The contribution of the swimbladder to buoyancy in the adult zebrafish (Danio rerio): a morphometric analysis

Many teleost fishes use a swimbladder, a gas-filled organ in the coelomic cavity, to reduce body density toward neutral buoyancy, thus minimizing the locomotory cost of maintaining a constant depth in the water column. However, for most swimbladder-bearing teleosts, the contribution of this organ to the attainment of neutral buoyancy has not been quantified. Here, we examined the quantitative contribution of the swimbladder to buoyancy and three-dimensional stability in a small cyprinid, the zebrafish (Danio rerio). In aquaria during daylight hours, adult animals were observed at mean depths from 10.1 +/- 6.0 to 14.2 +/- 5.6 cm below the surface. Fish mass and whole-body volume were linearly correlated (r(2) = 0.96) over a wide range of body size (0.16-0.73 g); mean whole-body density was 1.01 +/- 0.09 g cm(-3). Stereological estimations of swimbladder volume from linear dimensions of lateral X-ray images and direct measurements of gas volumes recovered by puncture from the same swimbladders showed that results from these two methods were highly correlated (r(2) = 0.85). The geometric regularity of the swimbladder thus permitted its volume to be accurately estimated from a single lateral image. Mean body density in the absence of the swimbladder was 1.05 +/- 0.04 g cm(-3). The swimbladder occupied 5.1 +/- 1.4% of total body volume, thus reducing whole-body density significantly. The location of the centers of mass and buoyancy along rostro-caudal and dorso-ventral axes overlapped near the ductus communicans, a constriction between the anterior and posterior swimbladder chambers. Our work demonstrates that the swimbladder of the adult zebrafish contributes significantly to buoyancy and attitude stability. Furthermore, we describe and verify a stereological method for estimating swimbladder volume that will aid future studies of the functions of this organ.     

Effects of simulated microgravity on the development of the swimbladder and buoyancy control in larval zebrafish (Danio rerio)

The gas-filled swimbladder of teleost fishes provides hydrodynamic lift which counteracts the high density of other body tissues, and thereby allows the fish to achieve neutral buoyancy with minimal energy expenditure. In this study, we examined whether the absence of a constant direction gravitational vector affects the ontogeny of the swimbladder and buoyancy control in zebrafish (Danio rerio). We exposed fertilized eggs to simulated microgravity (SMG) in a closed rotating wall vessel with control eggs placed in a similar but nonrotating container. All eggs hatched in both groups. At 96 hr of postfertilization (hpf), all larvae were removed from the experimental and control vessels. At this point, 62% of the control larvae, but only 14% of SMG-exposed larvae, were observed to have inflated their swimbladder. In addition, the mean volume of the inflated swimbladders was significantly greater in the control larvae compared with larvae raised in SMG. After transfer to open stationary observation tanks, larvae with uninflated swimbladders in both groups swam to the surface to complete inflation, but this process was significantly delayed in larvae exposed to SMG. Initial differences in swimbladder inflation and volume between groups disappeared by 144 hpf. Furthermore, there were no apparent changes in patterns of development and maturation of swimbladder musculature, vasculature, or innervation resulting from SMG exposure at later stages of ontogeny. These data indicate that, despite a transient delay in swimbladder inflation in zebrafish larvae exposed to SMG, subsequent swimbladder development in these animals proceeded similarly to that in normal larvae.     

Branchial arch muscle innervation by the glossopharyngeal (IX) and vagal (X) nerves in tetraodontiformes, with special reference to muscle homologies

Branchial arch muscle innervation by the glossopharyngeal (IX) and vagal (X) nerves in 10 tetraodontiform families and five outgroup taxa was examined, with special reference to muscle homologies. Basic innervation patterns and their variations were described for all muscle elements (except gill filament muscles). In the tetraodontids Takifugu poecilonotus and Canthigaster rivulata, diodontid Diodon holocanthus, and molid Mola mola, levator externus 4 was innervated by the 3rd vagal branchial trunk (BX3) in addition to BX2, owing to strong posterior expansion of the muscle. Based on nerve innervation, migrations of the muscle attachment sites (i.e., origins and insertions) were recognized in levator internus 2 (in Mola mola), obliquus dorsalis 3 (in Ostracion immaculatus and Canthigaster rivulata), and obliquus ventralis 2 (in Stephanolepis cirrhifer), muscle topologies not necessarily being indicative of homologies. Embryonic origin of the retractor dorsalis and parallel attainment of the swimbladder muscle within the order were also discussed.     

