Photon event distribution sampling: an image formation technique for scanning microscopes that permits tracking of sub-diffraction particles with high spatial and temporal resolutions.

In photon event distribution sampling, an image formation technique for scanning microscopes, the maximum likelihood position of origin of each detected photon is acquired as a data set rather than binning photons in pixels. Subsequently, an intensity-related probability density function describing the uncertainty associated with the photon position measurement is applied to each position and individual photon intensity distributions are summed to form an image. Compared to pixel-based images, photon event distribution sampling images exhibit increased signal-to-noise and comparable spatial resolution. Photon event distribution sampling is superior to pixel-based image formation in recognizing the presence of structured (non-random) photon distributions at low photon counts and permits use of non-raster scanning patterns. A photon event distribution sampling based method for localizing single particles derived from a multi-variate normal distribution is more precise than statistical (Gaussian) fitting to pixel-based images. Using the multi-variate normal distribution method, non-raster scanning and a typical confocal microscope, localizations with 8 nm precision were achieved at 10 ms sampling rates with acquisition of ~200 photons per frame. Single nanometre precision was obtained with a greater number of photons per frame. In summary, photon event distribution sampling provides an efficient way to form images when low numbers of photons are involved and permits particle tracking with confocal point-scanning microscopes with nanometre precision deep within specimens.

Dynamic responses of electrically coupled systems.

An identified pair of electrically coupled neurons in the buccal ganglion of the freshwater snail Helisoma trivolvis is an experimentally accessible model of electrical synaptic transmission. In this investigation, electrical synaptic transmission is characterized using sinusoidal frequency (Bode) responses computed by Laplace transforms and responses to brief stimuli. The frequency response of the injected neuron shows a 20-dB/decade attenuation and a phase shift from 0 degree at low frequencies to -90 degrees at high frequencies. The response of a coupled cell shows a 40-dB/decade attenuation and a phase shift from 0 degrees at low frequencies to -180 degrees at high frequencies. A simple mathematical model of electrical synaptic transmission is described that displays each of these crucial features of the measured frequency responses. Methods are described to estimate the frequency responses of coupled systems based on presynaptic measurements. The responses of the coupled system to brief pulses of current were computed using the principle of superposition. The electrical properties of coupled systems impose a minimum delay in reaching a peak in all postsynaptic responses. The delays in the postsynaptic responses to brief stimuli are related to the electrical and anatomical parameters of coupled networks.

Relationship between nitric oxide and vasoactive intestinal polypeptide in enteric inhibitory neurotransmission.

Although considerable evidence suggests that NO serves as a neurotransmitter in gastrointestinal muscles, it is unlikely to be the only substance involved in enteric inhibitory neurotransmission. Vasoactive intestinal polypeptide (VIP) is known to be expressed by inhibitory motor neurons in the gut, and it appears to be co-localized with nitric oxide synthase (NOS) in a subpopulation of enteric neurons. These data suggest that NO and VIP may be parallel neurotransmitters. Others have suggested that VIP is the primary inhibitory transmitter, and it stimulates production of NO in smooth muscle cells. In this "serial cascade" model NO is a paracrine substance. We performed experiments on circular muscles and cells from the canine proximal colon to further test the idea that NO and VIP are parallel neurotransmitters and to determine the validity of the serial cascade model in these muscles. We found that NO-independent inhibitory effects were unmasked when excitatory and NO-dependent inhibitory responses were blocked. NO-independent inhibitory effects were reduced by alpha-chymotrypsin and blocked by tetrodotoxin. NOS- and VIP-like immunoreactivities were co-localized in enteric neurons and varicose fibers in the circular muscle layer. Similar to several other reports we found no evidence for a constitutive NOS in smooth muscle cells. Several aspects of the serial cascade model were not supported by our results: (i) the electrical and mechanical effects of VIP did not depend upon NO synthesis; (ii) VIP-induced changes in [Ca2+]i did not depend upon NO synthesis; and (iii) VIP did not cause the release of NO from canine colonic muscles. These results are consistent with the hypothesis that NO and VIP are co-transmitters, released in parallel from enteric inhibitory nerves.

