Ontogeny of galanin-immunoreactive elements in the intrinsic nervous system of the chicken gut.

Galanin is a brain-gut peptide that is present in the central and peripheral nervous systems. In the gut, it is contained exclusively in intrinsic and extrinsic nerve supplies, and it is involved overall in the regulation of gut motility. To obtain information about the ontogeny of galanin, we undertook an immunohistochemical study of chicken embryos. The time of first appearance and the distribution patterns of galanin were investigated with fluorescence and streptavidin-biotin-peroxidase (ABC) immunohistochemical protocols by using a galanin polyclonal antiserum. The various regions of the gut and the pancreas were obtained from chicken embryos aged from 3 days of incubation to hatching. All specimens were fixed in buffered picric acid-paraformaldehyde, frozen, and cut with a cryostat. Galanin-immunoreactive neuroblasts were first detected at 4 days in the mesenchyme of the proventriculus/gizzard primordium and within the Remak ganglion. They then extended cranially and caudally, reaching all of the other gut regions at 6.5 days. Galanin-immunoreactive nerve elements mainly occupied the sites of myenteric and submucous plexuses. From day 15, galanin-immunoreactive nerve fibers tended to invade the circular muscular layer and part of the lamina propria of the mucosa. In the pancreas, weak galanin-immunoreactive nerve elements were detected at 5.5 days. They tended to be distributed among the glandular lobules according to the organ differentiation. The widespread distribution during the earlier embryonic stages represents evidence indicating that the neuropeptide galanin may have a role as a differentiating or growth factor. From late embryonic life, its predominant presence in sympathetic nerves and in muscular layers fits with the functions demonstrated previously in adults of other vertebrates for galanin as a modulator of intestinal motility.

Pharmacological properties of non-adrenergic, non-cholinergic inhibitory transmission in chicken gizzard.

1. Non-adrenergic, non-cholinergic (NANC) inhibitory transmission in chicken gizzard was studied by use of intracellular microelectrode techniques. Changes in membrane potential in response to NANC nerve stimulation were recorded in the gizzard smooth muscle pretreated with atropine (1 microM) and guanethidine (1 microM). 2. Field stimulation of the intramural nerves (FS) evoked inhibitory junction potentials (i.j.ps) which were abolished by tetrodotoxin (1 microM), but not inhibited at all by K+ channel blockers including apamin (0.5 microM), tetraethylammonium (TEA, 10 mM), charybdotoxin (0.2 microM) and glibenclamide (10 microM). 3. NG-nitro-L-arginine (3 mM), an inhibitor of nitric oxide (NO) synthase, inhibited i.j.ps. The effect was reversed by L-arginine (3 mM), but not by D-arginine (3 mM). 4. 8-Bromo cyclic GMP (100 microM), a membrane permeable analogue of cyclic GMP, produced a membrane hyperpolarization which was blocked by TEA (10 mM) or glibenclamide (10 microM). 5. NO at concentrations of up to 400 microM affected neither i.j.ps nor resting membrane potential. On the other hand, NO (80 microM) caused the membrane to hyperpolarize in the smooth muscle of guinea-pig ileum. 6. These results suggest that in the chicken gizzard, NANC i.j.ps may not arise from opening of conventional types of K+ channel and that NO seems unlikely to be involved in the generation of i.j.ps. A possible mechanism by which the inhibitory effect of NG-nitro-L-arginine on i.j.ps was brought about will be discussed.

Differential neural crest cell attachment and migration on avian laminin isoforms.

A number of laminin isoforms have recently been identified and proposed to exert different functions during embryonic development. In the present study, we describe the purification and partial characterization of several isoforms isolated from chick heart and gizzard, and provide data on the molecular mechanisms underlying the interaction of avian neural crest cells with these molecules in vitro. Laminins extracted from heart and gizzard tissues were separated by gel filtration and purified to homogeneity by sequential lectin and immunoaffinity chromatography by utilizing monoclonal antibodies directed against the avian alpha 2, beta 2 and gamma 1 laminin chains. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern of the polypeptide complexes obtained and immunoblotting with polyclonal antisera allowed the identification of Laminin-2 (alpha 2 beta 1 gamma 1), Laminin-4 (alpha 2 beta 2 gamma 1), and laminins comprising the beta 1, beta 2 and gamma 1 chains associated with a shorter alpha chain which, in SDS-PAGE, co-migrate with the beta/gamma complex in the 200 kDa region. These latter laminins, which are here arbitrarily denoted Laminin-alpha x (heart tissue) and Laminin-G (gizzard tissue), are somewhat distinct in their apparent molecular weight, are differentially associated with nidogen, and appear as "T"-shaped particles similar to Laminin-6 and Laminin-7 when analyzed by transmission electron microscopy following rotary shadowing. In contrast, the avian Laminin-2 and Laminin-4 isoforms exhibit the characteristic cruciform shape described previously for their mammalian counterparts. Isolated neural crest cells differentially attached and migrated on these laminin isoforms, showing a clear preference for Laminin-G. Similarly to the EHS Laminin-1, neural crest cells recognized all avian isoforms through their alpha 1 beta 1 integrin, shown previously to be the primary laminin-binding receptor on these cells. Neural crest cell interaction with the avian laminins was dependent upon maintenance of the secondary and tertiary structure of the molecules, as shown by the marked reduction in cell attachment and migration upon disruption of the alpha-helical coiled-coil structure of their constituent chains. The results demonstrate that different laminin isoforms may be differentially involved in the regulation of neural crest cell migration and suggest that this regulation operates through interaction of the cells with a structurally conserved cell binding site recognized by the alpha 1 beta 1 integrin.