Lateral line morphology and cranial osteology of the Rubynose Brotula,Cataetyx rubrirostris

Cranial osteology, canal neuromast distribution, superficial neuromast distribution and innervation, and cephalic pore structure were studied in cleared and stained specimens of the deep sea brotulid Cataetyx rubrirostris. The cranial bone structure of C. rubrirostris is similar to other brotulids (Dicrolene sp.) and zoarcids (Zoarces sp.), except for an unusual amount of overlapping of the bones surrounding the cranial vault. The superficial neuromasts are innervated by the anterodorsal, anteroventral, middle and posterior lateral line nerves and are organized similarly to those of the blind ophidioid cave fish Typhliasina pearsei. The cephalic pores open into a widened lateral line canal system. The canal is compartmentalized into a series of neuromast‐containing chambers that probably amplify signals received by the system. J. Morphol. 241:265–274, 1999. © 1999 Wiley‐Liss, Inc.     

Ontogeny of body density and the swimbladder in yellowtail kingfish Seriola lalandi larvae

The ontogeny of larval body density and the morphological and histological events during swimbladder development were investigated in two cohorts of yellowtail kingfish Seriola lalandi larvae to understand the relationship between larval morphology and body density. Larvae <3 days post hatch (dph) were positively buoyant with a mean ± s.d . body density of 1·023 ± 0·001 g cm^?3. Histological evidence demonstrated that S. lalandi larvae are initially transient physostomes with the primordial swimbladder derived from the evagination of the gut ventral to the notochord and seen at 2 dph. A pneumatic duct connected the swimbladder to the oesophagus, but degenerated after 5 dph. Initial swimbladder (SB) inflation occurred on 3 dph, and the inflation window was 3–5 dph when the pneumatic duct was still connected to the gut. The swimbladder volume increased with larval age and the epithelial lining on the swimbladder became flattened squamous cells after initial inflation. Seriola lalandi developed into a physoclist with the formation of the rete mirabile and the gas‐secreting gland comprised low‐columnar epithelial cells. Larvae with successfully inflated swimbladders remained positively buoyant, whereas larvae without SB inflation became negatively buoyant and their body density gradually reached 1·030 ± 0·001 g cm^?3 by 10 dph. Diel density changes were observed after 5 dph, owing to day time deflation and night‐time inflation of the swimbladder. These results show that SB inflation has a direct effect on body density in larval S. lalandi and environmental factors should be further investigated to enhance the rate of SB inflation to prevent the sinking death syndrome in the early life stage of the fish larvae.     

