The GAR-3 muscarinic receptor cooperates with calcium signals to regulate muscle contraction in the Caenorhabditis elegans pharynx

Muscarinic acetylcholine receptors regulate the activity of neurons and muscle cells through G-protein-coupled cascades. Here, we identify a pathway through which the GAR-3 muscarinic receptor regulates both membrane potential and excitation-contraction coupling in the Caenorhabditis elegans pharyngeal muscle. GAR-3 signaling is enhanced in worms overexpressing gar-3 or lacking GPB-2, a G-protein beta-subunit involved in RGS-mediated inhibition of G(o)alpha- and G(q)alpha-linked pathways. High levels of signaling through GAR-3 inhibit pharyngeal muscle relaxation and impair feeding--but do not block muscle repolarization--when worms are exposed to arecoline, a muscarinic agonist. Loss of gar-3 function results in shortened action potentials and brief muscle contractions in the pharyngeal terminal bulb. High levels of calcium entry through voltage-gated channels also impair terminal bulb relaxation and sensitize worms to the toxic effects of arecoline. Mutation of gar-3 reverses this sensitivity, suggesting that GAR-3 regulates calcium influx or calcium-dependent processes. Because the effects of GAR-3 signaling on membrane depolarization and muscle contraction can be separated, we conclude that GAR-3 regulates multiple calcium-dependent processes in the C. elegans pharyngeal muscle.     

Reconstruction of the muscle system in Antygomonas sp. (Kinorhyncha,Cyclorhagida) by means of phalloidin labeling and cLSM

In the present investigation the entire muscle system of the cyclorhagid kinorhynch Antygomonas sp. was three-dimensionally reconstructed from whole mounts by means of FITC-phalloidin labeling and confocal scanning microscopy. With this technique, which proved to be especially useful for microscopically small species, we wanted to reinvestigate and supplement the findings obtained by histological and electron microscopical methods. The organization of the major muscle systems can be summarized as follows: 1) All muscle fibers, apart from the intestinal ones, the spine, and the mouth cone muscles, show a cross-striated pattern; 2) Dorsal longitudinal muscle fibers as well as segmentally arranged dorsoventral fibers occur from segment III to XIII; 3) Diagonal muscle fibers are located laterally in segments III to X; 4) Two rings of circular fibers are present in segment II, forming the closing apparatus in Cyclorhagida. Further circular muscles are present in segment I, forming the mouth cone and the eversible introvert, and in the pharyngeal bulb.     

Anatomical and ultrastructural study of the pharyngeal bulb in Protodrilus (Polychaeta, Archiannelida). I. Muscles and myo-epithelial junctions

The pharyngeal bulb of Protodrilus is both a muscular and an epithelial organ whose function is the drawing up of food particles. The muscular system of the bulb is formed of tightly connected antagonistic muscles: the bulbus muscle and the sagittal and ‘grating plate’ muscles. All of them are composed of obliquely striated fibers whose ultrastructural characteristics are similar to those of Hirudina and even more to those of epitokous forms of Nereidae and Syllidae. Myo-epithelial cells do not exist in the pharyngeal bulb of Protodrilus contrary to what was previously thought; the muscles and the stomodeal epithelium are united by junction areas on both sides of the basal lamina. These myo-epithelial junctions may be compared to the myoepidermic junctions known in several Arthropods. A comparison of the ultrastructural features of the bulbus muscle fibers of Protodrilus (Protodrilidae) and Trilobodrilus (Dinophilidae) shows that the Protodrilus fiber clearly belongs to the obliquely striated type classically found in Polychaeta, while the Trilobodrilus fiber is a very peculiar type of obliquely striated fiber. These differences do not agree with Jägersten's hypothesis on the unity of the Archiannelida established on the basis of a structural similarity of the bulbus muscles.     