Development of a time-cycled volume-controlled pressure-limited respirator and lung mechanics system for total liquid ventilation.

Total liquid ventilation can support gas exchange in animal models of lung injury. Clinical application awaits further technical improvements and performance verification. Our aim was to develop a liquid ventilator, able to deliver accurate tidal volumes, and a computerized system for measuring lung mechanics. The computer-assisted, piston-driven respirator controlled ventilatory parameters that were displayed and modified on a real-time basis. Pressure and temperature transducers along with a lineal displacement controller provided the necessary signals to calculate lung mechanics. Ten newborn lambs (<6 days old) with respiratory failure induced by lung lavage, were monitored using the system. Electromechanical, hydraulic, and data acquisition/analysis components of the ventilator were developed and tested in animals with respiratory failure. All pulmonary signals were collected synchronized in time, displayed in real-time, and archived on digital media. The total mean error (due to transducers, analog-to-digital conversion, amplifiers, etc.) was less than 5% compared with calibrated signals. Components (tubing, pistons, etc.) in contact with exchange fluids were developed so that they could be readily switched, a feature that will be important in clinical settings. Improvements in gas exchange and lung mechanics were observed during liquid ventilation, without impairment of cardiovascular profiles. The total liquid ventilator maintained accurate control of tidal volumes and the sequencing of inspiration/expiration. The computerized system demonstrated its ability to monitor in vivo lung mechanics, providing valuable data for early decision making.

A technique to locate the pacemaker in smooth muscles.

A technique was developed to locate the site of slow-wave origin (pacemaker) in a sheet of smooth muscle tissue. Evoked slow waves were used to measure conduction velocities in the two dimensions of sheets of smooth muscle. These conduction velocities were used to "triangulate" to the pacemaker site by an iterative minimization process. The model was tested by triangulating to events evoked from known regions within sheets of canine gastric muscle. The technique was used to determine the sites of origin of spontaneous slow waves and the shift in the spontaneous pacemaker caused by localized injury. This technique will be useful in locating pacemaker regions and to study the factors that affect the origin and frequency of slow waves in syncytial tissues. The triangulation technique should be applicable to intact organs as well as isolated sheets of muscle.

Myogenic regulation of propagation in gastric smooth muscle.

Experiments were performed to test the effects of frequency and stretch on the velocity of slow wave propagation parallel and perpendicular to the long axis of circular muscle fibers in the canine gastric antrum. Slow waves were evoked from one corner of a rectangular sheet of muscle and propagated throughout the tissue. Mathematics were derived and are presented, which simultaneously compute conduction velocities in each direction, regardless of electrode positions. Increased rate of stimulation had no significant effect on conduction velocity in the circumferential axis, but propagation slowed in the axis perpendicular to the circular fibers by an average of 25% over interstimulus intervals between 12 and 60 s. Conduction velocity was also a function of the degree of stretch. The most rapid conduction velocity occurred when muscles were stretched to an average of 118% of the resting, fasted length found in situ in the axis parallel to the circular fibers and 140% in the axis perpendicular to the circular fibers. Propagation was blocked by stretching muscles past 200% of resting length. These results suggest that the frequency of slow waves and gastric distension are intrinsic mechanisms capable of regulating the spread of slow waves.

Effects of frequency on the wave form of propagated slow waves in canine gastric antral muscle.

Experiments were performed to test the effects of frequency on the wave form of electrical slow waves in canine antral circular muscle. At frequencies between 3.0 and 5.6 cycles per minute antral slow waves revealed an alternating wave form pattern. At physiological frequencies antral muscle was incapable of consistently propagating mechanically productive slow waves. Two components of the slow wave were identified on the basis of propagation refractory period. At inter-slow-wave intervals of 3-14 s, the amplitude and duration of the plateau phase wave decreased, but the upstroke phase of the slow wave was minimally affected. Intervals of 2.5-4 s resulted in a normal upstroke event but abolished the plateau. At shorter intervals the upstroke phase of the slow wave was greatly reduced or abolished. The absolute propagation refractory period averaged 2.8 +/- 0.9 s (n = 7) following repolarization of a 'conditioning' slow wave. Slow waves failed to propagate within the absolute propagation refractory period. Acetylcholine decreased the interval required for the plateau phase of the slow wave to recover and permitted conduction of mechanically productive slow waves at or above physiological frequencies. The data presented suggest that gastric motility is modulated by extrinsic and intrinsic factors which regulate slow-wave frequency.