Innervation of regenerated spindles in muscle grafts of the rat

Features of the nerve supply and the encapsulated fibers of muscle spindles were assessed in grafted and normal extensor digitorum longus (EDL) muscles of rats by analysis of serial 10-microns frozen transverse sections stained for enzymes which delineated motor and sensory endings, oxidative capacity and muscle fiber type. The number of fibers was significantly more variable, and branched fibers were more frequently observed in regenerated spindles than in control spindles. Forty-eight percent of regenerated spindles received sensory innervation. Spindles reinnervated by afferents had a larger periaxial space than did spindles which were not reinnervated by afferents. Regenerated fibers innervated by afferents had small cross-sectional areas, equatorial regions with myofibrils restricted to the periphery of fibers, unpredictable patterns of nonuniform and nonreversible staining along the length of the fiber for 'myofibrillar' adenosine triphosphatase (mATPase) after acid and alkaline preincubation. In contrast, regenerated fibers devoid of sensory innervation resembled extrafusal fibers in that they usually exhibited myofibrils throughout the length of the fiber, no central aggregations of myonuclei, uniform staining for mATPase and a reversal of staining for mATPase after preincubation in an acid or alkaline medium. Approximately thirty percent of encapsulated fibers devoid of sensory innervation stained analogous to a type I extrafusal fiber, a pattern of staining never observed in intrafusal fibers of normal spindles. Groups of encapsulated fibers all exhibiting this pattern of staining reflect that either these fibers may have been innervated by collaterals of skeletomotor axons that originally innervated type I extrafusal fibers or that fibers innervated by only fusimotor neurons express patterns of staining for mATPase similar to extrafusal fibers in the absence of sensory innervation. Sensory innervation may also influence the reestablishment of multiple sites of motor endings on regenerated intrafusal fibers. Those regenerated fibers innervated by afferents had more motor endings than did regenerated fibers devoid of sensory innervation. Differences in size, morphology, and patterns of staining for mATPase and numbers of motor endings between fibers innervated by afferents and fibers devoid of sensory innervation reflect that afferents can influence the differentiation of muscle cells and the reestablishment of motor innervation other than during the late prenatal/early postnatal period when muscle spindles form and differentiate in rats.     

Different on the inside: extreme swimbladder sexual dimorphism in the South Asian torrent minnows

The swimbladder plays an important role in buoyancy regulation but is typically reduced or even absent in benthic freshwater fishes that inhabit fast flowing water. Here, we document, for the first time, a remarkable example of swimbladder sexual dimorphism in the highly rheophilic South Asian torrent minnows (Psilorhynchus). The male swimbladder is not only much larger than that of the female (up to five times the diameter and up to 98 times the volume in some cases), but is also structurally more complex, with multiple internal septa dividing it into smaller chambers. Males also exhibit a strange organ of unknown function or homology in association with the swimbladder that is absent in females. Extreme sexual dimorphism of non-gonadal internal organs is rare among vertebrates and the swimbladder sexual dimorphisms that we describe for Psilorhynchus are unique among fishes.     

The ultrastructure of the swimbladder of the toadfish,Opsanus tau L.

Summary The anterior chamber of the swimbladder of the toadfish Opsanus tau L. is lined by a single layer of columnar gas gland cells, cuboidal cells that resemble gas gland cells but are located outside of the gas gland region, and squamous cells. Multilamellar bodies are numerous in the gas gland cells and the cuboidal cells and are present in smaller numbers in the squamous cells. Capillaries lie in the lamina propria directly below the epithelial lining. A thick continuous muscularis mucosae and a submucosa consisting of tightly packed cells, cell processes, and connective tissue may contribute to the impermeability to gases of the wall of the anterior chamber.The posterior chamber of the swimbladder is lined by a single type of squamous epithelial cell. Multilamellar bodies were occasionally observed in these cells also. Other types of cells frequently form a partial second layer between the epithelial lining and the basement lamina. A thin muscularis mucosae lies directly below the basement lamina and the capillaries of the posterior chamber are located in the submucosa. The tunica externa is a layer of dense connective tissue that surrounds both the anterior and posterior chambers. Collagen fibrils in the form of tactoids are present in this layer.Part of this work was submitted by S.M.M. in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Biology Department, Boston University. S.M.M. is grateful for a National Science Foundation Traineeship.     

A finite interval of initial swimbladder inflation in Latris lineata revealed by sequential removal of water‐surface films