Mapping the origin of the avian enteric nervous system with a retroviral marker.

The enteric nervous system is largely formed from the vagal neural crest which arises from the neuroaxis between somites 1-7. In order to evaluate the contribution of different regions of the vagal crest to the enteric nervous system, we marked crest cells by injecting somites 1-10 with a replication-defective spleen necrosis virus vector which contains the marker gene lacZ. After incubation in X-gal, lacZ-positive blue cells were found in the wall of the gut in three locations. Most were found at the peripheral edge of the developing circular muscle and within the developing submucosa, sites characteristic of developing ganglia. LacZ-positive cells in these ganglionic sites were always surrounded by HNK-1 immunostained cells, confirming their neural crest origin. LacZ-positive cells were also seen in a third location, the circular muscle layer of the esophagus and crop, and were separated from the HNK-1 positive ganglionic elements. These cells in the circular muscle are probably muscle cells derived from labeled mesodermal cells of the somite. Injection of somites 3, 4, 5, and 6 resulted in the largest percentage of preparations with lacZ-positive crest-derived cells and in the largest number of positive cells in the gut. After injection of these somites, lacZ-positive crest-derived cells were found in all regions of the gut from the proventriculus to the rectum. Very few positive crest-derived cells were found in the esophagus.(ABSTRACT TRUNCATED AT 250 WORDS)

Appearance and some neurochemical features of nitrergic neurons in the developing quail digestive tract.

Using immunocytochemistry, NADPH-diaphorase (NADPHd) histochemistry and electron microscopy, the appearance of nitrergic enteric neurons in different digestive tract regions of the embryonic, neonatal and adult quail was studied in whole mounts and sections. NADPHd was first expressed by embryonic day 4-5 in two distinct locations, namely the mesenchyme of the gizzard primordium and at the caeco-colonic junction. At embryonic day 6, nitrergic neurons had already begun to form a myenteric nerve network in the wall of the proventriculus, gizzard and proximal part of the large intestine and by embryonic day 9, a myenteric network was visualized along the entire digestive tract of the quail. At the level of the stomach, this network was confined to the area covered by the intermediate muscles. By embryonic day 12-13, the NADPHd-positive myenteric neurons in the wall of the distal parts of the blind-ending paired caeca also became organized into ganglia. From this developmental stage on, a submucous nitrergic nerve network, sandwiched between the lamina muscularis mucosae and the luminal side of the outer muscle layer, became prominent in the proventriculus and intestinal walls. In the adult quail, only a minority of the NADPHd-positive neurons stained for vasoactive intestinal polypeptide (VIP) along the intestine. VIP-immunoreactive (IR) cell bodies were frequent in the myenteric plexus but not in the submucous plexus, whereas there were considerable numbers of NADPHd-positive neurons in both these plexuses. Nitrergic fibres were also observed in the outer muscle layer, but were almost absent from the lamina muscularis mucosa and lamina propria, in contrast to the dense VIP-ergic innervation encircling the bases of the intestinal crypts.

Development of vagal innervation to the muscle of the avian gizzard.