Ultrastructure of muscle cells from a few selected turbellarians: possible correlations between form and function

A comparative ultrastructural study of muscle cells from several turbellarian species revealed a basic smooth type of construction, with thick and thin myofilaments. However, the distribution of contractile elements, "dense bodies", smooth reticulum, mitochondria, as well as junctional specializations was found to vary within different systems. This variability is probably related to greater or lesser physical demands on the musculature. From a mechanical viewpoint, the most ‘powerful’ muscular system studied was represented by the pharyngeal bulb of Geocentrophora applanata, in which muscle fibers were organized into a compact, square reticulum, hemidesmosomes occurred in large numbers, and myofilaments were grouped in patches reminiscent of A and I bands of striated muscles. At the other extreme, muscular fibrils of the catenulids studied were thin and regularly spaced along the body wall, and the branched ends of radial myofibrils in the parenchyma were connected to the epithelial layer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immunochemical localization of myosin heavy chain isoforms and paramyosin in developmentally and structurally diverse muscle cell types of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans contains two major groups of muscle cells that exhibit organized sarcomeres: the body wall and pharyngeal muscles. Several additional groups of muscle cells of more limited mass and spatial distribution include the vulval muscles of hermaphrodites, the male sex muscles, the anal-intestinal muscles, and the gonadal sheath of the hermaphrodite. These muscle groups do not exhibit sarcomeres and therefore may be considered smooth. Each muscle cell has been shown to have a specific origin in embryonic cell lineages and differentiation, either embryonically or postembryonically (Sulston, J. E., and H. R. Horvitz. 1977. Dev. Biol. 56:110-156; Sulston, J. E., E. Schierenberg, J. White, and J. N. Thomson. 1983. Dev. Biol. 100:64- 119). Each muscle type exhibits a unique combination of lineage and onset of differentiation at the cellular level. Biochemically characterized monoclonal antibodies to myosin heavy chains A, B, C, and D and to paramyosin have been used in immunochemical localization experiments. Paramyosin is detected by immunofluorescence in all muscle cells. Myosin heavy chains C and D are limited to the pharyngeal muscle cells, whereas myosin heavy chains A and B are localized not only within the sarcomeres of body wall muscle cells, as reported previously, but to the smooth muscle cells of the minor groups as well. Myosin heavy chains A and B and paramyosin proteins appear to be compatible with functionally and structurally distinct muscle cell types that arise by multiple developmental pathways.     

Morphology and ultrastructure of the anterior end of Diplocirrus longisetosus Marenzeller, 1890 (Flabelligeridae, Polychaeta, Annelida)

The morphology and ultrastructure of the sedentary polychaete Diplocirrus longisetosus Marenzeller, 1890, collected from the White Sea, were studied using dissection, histological methods, light microscopy, and both scanning and transmission electron microscopy. The prostomium and peristomium carry a pair of palps, eight branchiae, a pair of nuchal organs and two nephridiopores, ciliated folds and the mouth. The prostomium, peristomium and the first chaetigerous segment with all appendages comprise the so-called siphon complex. The mouth leads to a pharyngeal organ that is closed ventrally and composed of a ventral muscle bulb adjoined dorsally by two folds projecting into the pharyngeal lumen. These parts are connected and enveloped by the longitudinal investing muscle. No tongue-like organ is present. The nervous system of the siphonal part comprises the brain, the circum-oesophageal connectives and the ganglia of the peristomium and first chaetigerous segment.     

The Efferent Connections of the Olfactory Bulb and Accessory Olfactory Bulb in the Snakes,Thamnophis sirtalis and Thamnophis radix

The efferent connections of the olfactory bulb and accessory olfactory bulb of two species of garter snakes, Thamnophis sirtalis and T. radix were studied with experimental anterograde degeneration techniques. Axons of cells located in the olfactory bulb terminate ipsilaterally in all parts of the anterior olfactory nucleus, olfactory tubercle and lateral pallium. In addition, some axons enter the ipsilateral stria medullaris thalami, cross the midline in the habenular commissure, enter the contralateral stria medullaris thalami and terminate in the contralateral lateral pallium. The axons of cells in the accessory olfactory bulb course through the telencephalon completely separated from the fibers of olfactory bulb origin and terminate predominantly in the nucleus sphericus. These results confirm previous reports of the separation between the central projections of the olfactory and vomeronasal systems in a variety of vertebrates. The totality of the separation between these two systems coupled with the extensive development of the vomeronasal-accessory bulb system in these snakes suggests that they may be ideal subjects for further research on the functional significance of the vomeronasal system.     

Histochemical study of distribution of esterases in the pharyngeal bulb of earthworms.

The esterases are studied histochemically in the pharyngeal bulb of local earthworms using tweens, naphthols and indoxyl substrates. Lipase and esterases are located mainly in the pharyngeal epithelial cells and chromophill cells. No activity is seen in the nonchromophill cells. The connective tissue and musculo-vascular tissue contain some esterases activity. Possible role of the esterases in the cellular elements of pharynx has been discussed.     