A finite interval of initial swimbladder inflation in striped trumpeter Latris lineata larvae occurred over 4 days at 16° C. Water‐surface films were removed on different days to form treatments: 4, 8, 9, 10, 11 and 12 days post hatching, dph (day 4, 8, 9, 10, 11 and 12 treatments, respectively). No swimbladder inflation was recorded prior to water‐surface film removal. When the water‐surface films were removed in day 4 and 8 treatments, initial swimbladder inflation was first recorded in larvae 9 dph at mean ± s .e . 35·0 ± 5·4%(n = 4) and 45·0 ± 7·9%, respectively. Water‐surface film removal at days 9, 10 and 11, resulted in initial swimbladder inflation the following day at 62·5 ± 2·5, 62·5 ± 7·2 and 11·3 ± 5·5% in larvae 10, 11 and 12 dph, respectively. No swimbladder inflation was recorded following water‐surface film removal on day 12. There was no significant difference in initial inflation among larvae in day 4, 8, 9 and 10 treatments, ranging from 65·0 ± 4·1 to 73·8 ± 6·9%(P > 0·05). Initial inflation was significantly lower in the day 11 treatment (11·3 ± 5·5%)(P < 0·05). During the inflation interval (9–12 dph) swimbladders displayed one of three morphologies; liquid dilation, gas inflated and collapsed. Collapse of the swimbladder lumen was first apparent in larvae without swimbladder inflation from 11 dph and progressively developed thereafter in all larvae with non‐inflated swimbladders. Larvae >6·1 mm standard length lost the ability to undergo initial swimbladder inflation. This study demonstrates that the interval for initial swimbladder inflation in striped trumpeter is short, finite and related to larval size. The end of the inflation interval was marked by onset of abnormal swimbladder morphologies, but not to closure of the pneumatic duct.     

Role of nerve and muscle factors in the development of rat muscle spindles

The soleus muscles of fetal rats were examined by electron microscopy to determine whether the early differentiation of muscle spindles is dependent upon sensory innervation, motor innervation, or both. Simple unencapsulated afferent-muscle contacts were observed on the primary myotubes at 17 and 18 days of gestation. Spindles, encapsulations of muscle fibers innervated by afferents, could be recognized early on day 18 of gestation. The full complement of spindles in the soleus muscle was present at day 19, in the region of the neuromuscular hilum. More afferents innervated spindles at days 18 and 19 of gestation than at subsequent developmental stages, or in adult rats; hence, competition for available myotubes may exist among afferents early in development. Some of the myotubes that gave rise to the first intrafusal (bag2) fiber had been innervated by skeletomotor (alpha) axons prior to their incorporation into spindles. However, encapsulated intrafusal fibers received no motor innervation until fusimotor (gamma) axons innervated spindles 3 days after the arrival of afferents and formation of spindles, at day 20. The second (bag1) intrafusal fiber was already formed when gamma axons arrived. Thus, the assembly of bag1 and bag2 intrafusal fibers occurs in the presence of sensory but not gamma motor innervation. However, transient innervation of future bag2 fibers by alpha axons suggests that both sensory and alpha motor neurons may influence the initial stages of bag2 fiber assembly. The confinement of nascent spindles to a localized region of the developing muscle and the limited number of spindles in developing muscles in spite of an abundance of afferents raise the possibility that afferents interact with a special population of undifferentiated myotubes to form intrafusal fibers.     

Distribution and origin of vasoactive intestinal polypeptide (VIP)-immunoreactive,acetylcholinesterase-positive and adrenergic nerves of the cerebral arteries in the bent-winged bat (Mammalia: Chiroptera)

Summary The overall distribution and origins of vasoactive intestinal polypeptide (VIP)-immunoreactive (IR), acetylcholinesterase (AChE)-positive and adrenergic nerves in the walls of the cerebral arteries were investigated in the bent-winged bat. VIP-IR and AChE-positive nerves innervating the bat cerebral vasculature appear to arise mainly from VIP-IR and AChE-positive cell bodies within microganglia found in the nerve bundle accompanying the sympathetic nerve bundle within the tympanic cavity. These microganglia, as well as the nerve bundle containing them, do not emit catecholamine fluorescence, suggesting that they are of the cranial parasympathetic outflow, probably the facial or glossopharyngeal one. The axons from VIP-IR and AChE-positive microganglia run intermingled with sympathetic adrenergic nerves in the same thick fiber bundles, and reach the cranial cavity through the carotid canal. In addition, some of the VIP-IR fibers innervating the vertebro-basilar system, at least the basilar artery, originate from VIP-IR nerve cells located in the wall of this artery.The supply of VIP-IR fibers to the bat major cerebral arteries is the richest among mammals that have been studied, and differs from other mammals in that it is much greater in the vertebro-basilar system than in the internal carotid system: plexuses of VIP-IR nerves are particularly dense along the walls from the posterior ramus to posterior cerebral and basilar arteries. Small pial and intracerebral arteries of the vertebro-basilar system, especially those of the posterior cerebral artery which supply most parts of the diencephalon and cerebrum, are also richly innervated by peripheral VIP-IR fibers. This pattern corresponds well with the innervation pattern of adrenergic and AChE-positive nerves.     