The development of the vagal innervation to the gizzard has been investigated in chick embryos and young chicks. The membrane potential, first measurable on the 15th day of incubation, was -54 +/- 0.5 mV and increased with development to -67 +/- 0.4 mV. The latter value was attained 5 days after hatching and persisted thereafter. Stimulation of intramural nerves elicited a cholinergic, excitatory junction potential (EJP) for the first time, only in a small fraction of cells, on the 20th day of incubation. Within 3 days after hatching, cholinergic transmission showed the same features as in older chicks. Stimulation of the vagus nerve elicited no membrane potential responses before hatching but as early as 4 days after hatching, non-adrenergic, inhibitory junction potentials (IJPs) were evoked. In the next 10 days or so, the IJP was replaced with a cholinergic EJP as seen in mature tissues. After atropine (0.1-1 microM) treatment, both vagal and intramural nerve stimulation evoked a non-adrenergic IJP in a small fraction of cells immediately after hatching. The fraction of cells exhibiting the IJP increased with growth and reached 100% 5 days after hatching. Hexamethonium (50 or 100 microM) abolished the vagally-evoked EJPs. The vagally-evoked IJPs remained unchanged after application of hexamethonium in the early days after hatching, but later they were abolished in about half of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

On the nerve plexus of the chicken gizzard.

The Auerbach's plexus of the gizzard was stained in toto in adult chicken and in young and newly-hatched chicks. The plexus lies immediately beneath the serosa and extends over 55% of the surface of the organ, covering its cranial and caudal poles and the two curvatures. The areas into which the plexus does not extend (i.e., most of the ventral and the dorsal surface) are those where the muscle is covered by the laminar tendon of the gizzard. The ganglia are large, often with hundred of neurons, and have short and broad connecting strands. They are surrounded by a capsule of connective tissue. The ganglion neurons are discoidal and in the adult they measure up to 50 microns in diameter, each being surrounded by a set of glial cells. A few small neurons persist in the adult; in the newly-hatched chick these are predominant, but some large neurons up to 25 microns in diameter are already present. The ultra-structural features of the ganglia of the Auerbach's plexus include the abundance of axo-somatic synapses, as well as numerous axo-dendritic synapses, the presence of intra-ganglionic bundles of collagen fibrils and blood vessels, the abundance of glial cells. In addition to the plexus beneath the serosa, the gizzard has many small intramuscular ganglia located throughout the musculature (which is exclusively circular). These ganglia do not have a connective tissue capsule and are made of small and tightly packed neurons.

Topographic representation of visceral target organs within the dorsal motor nucleus of the vagus nerve of the pigeon Columba livia.

Our previous work (Katz and Karten, '83a J. Comp. Neurol. 217:31-46 demonstrated that the dorsal motor nucleus of the vagus nerve (DMN complex) in the pigeon is composed of cytoarchitecturally distinct subnuclei that are distinguished by the size, shape, position, and cytochemical characteristics of their constituent neurons. In view of the diversity of target organs innervated by the vagus nerve, we sought to determine whether the subnuclear heterogeneity of the DMN complex is related to the pattern of target innervation. To test this possibility, retrograde tracing techniques were used to define the subnuclear localization of vagal motoneurons that innervate individual vagal target organs. The distribution of horseradish peroxidase (HRP)-labeled motoneurons within the DMN complex was studied following application of HRP to the cut central end of individual vagal nerve branches and after injection of the tracer into vagal target tissues. In addition, we examined the distribution of acetylcholinesterase depletion within the DMN complex following transection of individual vagal branches. Our data demonstrate that individual vagal target organs have discrete and topographic representations within cytoarchitecturally distinct subnuclei of the DMN complex. Therefore, in the pigeon, the subnuclear distribution of vagal motoneurons plays a critical role in the organization of descending vagal motor pathways. Segregation of visceral representations within the DMN complex may provide a mechanism for organizing functionally diverse afferent inputs to target-specific populations of vagal motoneurons.

Chicken gizzard. The muscle, the tendon and their attachment.

The fine structure and the organization of muscle and connective tissue in the middle portion of the chicken gizzard (muscular stomach) has been studied by light and electron microscopy. The musculature is divided into long, well-defined bundles arranged circularly and concentrically and extending between the two tendons (tendinous aponeurosis). The muscle bundles are inserted onto the inner surface of the tendon at an angle of about 45 degrees. In addition to muscle cells (which are ultrastructurally similar to those of the small intestine) the musculature contains fibroblasts and interstitial cells and a small number of nerve bundles and capillaries. The gizzard tendons are very compact, made of parallel fascicles of collagen fibrils with interposed stellate tendon cells; ultrastructurally they are very similar to the tendon of skeletal muscles of this and other species. Their collagen fibrils range in size from 30 to 160 nm. The muscle cells that approach the tendon develop longitudinal invaginations of the cell membrane and then break into finger-like terminal processes heavily encrusted with dense bands. The membrane of the invaginations and the terminal processes are surrounded by a basal lamina material which embeds a conspicuous web of small collagen fibrils. The boundary between tendon and muscle is sharp, without interpenetration of the two tissues. A novel type of cell is found at the interface of muscle and tendon (junctional cells), filled with intermediate filaments and some rough endoplasmic reticulum and displaying a trace of a basal lamina.