Excitation-contraction coupling in gastric muscles.

Several mechanisms contribute to the regulation of force generated by gastric muscles. Phasic contractions in the stomach are triggered by the propagation of electrical slow waves. These events are associated with an influx of Ca2+ and an increase in intracellular Ca2+ sufficient to elicit contraction. Entry of Ca2+ may be supplemented by the release of Ca2+ from intracellular stores. Excitatory agonists enhance the amplitude of slow waves, increase the amplitude of Ca2+ transients, and increase the force of phasic contractions. Inhibitory agonists have opposite effects. Excitatory agonists may also enhance release of Ca2+ from stores via the production of IP3. Excitatory and inhibitory agonists may also regulate the sensitivity of the contractile apparatus for Ca2+ and therefore alter the contractile response to a given change in intracellular Ca2+.

Are relaxation oscillators an appropriate model of gastrointestinal electrical activity?

Mathematical models based on relaxation oscillators have heavily influenced the terminology and experimental designs of investigations in gastrointestinal motility for nearly two decades. Relaxation oscillator equations have been used to stimulate the electrical activities of the esophagus, stomach, small intestine, colon, and rectosigmoid region. It has been suggested that many attributes of gastrointestinal electrical activity cannot be adequately explained by classic "core-conductor" or "cable" models of excitation and conduction. This article critically reviews the relaxation oscillator model and provides an explanation for each of the putative inadequacies of core-conductor theory. Furthermore, we question whether relaxation oscillator equations are able to simulate the waveforms of gastrointestinal slow waves, alterations in waveform in response to drugs or electrical stimulation, patterns of slow-wave activity when stimulated at physiological frequencies, prolonged periods of constant resting membrane potential between gastric slow waves and electrotonic spread into inactive regions. We conclude that the relaxation oscillator equations do not fully describe gastrointestinal electrical activity; excitation and propagation can be modeled by a theory that provides for morphological features, ionic conductances, and other elements included in the cable equations.

Origin and spread of slow waves in canine gastric antral circular muscle.

Electrical slow waves recorded from circular muscle cells near the myenteric and submucosal plexuses were found to be significantly different. By measuring the latencies between the arrival of evoked events at two recording sites, slow wave conduction velocities were determined in the three dimensions of circular muscle. Slow waves propagated more rapidly in the axis parallel to the circular fibers than in the axes perpendicular to the circular fibers. The rates of slow wave propagation were also determined in axes parallel and perpendicular to fibers in myenteric and submucosal circular muscles. Slow waves conducted more slowly in the circular muscle near the submucosa than in circular muscle near the myenteric plexus. From conduction velocity measurements, a technique was developed to determine the pacemaker site of spontaneous slow waves in a muscle strip. These data demonstrate that slow waves originate from multiple discrete foci; in muscle strips cut along the long axis of the stomach, these foci are found predominantly in the orad region of the muscle strip; and slow waves originate in the outer myenteric half of the muscle.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle.

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Propagation and neural regulation of calcium waves in longitudinal and circular muscle layers of guinea pig small intestine.