Histochemistry of the lung of the Australian snake-necked tortoise chelodina longicollis

The innervation and structure of the lung of the Australian snake-necked tortoise, Chelodina longicollis, was examined by using light microscopy including fluorescence histochemical techniques. The anterior lung was divided into a number of compartments with numerous alveolar spaces. The posterior lung was simpler and saclike in structure and alveolar spaces were absent. Smooth muscle fibers occurred in discrete muscle bands and in the walls of the septal bands. Ganglion cells occurred along nerve trunks throughout the lung but were more numerous in the posterior lung. Smooth muscle bands, the extrinsic pulmonary artery, and the arteries within the lung were sparsely innervated by adrenergic fibers. Substance P-containing sensory fibers were not demonstrated. The innervation and structure of the lung are compared to published work on other reptiles.     

The gas bladder of Argentina silus L., with special reference to the ultrastructure of the gas gland cells and the countercurrent vascular bundles

Summary The orifice between the two chambers of the gas bladder in Argentina silus is surrounded by a sphincter muscle. Gas analyses of the gas bladder contents of fish from 400 meters depth give 0–1% carbon dioxide and 9–72% oxygen. Micro-retia mirabilia form a countercurrent vascular system, and the arterial component has peripherally a sphincter mechanism. The function of the glandular layer of the anterior chamber remains uncertain, but the structure indicates secretion into blood capillaries. The lining epithelium of the anterior chamber may secrete some substance into blood or directly into the lumen, which may be involved in a secretory mechanism. This conclusion is not supported by our histochemical tests. The posterior chamber has no micro-retia and the blood vessels have a different origin from those of the anterior chamber. The blood vessels form a plexus of capillaries or sinuses in contact with the flat lining epithelium, thus allowing gases to pass freely by diffusion. — The muscular layers of both chambers are innervated by catecholamine-containing nerve fibres.The investigation was supported by grants from the Swedish Natural Research Council (No. 99-35) and by the Faculty of Mathematics and Science, University of Lund.     

Sensory innervation of the canine esophagus, stomach, and duodenum.

The sensory innervation of the postpharyngeal foregut was investigated by injecting the enzyme horseradish peroxidase (HRP) into the walls of the esophagus, stomach, or duodenum. The transported HRP was identified histochemically, labeled neurons in the spinal and vagal ganglia were counted, and the results were plotted using an SAS statistical program. The spinal sensory fields of each viscus were defined using three determinations: craniocaudal extent, principal innervation field, and peak innervation field. The data revealed that innervation fields are craniocaudally extensive, the sensory field of each viscus overlaps significantly with its neighbor, yet each viscus can be characterized by a field of peak innervation density. Craniocaudal innervation of the esophagus spans as many as 22-23 paired spinal ganglia (C1-L2). There are two peak innervation fields for the cervical (C2-C6 and T2-T4) and for the thoracic (T2-T4 and T8-T12) sectors of the esophagus. The sensory innervation of the stomach extends craniocaudally over as many as 25 paired spinal ganglia (C2-L5). The peak innervation field of the stomach spans a large area comprising the cranial, middle, and the immediately adjoining caudal thoracic ganglia (T2-T10). The duodenum is innervated craniocaudally by as many as 15 paired thoracolumbar ganglia (T2-L3). Peak innervation originates in the middle and caudal thoracic ganglia and cranial lumbar (T6-L1) ganglia. There is a recognizable viscerotopic organization in the sensory innervation of the postpharyngeal foregut; successively more caudal sectors of this region of the alimentary canal are supplied with sensory fibers from successively more caudal spinal dorsal root ganglia. Vagal afferent innervation of the esophagus, stomach, and duodenum is bilateral and originates predominantly, but not exclusively, from vast numbers of neurons in the nodose (distal) ganglia. The esophagus is innervated bilaterally and more abundantly by jugular (proximal) ganglia neurons than is either the stomach or duodenum. The physiological significance of the findings are discussed in relation to the phenomena of visceral pain and referred pain.     

