New tools for resolving phylogenies: a systematic revision of the Convolutidae (Acoelomorpha, Acoela)

We report about the muscular system and the serotonergic and FMRFamidergic components of the nervous system of the Bucephalidae trematode, Rhipidocotyle campanula, an intestinal parasite of the pike. We use immunocytochemical methods and confocal scanning laser microscopy (CLSM). The musculature is identified by histochemical staining with fluorescently labeled phalloidin. The body wall musculature of R. campanula contains three layers of muscle fibres - the outer thin circular, intermediate longitudinal and inner diagonal muscle fibres running in two opposite directions. The digestive system of R. campanula possess of a well-developed musculature: radial, longitudinal and circular muscle elements are detected in the pharynx, circular and longitudinal muscle filaments seen in the oesophagus, and longitudinal and the circular muscle fibres were found in the intestinal wall. Specific staining indicating the presence of actin muscle filaments occurs in the cirrus sac localized in the posterior body region. The frontal region of anterior attachment organ, the rhynchus, in R. campanula is represented by radial muscle fibres. The posterior part of the rhynchus comprise of radial muscles forming the organ's wall, and several strong longitudinal muscle bundles. Serotonergic and FMRFamidergic structures are detected in the central and peripheral compartments of the nervous system of R. campanula, that is, in the paired brain ganglia, the brain commissure, the longitudinal nerve cords, and connective nerve commissures. The innervations of the rhynchus, pharynx, oesophagus and distal regions of the reproductive system by the serotonergic and FMRFamidergic nervous elements are revealed. We compare our findings obtained on R. campanula with related data for other trematodes.

The network of interstitial cells of Cajal associated with Auerbach's (myenteric) plexus in the canine colon was investigated to determine its role in facilitating communication between circular and longitudinal muscle layers. Electrical coupling between the muscle layers was demonstrated by propagating extracellularly evoked electrotonic pulses from circular muscle cells to nearby longitudinal muscle cells. The likelihood of cytoplasmic continuity across Auerbach's plexus was further demonstrated by the ability of neurobiotin to spread between the interstitial cells and the circular and longitudinal muscle cells. Importantly, direct neurobiotin spread between circular and longitudinal muscle cells was not observed even when they were in close proximity as determined by confocal microscopy. When neurobiotin did spread across the two muscle layers, the intervening interstitial cells were always neurobiotin-positive. In regions where circular and longitudinal muscle cells approach each other closely, electron microscopy revealed the presence of close appositions between interstitial cells and smooth muscle cells. Gap junctions between interstitial cells and smooth muscle cells of both layers, as judged by electron microscopy, were extremely rare. Neither gap junctions nor close appositions were observed between longitudinal and circular muscle cells. The special arrangement for electrotonic coupling across Auerbach's plexus through interstitial cells of Cajal suggests controlled coupling between the two muscle layers, explaining the preservation of their distinct electrical activities.

Using the filamentous actin marker, FITC-conjugated phalloidin, the major muscle systems of adult male and female schistosomes have been examined. The body wall musculature comprises an outer sheath of circular fibres, within which there is a compact layer of short, spindle-shaped longitudinal fibres and a lattice-like arrangement of inner diagonal fibres. Within the oral sucker and acetabulum 3 fibre types, circular, radial and longitudinal can be distinguished. The wall of the oesophagus is lined by a grid-like array of circular and longitudinal fibres, whereas the walls of the intestinal caeca contain only comparably broad circular fibres. Within the female reproductive system, only circular fibres are present in the oviduct, vitelline duct and uterus. In contrast, the wall of the ootype displays closely arranged circular and longitudinal muscle fibres. Antisera to previously identified myoactive compounds (serotonin [5-hydroxytryptamine, 5-HT], neuropeptide F [Moniezia expansa] and GYIRFamide [Bdelloura candida, Dugesia tigrina]) were used as neuronal markers in a preliminary study of the spatial inter-relationships of specific nerve fibres and various muscle systems. Serotoninergic fibres innervate both suckers and also constitute a subtegumental nerve net. In males they provide innervation to the dorso-ventral muscle fibres of the gynaecophoric canal, and in females they innervate the circular and longitudinal muscle fibres of the ootype. Neuropeptide F and the FMRFamide-related peptide, GYIRFamide are both localized within nerve plexuses associated with the dorso-ventral fibres of the gynaecophoric canal, and are evident in the innervation of the ventral and oral sucker.