BACKGROUND & AIMS: The relative movements of longitudinal muscle (LM) and circular muscle (CM) and the role that nerves play in coordinating their activities has been a subject of controversy. We used fluorescent video imaging techniques to study the origin and propagation of excitability simultaneously in LM and CM of the small intestine. METHODS: Opened segments of guinea pig ileum were loaded with the Ca(2+) indicator fluo-3. Mucosal reflexes were elicited by lightly depressing the mucosa with a sponge. RESULTS: Spontaneous Ca(2+) waves occurred frequently in LM (1.2 s(-1)) and less frequently in CM (3.2 min(-1)). They originated from discrete pacing sites and propagated at rates 8-9 times faster parallel (LM, 87 mm/s; CM, 77 mm/s) compared with transverse to the long axis of muscle fibers. The presence of Ca(2+) waves in one muscle layer did not affect the origin, rate of conduction, or range of propagation in the other layer. The extent of propagation was limited by collisions with neighboring waves or recently excited regions. Simultaneous excitation of both muscle layers could be elicited by mucosal stimulation of either ascending or descending reflex pathways. Neural excitation resulted in an increase in the frequency of Ca(2+) waves and induction of new pacing sites without eliciting direct coupling between layers. CONCLUSIONS: Localized, spontaneous Ca(2+) waves occur independently in both muscle layers, promoting mixing (pendular or segmental) movements, whereas activation of neural reflexes stimulates Ca(2+) waves synchronously in both layers, resulting in strong peristaltic or propulsive movements.

Calcium oscillations in freshly dispersed and cultured interstitial cells from canine colon.

Spontaneous oscillations in intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were used to monitor rhythmicity in freshly dispersed and cultured interstitial cells (IC) from the canine colon. The frequency of oscillations and responses to a number of channel blockers, agonists, ionic substitutions, and temperature were similar in freshly dispersed and cultured cells. An increase in the amplitude of Ca2+ oscillations after 3-6 days in culture and an increase in the rate of decline of [Ca2+]i in cultured IC were two differences noted between freshly dispersed and cultured cells. The frequency and amplitude of oscillations were a function of extracellular Ca2+ concentrations, and oscillations were abolished when the transmembrane flux of Ca2+ was reduced by nicardipine, La3+, or removal of Ca2+ from the extracellular medium. Oscillations persisted in the presence of ryanodine and ouabain. Lowered temperatures or a reduction in the concentration of Ca2+ in the medium reduced the frequency of spontaneous oscillations. Carbachol and substance P caused a transient increase in [Ca2+]i. Substance P then abolished spontaneous events. ATP and calcitonin gene-related peptide increased the frequency of spontaneous activity. Vasoactive intestinal peptide caused a temporary delay in spontaneous oscillations when added to the medium. Results indicate that freshly dispersed and cultured IC may be useful in studies of the mechanisms of rhythmicity in the gastrointestinal system.

Charybdotoxin block of Ca(2+)-activated K+ channels in colonic muscle depends on membrane potential dynamics.

Charybdotoxin (ChTX) is a specific blocker of Ca(2+)-activated K+ channels. The voltage- and time-dependent dynamics of ChTX block were investigated using canine colonic myocytes and the whole cell patch-clamp technique with step and ramp depolarization protocols. During prolonged step depolarizations, K+ current slowly increased in the continued presence of ChTX (100 nM). The rate of increase depended on membrane potential with an e-fold change for every 60 mV. During ramp depolarizations, the effectiveness of ChTX block depended significantly on the rate of the ramp (50% at 0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanism in which ChTX slowly "unbinds" in a voltage-dependent manner. A simple kinetic model was developed in which ChTX binds to both open and closed states. Slow unbinding is consistent with ChTX having little effect on electrical slow waves recorded from circular muscle while causing depolarization and contraction of longitudinal muscle, which displays more rapid "spikes." Resting membrane potential and membrane potential dynamics are important determinants of ChTX action.

Bursting calcium rotors in cultured cardiac myocyte monolayers.

Rotating waves (rotors) of cellular activity were observed in nonconfluent cultures of embryonic chick heart cells by using a macroscopic imaging system that detected fluorescence from intracellular Ca2+. Unlike previous observations of rotors or spiral waves in other systems, the rotors did not persist but exhibited a repetitive pattern of spontaneous onset and offset leading to a bursting rhythm. Similar dynamics were observed in a cellular automaton model of excitable media that incorporates spontaneous initiation of activity, and a decrease of excitability as a consequence of rapid activity (fatigue). These results provide a mechanism for bursting dynamics in normal and pathological biological processes.