ANATOMY OF THE SWIMBLADDERS OF SEVEN FAMILIES OF TRANSVAAL FRESHWATER FISHES

SUMMARY

IN the genera Barbus and Labeo of the family Cyprinidae there is a typical twolobed, cylindrical swimbladder: a shorter anterior and a longer posterior lobe, connected by an isthmus. The pneumatic duct passes from the anteroventral end of the posterior lobe to the oesophagus. In the genus Labeo two spiral bands encircle the posterior lobe twice. No rete mirabile, nor any indication of a gas gland, was observed.

The species Hydrocynus vittutus of the family Characidae has a very similarly shaped swimbladder to that of the Cyprinidea. Inside the anterior lobe, however, there is a peculiar structure, which is evidently the gas glad, although a rete mirabile was not observed.

In the families of the Siluriformes, studied, with the exception of the Clariidae, a single lobed, heartshaped swimbladder is present. It is divided by a longitudinal and a transverse. septum into three chambers: an anterior, a right and a left posterior chamber. The pneumatic duct originates from the medial posteroventral part of the anterior chamber. In Clarias gariepinus the two-lobed, right and left lobed, swimbladder lies in a bony capsule, which is attached transversely to the posteroventral part of the skull. In all the Siluriformes, studied, no trace of a gas gland, nor of a rete mirabile was found.

The Cichlid swimbladder has no pneumatic duct, nor any other exit, hence it is physoclistic. In the Cichlids the retroperitoneal position of the swimbladder is accentuated, as the peritoneum and the outer tectum of the swimbladder have united to form a thick, tough membrane, which divides the body cavity into a distinct ventral, or visceral cavity, and a dorsal, or swimbladder cavity. The swimbladder cavity acts as an outer swimbladder. It contains an inner, smaller bladder whose internal ventro-anterior surface is covered with arborescently arranged patches of gas glands.

The attachment of the swimbladder to the tripus and also to the ossa suspensoris is discussed.     

Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers

The swimbladder is a hydrostatic organ in fish postulated as a homolog of the tetrapod lung. While lung development has been well studied, the molecular mechanism of swimbladder development is essentially uncharacterized. In the present study, swimbladder development in zebrafish was analyzed by using several molecular markers: hb9 (epithelium), fgf10a and acta2 (mesenchyme), and anxa5 (mesothelium), as well as in vivo through enhancer trap transgenic lines Et(krt4:EGFP)^sq33-2 and Et(krt4:EGFP)^sqet3 that showed strong EGFP expression in the swimbladder epithelium and outer mesothelium respectively. We defined three phases of swimbladder development: epithelial budding between 36 and 48 hpf, growth with the formation of two additional mesodermal layers up to 4.5 dpf, and inflation of posterior and anterior chambers at 4.5 and 21 dpf respectively. Similar to those in early lung development, conserved expression of Hedgehog (Hh) genes, shha and ihha, in the epithelia, and Hh receptor genes, ptc1 and ptc2, as well as fgf10a in mesenchyme was observed. By analyzing several mutants affecting Hh signaling and Ihha morphants, we demonstrated an essential role of Hh signaling in swimbladder development. Furthermore, time-specific Hh inhibition by cyclopamine revealed different requirements of Hh signaling in the formation and organization of all three tissue layers of swimbladder.     