The paper presents data of the histochemical study of the muscular system of trematode Acrolichanus auriculatus, an intestinal parasite of the sterlet. Using fluorophore-conjugated phalloidin and confocal scanning laser microscopy, the musculature of the body wall, oral and ventral suckers, and digestive system of the parasite were described. The body wall musculature of A. auriculatus contains three layers of muscle fibers: circular, longitudinal, and diagonal muscle fibres. The distribution of muscle fibers along the parasite body were irregular; there were no diagonal muscle fibers in the posterior part of the body. In the region of the lower edge of the genital pore, a complex of muscle fibers was identified which extended from the lower edge of the genital pore to different directions. Several types of muscle fibers were found in oral and abdominal suckers. In the (ventral, dorso-lateral, and dorso-medial) lobes of the oral sucker, the transverse and longitudinal muscle fibers were identified. We described muscles of the pharynx that contained longitudinal, circular, and radial muscle fibers, and the intestine including longitudinal and circular muscles. A comparative analysis confirms the thesis of great diversity in the organization of the trematode muscles and shows the presence in various trematode representatives of both general (conservative) and specific characteristics typical for one or another attribute type.

The distribution of muscle elements in adult Fasciola hepatica was studied on frozen sections using histochemical staining and fluorescent microscopy. Measurements were taken from the stained sections on photomicrograms. The histochemical staining detected actin in muscle filaments of the muscle system. The body wall consisted of circular, diagonal and longitudinal muscle fibers. The thickness of the dorsal muscle layer of the body was 25–50 µm, and the ventral muscle layer was 18– 25 µm. Numerous dorsal and ventral muscle bundles of 10–28 µm thick connected the dorsal and abdominal walls of the body. The musculature thickness of the oral sucker was 196 to 238 µm. The thickness of the pharyngeal wall was 64–98 µm; it was represented by circular, longitudinal and radial muscle fibers. The round and oval lumens of the branched blind intestine had a diameter of 63–119 µm. The intestine was surrounded by thin circular and diagonal muscle fibers of 2–3 µm thick, with a distance of 5-7 µm between them. The ventral sucker consisted of three types of muscle fibers, namely, circular, longitudinal and radial fibers. The maximum thickness of its musculature was about 340 µm.

1. Electrical communication between circular muscle, longitudinal muscle and interstitial cells of Cajal (ICC) was investigated; the hypothesis was tested that the resting membrane potential (RMP) gradient in the circular muscle of canine colon is caused by electrical coupling to neighbouring cells. 2. Isolated longitudinal muscle exhibited spike-like action potentials at a RMP of -45 mV with a frequency and amplitude of 20 cycles/min and 12 mV, respectively. 3. The circular muscle (CM), devoid of longitudinal muscle, myenteric plexus and submuscular ICC-smooth-muscle network, was electrically quiescent at a uniform RMP of -62 mV across the entire circular muscle layer. 4. Preparations consisting of only the submuscular ICC network and a few adjacent layers of circular muscle cells exhibited slow wave-type action potentials at a RMP of about -80 mV. 5. In ICC-CM preparations, consisting of the submuscular ICC network and circular muscle, a RMP gradient of 10 mV was observed near the submucosal border, whereas the RMP was constant at -62 mV in the myenteric half of the circular muscle. 6. In full thickness (FT) preparations, a RMP gradient of 23 mV was observed. The RMP decreased gradually from -71 mV at the submucosal border to -48 mV at the myenteric border of the circular muscle. 7. Coupling of longitudinal muscle to circular muscle caused circular muscle cells at the myenteric surface to depolarize by 14 mV and longitudinal muscle cells to hyperpolarize by 3 mV. 8. In the ICC-CM preparations, the slow wave amplitudes did not decay exponentially away from the ICC network indicating that slow waves propagated actively into the circular muscle; in the FT preparations there was an apparent exponential decay but this was due to the RMP gradient. 9. Spike-like action potentials (SLAPs) superimposed on the plateau phase of slow waves did not decay exponentially away from the myenteric border suggesting that SLAPs were generated within the circular muscle layer. 10. In summary, circular muscle cells possess a uniform intrinsic RMP of -62 mV. The RMP gradient in situ is caused by electrical coupling of circular muscle cells to longitudinal muscle cells and the submuscular network of ICC. In situ, slow wave-type action potentials propagate actively into the circular muscle layer, and, dependent on the level of excitation, circular muscle cells actively generate spikes.