On the median pharyngeal valve of the American alligator (Alligator mississippiensis)

The middle ear cavities of crocodilians have complex connections with the pharyngeal lumen, including lateral and median components which both open into a single chamber located on the dorsal midline of the pharynx. This chamber and the surrounding soft-tissue is herein termed the median pharyngeal valve. In the American alligator (Alligator mississippiensis) this valve opens, for a duration of 0.3 s, approximately every 120 s; the patency of the median pharyngeal valve was not influenced by either auditory stimuli or by submersing the alligator underwater. The median pharyngeal valve has an outer capsule of dense connective tissue and fibrocartilage and an inner “plug” of loose connective tissue. These opposing surfaces are lined by respiratory epithelium and separated by a cavity that is continuous with the middle ear cavities and the pharyngeal lumen (through a central opening in the capsule termed the pore). The inner plug of the median pharyngeal valve is contacted by skeletal muscles positioned to serve as both elevators/retractors (which would open the valve) and elevators/protractors (which, in conjunction with gravity, would close the valve). Unlike other vertebrate valve systems, the median pharyngeal valve appears to function as a deformable ball check valve.     

Physiological and pharmacological studies on nematodes

Classical transmitters and neuroactive peptides act as transmitters or modulators within the central and peripheral nervous systems of nematodes, for example Ascaris suum and Caenorhabditis elegans. Acetylcholine (ACh) and gamma-aminobutyric acid (GABA) are respectively the excitatory and inhibitory transmitters onto somatic body wall muscle while 5-hydroxytrypamine (5-HT) is the excitatory transmitter onto pharyngeal muscle. 5-HT also reduces ACh-induced contractions of somatic muscle and this action of 5-HT is mediated through activation of adenylate cyclase while that on pharyngeal muscle is mediated through inositol phosphate activation. Glutamate, dopamine and octopamine also have transmitter roles in nematodes. Neuroactive peptides of the RFamide family can excite somatic muscle, for example, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-3 (AVPGVLRFamide) and AF-4 (GDVPGVLRFamide) or inhibit and relax this muscle, for example, PF-1 (SDPNFLRFamide), PF-2 (SADPNFLRFamide) and PF-4 (KPNlRFamide). In addition PF-3 (AF-8) (KSAYMRFamide) has a biphasic action on pharyngeal muscle, excitation followed by inhibition while AF-1 only inhibits this muscle. The peptide effects can be either pre- or postsynaptic or both and are likely to be mediated through second messenger systems. In addition these peptides modulate the action of classical transmitters, particularly ACh.     

Functional design and evolution of the pharyngeal jaw apparatus in euteleostean fishes

Functional and structural patterns in the pharyngeal jaw apparatus of euteleostean fishes are described and analysed as a case study of the transformation of a complex biological design. The sequential acquisition of structural novelties in the pharyngeal apparatus is considered in relation to both current hypotheses of euteleostean phylogeny and patterns of pharyngeal jaw function. Several euteleostean clades are corroborated as being monophyletic, and morphologically conservative features of the pharyngeal jaw apparatus are recognized. Functional analysis, using cinematography and electromyography, reveals four distinct patterns of muscle activity during feeding in primitive euteleosts (Esox) and in derived euteleostean fishes(Perca, Micropterus, Ambloplites, Pomoxis). The initial strike, buccal manipulation, pharyngeal manipulation, and the pharyngeal transport of prey into the oesophagus all involve unique muscle activity patterns that must be distinguished in analyses of pharyngeal jaw function. During pharyngeal transport, the upper and lower pharyngeal jaws are simultaneously protracted and retracted by the action of dorsal and ventral musculoskeletal gill arch couplings. The levator externus four and retractor dorsalis muscles, anatomical antagonists, overlap for 70–90°of their activity period. Levatores externi one and two are the main protractors of the upper pharyngeal jaws in the acanthopterygian fishes studied. The major features of pharyngeal jaw movement in primitive euteleosts are retained in many derived clades in spite of a dramatic structural reorganization of the pharyngeal region. Homologous muscles have radically changed their relative activity periods while pharyngeal jaw kinematics have been modified relatively little. Patterns of transformation of activity may thus bear little direct relationship to the sequence of structural modification in the evolution of complex designs. Overall function of a structural system may be maintained, however, through co-ordinated modifications of the timing of muscle activity and anatomical reorientation of the musculoskeletal system. Deeper understanding of the principles underlying the origin and transformation of functional design in vertebrates awaits further information on the acquisition of both structural and functional novelties at successive hierarchical levels within monophyietic clades. This is suggested as a key goal of future research in functional and evolutionary morphology.     

