS: Short-term treatment with rhIL-11 is well tolerated in patients with active Crohn's disease. The thrombocytopoietic effect of rhIL-11 seems to be both dose and schedule dependent and may be minimized with retained clinical benefit in Crohn's disease at 16 microgram. kg-1. wk-1 given in 2 equal doses.

Spatiotemporal patterns of activity in an intact mammalian network with single-cell resolution: optical studies of nicotinic activity in an enteric plexus.

Multiple Site Optical Recording of Transmembrane Voltage (MSORTV) has been used to measure, continuously and simultaneously, the spontaneous electrical activity from all of the neurons in individual ganglia or up to five interconnected ganglia of the submucous plexus of the guinea pig small intestine. These are the first optical recordings of electrical activity with single-cell resolution from a mammalian nervous system. They are used to investigate the effects of acute and chronic application of nicotine on the firing patterns of this neural network containing important cholinergic components. After washout of acutely applied nicotine, the firing rates of selected neurons were dramatically elevated. These results suggest that nAChRs that reversibly desensitize after exposure to nicotine may be responsible for the enhancement of activity that is observed after a brief application of this agonist. In addition, immunostaining with monoclonal antibodies was used to localize alpha3/alpha5, alpha7, and beta2 nAChR subunits, and the results demonstrate the prevalence of alpha3/alpha5. It is this alpha3-containing nAChR subtype that probably accounts for most of the excess activity elicited by nicotine application.

Micromolar 4-aminopyridine enhances invasion of a vertebrate neurosecretory terminal arborization: optical recording of action potential propagation using an ultrafast photodiode-MOSFET camera and a photodiode array.

Modulation of the amount of neuropeptide released from a neurosecretory tissue may be achieved by different means. These include alterations in the quantity secreted from each active nerve terminal or in the actual number of terminals activated. From the vertebrate hypothalamus, magnocellular neurons project their axons as bundles of fibers through the median eminence and infundibular stalk to arborize extensively and terminate in the neurohypophysis, where the neurohypophysial peptides and proteins are released into the circulation by a Ca-dependent mechanism. Elevating [Ca2+]o increases the magnitude of an intrinsic optical change in the neurohypophysial terminals that is intimately related to the quantity of neuropeptide released. Similarly, the addition of micromolar concentrations of 4-aminopyridine to the bathing solution enhances this change in large angle light scattering. However, we show here that, while these effects are superficially similar, they reflect different mechanisms of action. Evidence from intrinsic optical signals (light scattering) and extrinsic (potentiometric dye) absorption changes suggests that calcium increases the amount of neuropeptide released from each active terminal in the classical manner, while 4-aminopyridine exerts its secretagogue action by enhancing the invasion of action potentials into the magno-cellular neuron's terminal arborization, increasing the actual number of terminals activated. Physiologically, electrical invasion of the complex terminal arborization in the neurohypophysis may represent an extremely sensitive control point for modulation of peptide secretion. This would be especially effective in a neurohaemal organ like the posterior pituitary, where, in contrast with a collection of presynaptic terminals, the precise location of release is less important than the quantity released.

Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale.

We have applied multiple site optical recording of transmembrane voltage (MSORTV) to patterned growth cultures of heart cells to analyze the effect of geometry per se on impulse propagation in excitable tissue, with cellular and subcellular resolution. Extensive dye screening led to the choice of di-8-ANEPPS as the most suitable voltage-sensitive dye for this application; it is internalized slowly and permits optical recording with signal-to-noise ratios as high as 40:1 (measured peak-to-peak) and average fractional fluorescence changes of 15% per 100 mV. Using a x 100 objective and a fast data acquisition system, we could resolve impulse propagation on a microscopic scale (15 microns) with high temporal resolution (uncertainty of +/- 5 microseconds). We could observe the decrease in conduction velocity of an impulse propagating along a narrow cell strand as it enters a region of abrupt expansion, and we could explain this phenomenon in terms of the micro-architecture of the tissue. In contrast with the elongated and aligned cells forming the narrow strands, the cells forming the expansions were aligned at random and presented 2.5 times as many cell-to-cell appositions per unit length. If the decrease in conduction velocity results entirely from this increased number of cell-to-cell boundaries per unit length, the mean activation delay introduced by each boundary can be estimated to be 70 microseconds. Using this novel experimental system, we could also demonstrate the electrical coupling of fibroblasts and endotheloid cells to myocytes in culture.