Swimming behavior in relation to buoyancy in an open swimbladder fish, the Chinese sturgeon

The swimbladder of fishes is readily compressed by hydrostatic pressure with depth, causing changes in buoyancy. While modern fishes can regulate buoyancy by secreting gases from the blood into the swimbladder, primitive fishes, such as sturgeons, lack this secretion mechanism and rely entirely on air gulped at the surface to inflate the swimbladder. Therefore, sturgeons may experience changes in buoyancy that will affect their behavior at different depths. To test this prediction, we attached data loggers to seven free-ranging Chinese sturgeons Acipenser sinensis in the Yangtze River, China, to monitor their depth utilization, tail-beating activity, swim speed and body inclination. Two distinct, individual-specific, behavioral patterns were observed. Four fish swam at shallow depths (7–31 m), at speeds of 0.5–0.6 m s⁻¹, with ascending and descending movements of 1.0–2.4 m in amplitude. They beat their tails continuously, indicating that their buoyancy was close to neutral with their inflated swimbladders. In addition, their occasional visits to the surface suggest that they gulped air to inflate their swimbladders. The other three fish spent most of their time (88–94%) on the river bottom at a depth of 106–122 m with minimum activity. They occasionally swam upwards at speeds of 0.6–0.8 m s⁻¹ with intense tailbeats before gliding back passively to the bottom, in a manner similar to fishes that lack a swimbladder. Their bladders were probably collapsed by hydrostatic pressure, resulting in negative buoyancy. We conclude that Chinese sturgeons behave according to their buoyancy, which varies with depth due to hydrostatic compression of the swimbladder.     

Expression of muscle-gene-specific isozymes of phosphorylase and creatine kinase in innervated cultured human muscle

Isozymes of creatine kinase and glycogen phosphorylase are excellent markers of skeletal muscle maturation. In adult innervated muscle only the muscle-gene-specific isozymes are present, whereas aneurally cultured human muscle has predominantly the fetal pattern of isozymes. We have studied the isozyme pattern of human muscle cultured in monolayer and innervated by rat embryo spinal cord explants for 20-42 d. In this culture system, large groups of innervated muscle fibers close to the ventral part of the spinal cord explant continuously contracted. The contractions were reversibly blocked by 1 mM d-tubocurarine. In those innervated fibers, the total activity and the muscle-gene-specific isozymes of both enzymes increased significantly. The amount of muscle-gene-specific isozymes directly correlated with the duration of innervation. Control noninnervated muscle fibers from the same dishes as the innervated fibers remained biochemically immature. This study demonstrated that de novo innervation of human muscle cultured in monolayer exerts a time-related maturational influence that is not mediated by a diffusable neural factor.     

Innervation of the cat pineal gland by neuropeptide Y-immunoreactive nerve fibers: an experimental immunohistochemical study

An immunohistochemical study of the cat pineal gland was performed using a rabbit polyclonal antibody directed against neuropeptide Y (NPY) and an antibody directed against the C-terminal flanking peptide of neuropeptide Y (CPON). Numerous NPY- and CPON-immunoreactive (IR) nerve fibers were demonstrated throughout the gland and in the pineal capsule. The number of IR nerve fibers in the capsule was high and from this location fibers were observed to penetrate into the gland proper via the pineal connective tissue septa, often following the blood vessels. From the connective tissue septa IR fibers intruded into the parenchyma between the pinealocytes. Many IR nerve fibers were observed in the pineal stalk and in the habenular as well as the posterior commissural areas. The number of NPY/CPON-IR nerve fibers in pineal glands from animals bilaterally ganglionectomized two weeks before sacrifice was low. The source of most of the extrasympathetic NPY/CPONergic nerve fibers is probably the brain from where they enter the pineal via the pineal stalk. However, an origin of some of the fibers from parasympathetic ganglia cannot be excluded due to the presence of a few IR fibers in the pineal capsule of ganglionectomized animals. It is concluded that the cat pineal is richly innervated with NPYergic nerve fibers mostly of sympathetic origin. The posttranslational processing of the NPY promolecule results in the presence of both NPY and CPON in intrapineal nerve fibers.