Neuro-muscular junctions of longitudinal and circular muscle fibers of the guinea-pig esophagus and their relation to myenteric plexus

The aim of this study was to evaluate the difference in the expression of mRNA of two types of calcium channels between longitudinal and circular muscle layer of rat myometrium during pregnancy. Changes in the expressions of the mRNA encoding L-type (α1C) and T-type (α1G, α1H, and α1I) calcium channels in longitudinal and circular smooth muscle cells of the rat myometrium were examined using a comparative kinetic RT/PCR method. During the course of pregnancy, α1C mRNA expression showed an N-shaped change in longitudinal muscle, but simply increased after mid-pregnancy in circular muscles. The mRNAs for α1G and α1H, but not that for α1I, were expressed in both longitudinal and circular smooth muscle. In longitudinal muscle, the change in α1H mRNA was similar to that in α1C mRNA during gestation, but the expression of α1G mRNA changed significantly only at term (day 22). In circular muscle, α1H mRNA expression was stable at any stage during pregnancy, but α1G mRNA significantly increased on day 15 and at term. No relationship was observed between voltage-dependent calcium-channel mRNA expressions and either proliferation or hypertrophy of circular muscle during pregnancy. These results show (a) that during pregnancy, the expression levels of L-type channels change dynamically, and it may contribute directly to the regulation of cell excitability, and (b) that the T-subtype that increases during pregnancy differs between longitudinal and circular muscle cells, although their functions remain unclear.

Embryonic Muscle Development of Convoluta pulchra (Turbellaria–Acoelomorpha, Platyhelminthes)

The mechanical and electrical properties of the longitudinal (fundus and corpus) and circular (antrum) muscle fibres of the guinea-pig stomach were investigated. 1. In the longitudinal but not in the circular muscle isotonic K Krebs and Na-free (sucrose) Krebs solutions produced a contracture with a tonic component. The different mechanical responses were not accompanied by different membrane responses. Verapamil abolished both phasic and tonic components of K-induced contracture. 2. During the tonic response of the K-induced contracture, repolarization of the membrane by current pulses relaxed the tissue; after cessation of the current pulse, rebound contracture occurred. In the circular muscle, the Q10 value for the rate of relaxation induced by inward current pulse was 3-1 and for the development of rebound contracture was 2-4. 3. After the tissue had been immersed in Ca-free isotonic K Krebs solution, application of Ca produced a large contracture in the longitudinal muscle, but contracture in the circular muscle was small or absent. However, the amplitude of subsequent carbachol-induced contracture in the above solution was enlarged in proportion to the durations of Ca treatment in both tissues. 4. Direct tetanic electrical stimulation could produce tension in both tissues. With low frequency of stimulation (0-1 Hz) a positive staircase was observed in the circular but not in the longitudinal muscle. 5. It is concluded from these results that topical differences of the motility in the stomach may be due not only to the activity of nervous elements, but also to differences in the properties of the muscle fibres themselves.

We summarize from previous works the functions of circular vs. longitudinal muscle in esophageal peristaltic bolus transport using a mix of experimental data, the conservation laws of mechanics and mathematical modeling. Whereas circular muscle tone generates radial closure pressure to create a local peristaltic closure wave, longitudinal muscle tone has two functions, one physiological with mechanical implications, and one purely mechanical. Each of these functions independently reduces the tension of individual circular muscle fibers to maintain closure as a consequence of shortening of longitudinal muscle locally coordinated with increasing circular muscle tone. The physiological function is deduced by combining basic laws of mechanics with concurrent measurements of intraluminal pressure from manometry, and changes in cross sectional muscle area from endoluminal ultrasound from which local longitudinal shortening (LLS) can be accurately obtained. The purely mechanical function of LLS was discovered from mathematical modeling of peristaltic esophageal transport with the axial wall motion generated by LLS. Physiologically, LLS concentrates circular muscle fibers where closure pressure is highest. However, the mechanical function of LLS is to reduce the level of pressure required to maintain closure. The combined physiological and mechanical consequences of LLS are to reduce circular muscle fiber tension and power by as much as 1/10 what would be required for peristalsis without the longitudinal muscle layer, a tremendous benefit that may explain the existence of longitudinal muscle fiber in the gut. We also review what is understood of the role of longitudinal muscle in esophageal emptying, reflux and pathology.