Anatomy and ultrastructure of ventral pharyngeal organs and their phylogenetic importance in Polychaeta (Annelida)

Summary Transmission electron microscopic studies were carried out on the ventral pharyngeal organs in Ctenodrilus serratus and Scoloplos armiger. The pharyngeal organs are composed of a muscle bulbus and a tongue-like organ. In both species the muscle bulbus consists of transverse muscle fibres and interstitial cells with voluminous cell bodies and dorsoventral tonofilaments; the investing muscle runs into the tongue-like organ; the nuclei of the investing muscle fibres are located in caudal bulges; salivary glands are not present, but numerous gland cells occur in the bulbus epithelium. The tongue-like organ, however, is formed by lateral folds (C. serratus) or a bridge-like structure (S. armiger). The specific structure of the bulbus muscle is probably a homologous characteristic also occurring in several other polychaete families. The phylogenetic importance of this ventral pharynx is discussed and a hypothesis is suggested to explain the differentiation of certain other ventral pharyngeal organs from this probably primitive type.     

大鼠脑内5-HT能神经元对咽肌的支配及调控

用ＰＲＶ和５－ＨＴ免疫组织化学双标记方法研究脑内５－ＨＴ能神经元对咽肌的神经支配及调控。观察到中缝核群的中缝苍白核、中缝隐核、中缝大核、中缝桥核、中缝正中核、中缝背核、和中缝尾侧线形核等部位有ＰＲＶ和５－ＨＴ双标记细胞,直接证明中缝核群的５－ＨＴ能神经元投射到支配咽肌的疑核运动神经元和孤束核中的前运动神经元,调控咽肌的运动。并推测脑干中缝核群中的５－ＨＴ能神经元对咽肌运动的调控可能经由５ＨＴ３和５ＨＴ１Ａ两种受体介导。     

Anatomy and ultrastructure of the ventral pharyngeal organs of Saccocirrus(Saccocirridae) and Protodriloides(Protodriloidae fam. n.) with remarks on the phylogenetic relationships within the Protodrilida (Annelida: Polychaeta)

General anatomy and ultrastructure of the ventral pharyngeal organs were investigated in Succocirrus krusudensis, Protodriloides chaetifer and P. symbioticus. Succocirrus papillocercus, a species without a ventral pharyngeal organ, was included for comparison. The two genera show homologous similarities in their pharynges: bulbus muscle composed of transverse muscle fibres and interstitial cells, those cells with small cell bodies and processes containing prominent tonofilaments which are orientated dorsoventrally and connect the bulbus epithelium with the investing muscle; bulbus muscle fibres circomyarian with nuclei and mitochondria located basally, investing muscle also with interstitial cells, which lack tonofilaments; oesophagus surrounded by gland cells opening into the pharynx. Most likely, a ventral pharynx with these characters was already present in the stem species of Saccocirridae, Protodriloidae fam. n. and Protodrilidae and evolutionary processes led to several changes: a tongue-like organ with a prominent tip and supporting elements is a synapomorphic character of Protodrilus and Succocirrus; the pharyngeal organ is reduced to stomodeal pouches and salivary glands in S. papillocercus and completely lost in Asromus raenioides. These results and data from previous studies are summarized in a tentative phylogenetic dendrogram and allow the introduction of a new family, Protodriloidae.     

The structure and function of a muscle articulation‐type jaw joint of a polychaete worm