The ionic dependence of action potentials evoked in giant smooth muscle fibres isolated by enzymatic digestion from the body wall of the marine invertebrate Beroe ovata (Ctenophora) has been investigated using conventional electrophysiological techniques. Differences were observed in the two fibre types studied. The resting membrane potential was –60 ± 1·35 mV (N = 25) in longitudinal muscle fibres and –66 ± 1·37 mV (N =32) in radial fibres. Action potentials had a short plateau in longitudinal fibres but not in radial fibres. The action potential overshoot of both fibre types was decreased in Ca2+-free artificial sea water (ASW). In Na+-deficient ASW, action potentials could not be generated in radial fibres and showed a reduced overshoot in longitudinal fibres. Tetrodotoxin (10−smoll−1) added to ASW or Ca2+-free ASW did not affect the action potentials of either type of fibre. Action potentials of both fibres were partially blocked by Co2+ (20-50 mmol 1−1) or Cd2+ (1-2 mmol 1−1). Action potentials of longitudinal fibres in Na+-deficient ASW were abolished by Co2+ (20 mmol 1−1). In Ca2+-free ASW, the action potential overshoots of both sets of fibres were restored following the addition of Sr2+ or Ba2+. In longitudinal fibres, Sr2+ increased the duration of the action potential plateau. In both longitudinal and radial muscle fibres, Ba2+ prolonged the action potential. In longitudinal fibres exposed to tetraethylammonium chloride (TEACI) or 4-aminopyridine (4AP), the action potential was slightly prolonged. In these fibres, TEA+ or 4AP added to Ca2+-free ASW induced only a long-lasting depolarizing plateau. In radial fibres, the action potential duration was slightly increased in the presence of TEA+; it was unaffected by 4AP. In Ca2+-free ASW, TEA+ and 4AP induced an oscillating membrane response which appeared to be dependent on the intensity of the injected current pulse. It is concluded that (a) there are significant differences between the action potentials of longitudinal and radial muscle fibres but that both are dependent on Na+ and Ca2+, (b) in longitudinal fibres, a Ca2+-activated K+ conductance and a TEA+-sensitive voltage-activated K+ conductance contribute to the repolarizing phase of the action potential, the former being predominant, (c) in radial fibres, the repolarizing phase of action potentials probably involves different membrane K+ conductances among which is a TEA+-sensitive K+ conductance.

We examined the intrinsic motor innervation of the guinea pig choledochoduodenal junction and actions of cholecystokinin octapeptide (CCK-OP) on contractile and membrane activity of circular and longitudinal smooth muscles from three different areas: close to the choledochal sphincter (I); central area in the ampulla (II); and close to the duodenal papilla (III). In response to electrical field stimulation, circular muscle strips showed an initial twitchlike contraction followed by relaxation in areas I and II and only a transient relaxation in the muscle strips prepared from area III. In the longitudinal strips, the regional differences in response to the field stimulation were not prominent, and biphasic twitchlike contractions were observed in areas I, II, and III. Electric field stimulation evoked excitatory junction potentials (EJPs), inhibitory junction potentials (IJPs), or biphasic membrane response (initial EJP followed by an IJP) in the circular and longitudinal smooth muscle cells. Prominent regional differences were observed in areas I, II, and III. Namely, in area III both the circular and longitudinal muscle layers IJPs predominated, whereas in area I the response was predominantly excitatory. CCK-OP (greater than 10-8M) evoked repetitive action potentials in the circular muscle cells, and CCK-OP increased the frequency of slow waves or the spontaneous action potentials in longitudinal muscle cells. CCK-OP enhanced the amplitude of the IJPs and EJPs in both muscle layers. It would thus appear that bile flow is controlled by complex combinations of contraction and relaxation of the smooth muscle that may be due to regional differences in excitatory and inhibitory innervations.

A body musculature of the planarian Polycelis tenuis (Turbellaria, Platyhelminthes) has been investigated by fluorescence microscopy using histochemical staining of whole preparations with fluorescently-labeled phalloidin, which stains muscle cells due to irreversible binding to actin filaments. The results showed that the musculature of the body wall contains circular, diagonal and longitudinal muscle fibers. The circular fibers are the thinnest ones and densely located within the outer layer of the muscle. The longitudinal fibers are thick, gathered into bundles. Individual diagonal muscle fibers are located at a significant distance, in two directions and at an angle to each other. In the work, the process of muscle tissue regeneration in P. tenuis is considered after removal of the planarian’s head. The current study investigates tissue regeneration on days 3, 5, 7, 10 and 13 following tissue amputation. The microscopy images provided valuable information about the main stages of muscle tissue regeneration and their characteristic features. It has been shown that the muscular system in P. tenuis has awesome regenerative abilities and tissue is regenerated within 10–13 days.

The ultrastructure of the longitudinal and circular muscle cells of the guinea pig stomach which show different contractile responses was compared. The extracellular space within the muscle bundles is about 12.1% in the longitudinal layer and about 4.4% in the circular layer. Nexuses were sonsistently found in the circular muscle layer but not in the longitudinal muscle layer. Numbers of both mitochondria and microtubules per unit area of smooth muscle cell are larger in the longitudinal than in the circular muscle. Cellular area occupied by sarcoplasmic reticulum is about 4.7% in the longitudinal muscle, 2.3% in the circular muscle. The numbers of caveolae are almost the same in both tissues. The most distinct difference between the two types of smooth muscle is the appearance of the thick filaments. The circular muscle cell contains approximately 50 thick filaments per 0.5 micron 2 of cytoplasmic area, while the longitudinal muscle cell only about 25 filaments which were usually much thinner than those of the circular muscle. These results indicate that the contractile apparatus itself is different in longitudinal and circular smooth muscles of the guinea pig stomach.