The arrangement of the musculature and the fibers of the extracellular matrix (ECM) in the flexible jaw joint of the sandworm Alitta virens (Annelida, Polychaeta) was studied using dissection and histology. The jaws are capable of a wide range of motions principally related to defense and feeding. The left and right jaws are embedded in and moved by a compact pharyngeal bulb of muscle and ECM that also forms the mouth and esophagus. Eight pharyngeal bulbs were removed and dissected to document gross anatomical features or preserved and embedded in plastic for sectioning in multiple planes. The sections were stained with toluidine blue and basic fuchsin to differentiate muscle and ECM. The sections were then digitized and used to develop a three‐dimensional computer illustration. We hypothesize that the muscle and fibers in the ECM are arranged as a muscular hydrostat to support the movement of the jaws. Four specimens were recorded using a digital video camera and a tank with an angled mirror to record lateral and ventral views of jaw movements during locomotion and biting associated with burrow guarding and feeding. Frame by frame kinematic analysis of this video showed that the jaws move symmetrically in a roughly horizontal plane. Although the angle between the jaws increases and then decreases after maximum gape has been reached, the jaws also translate relative to each other such that the axis of rotation is not fixed. Together, these functional morphological and behavioral data identify the jaw mechanism as a flexible joint known as a muscle articulation. As muscle articulations have been previously described only in the beaks of cephalopods and flatworms, this study implies that this type of joint is more common and important than previously recognized. J. Morphol. 276:403–414, 2015. © 2014 Wiley Periodicals, Inc.     

Endothelin regulates neural crest deployment and fate to form great vessels through Dlx5/Dlx6-independent mechanisms

Endothelin-1 (Edn1), originally identified as a vasoconstrictor peptide, is involved in the development of cranial/cardiac neural crest-derived tissues and organs. In craniofacial development, Edn1 binds to Endothelin type-A receptor (Ednra) to induce homeobox genes Dlx5/Dlx6 and determines the mandibular identity in the first pharyngeal arch. However, it remains unsolved whether this pathway is also critical for pharyngeal arch artery development to form thoracic arteries. Here, we show that the Edn1/Ednra signaling is involved in pharyngeal artery development by controlling the fate of neural crest cells through a Dlx5/Dlx6-independent mechanism. Edn1 and Ednra knock-out mice demonstrate abnormalities in pharyngeal arch artery patterning, which include persistent first and second pharyngeal arteries, resulting in additional branches from common carotid arteries. Neural crest cell labeling with Wnt1-Cre transgene and immunostaining for smooth muscle cell markers revealed that neural crest cells abnormally differentiate into smooth muscle cells at the first and second pharyngeal arteries of Ednra knock-out embryos. By contrast, Dlx5/Dlx6 knockout little affect the development of pharyngeal arch arteries and coronary arteries, the latter of which is also contributed by neural crest cells through an Edn-dependent mechanism. These findings indicate that the Edn1/Ednra signaling regulates neural crest differentiation to ensure the proper patterning of pharyngeal arch arteries, which is independent of the regional identification of the pharyngeal arches along the dorsoventral axis mediated by Dlx5/Dlx6.     

Slow Ca2+ dynamics in pharyngeal muscles in Caenorhabditis elegans during fast pumping

The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding. We used a new ratiometric fluorescent calcium indicator and a new optical system that allows simultaneous illumination and detection at any two wavelengths. Pumping was observed with fast, repetitive and synchronous spikes in calcium concentrations in the corpus and terminal bulb, indicative of electrical coupling throughout the muscles. The posterior isthmus, however, responded to only one out of several pumping spikes to produce broad calcium transients, leading to peristalsis, the slow and gradual motion needed for efficient swallows. The excitation-calcium coupling may be uniquely modulated in this region at the level of calcium channels on the plasma membrane.     

The Drosophila wing hearts consist of syncytial muscle cells that resemble adult somatic muscles

In Drosophila, hemolymph circulation in the wings is accomplished by a pair of wing hearts located in the thorax. The embryonic progenitors of these organs were only recently discovered and found to belong to the cardiac mesoderm. In this study, the functional morphology and the structure of mature organs were studied by light and electron microscopy to characterize the tissues arising from this new set of progenitors. Each wing heart consists of 7-8 muscle cells providing the pumping force, a thin layer of non-contractile mononucleated cells separating the muscle cells from the body cavity, and acellular suspending strands opposing the muscle contractions. The muscle cells are multinucleated syncytia attached to the cuticle via epidermal tendon cells. They have central nuclei and sarcomeres with discontinuous Z-discs, A-bands, and I-bands, whereas H-bands and M-bands are indiscernible. From 9 to 11 actin filaments surround each myosin filament. Mitochondria are abundantly interspersed between myofibrils and accumulated in characteristic outpockets of the plasma membrane. The analysis revealed that the wing heart muscles resemble in their ultrastructure and their mode of attachment adult somatic muscles. This suggests that, despite their origin in the cardiac mesoderm, wing heart progenitors are functionally related to somatic adult muscle precursors.