Calbindin-immunopositive cells are cholinergic interneurons in the myenteric plexus of rabbit ileum

The 28-kDa calcium-binding protein (calbindin) is a widely studied neuronal marker in the enteric nervous system of numerous species. Calbindin has previously been detected in myenteric neurons of rabbit ileum in which 3% of all myenteric neurons are calbindin-immunopositive. We have studied the detailed morphology and chemical coding of calbindin-immunopositive neurons in this segment of the gut. We have found calbindin immunoreactivity in both strongly and weakly stained neurons. Of these, the strongly immunoreactive neurons belong to the Dogiel type I category. These neurons project only to other ganglia and primary strands of the plexus and their processes never run to the muscle or mucosal layers. The neurons within this group are 29.5±6.6 m in length and 14.7±3.8 m in width. The second smaller group of immunoreactive cells (27%) label faintly and have different morphological properties. They are characterized by their round medium-sized cell bodies (long axis: 24.4±5.2 m; short axis: 15.5±2.9 m) and do not exhibit immunoreactivity either in their dendrites or in their axonal processes. Double-label studies show that all calbindin-immunopositive neurons lack immunoreactivity for nitric oxide synthase, vasoactive intestinal peptide and substance P but all are immunoreactive for the synthesizing enzyme of acetylcholine, choline acetyltransferase. Thus, populations of neurons containing calbindin are cholinergic interneurons in the myenteric plexus of rabbit ileum.This study was supported by grant OTKA T 34160     

Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse

Immunohistochemical techniques were used to examine the presence and co-localisation of a range of putative neurotransmitters and other neuronal markers in the myenteric plexus of the small and large intestine of the mouse. Distinct sub-populations of myenteric neurons were identified, based on the combinations of substances they contained and the distribution of their fibres. In the small intestine, there were two major classes of circular muscle motor neurons; one class was characterised by the presence of nitric oxide synthase, vasoactive intestinal peptide plus neuropeptide Y (NOS/VIP/NPY), and the second class contained calretinin plus substance P (CalR/SP). There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS/VIP, NPY, CalR/calbindin (CalB), SP or 5-HT. In the large intestine, there were five major classes of motor neurons that contained NOS, NOS/VIP, GABA, SP, or CalR/SP, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, CalR/CalB, CalR, SP, GABA or 5-HT. Although some aspects of the patterns of co-localisation are similar to those in other species, this study re-inforces recent analyses that indicate significant species differences in neurochemical patterns in the enteric neurons of different species. Received: 28 August 1995 / Accepted: 30 November 1995     

Identification of neuron types in the submucosal ganglia of the mouse ileum

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found. This work was supported by a grant from the National Health and Medical Research Council of Australia (grant no. 400020) and an Australian Research Council international linkage grant (no. LZ0882269) for collaboration between the Melbourne and Bologna laboratories.     

Immunohistochemical localisation of pre-synaptic muscarinic receptor subtype-2 (M2r) in the enteric nervous system of guinea-pig ileum

The cholinergic muscarinic 2 receptor (M2r) is known to be present on smooth muscle cells in the intestine. Pharmacological studies also suggest that M2rs regulate transmitter release from nerves in the enteric nervous system. This study localised M2rs in the guinea-pig ileum using different antibodies and fluorescence immunohistochemistry. Double labelling with antibodies against neurochemical markers was used to identify the type of nerves bearing M2r. Guinea-pig ileum were fixed, prepared for sections and wholemounts and incubated with antisera against the M2r sequence. Tissue was double labelled with antibodies against neuronal nitric oxide synthase (nNOS), common choline acetyltransferase (cChAT), substance P (SP), synaptophysin and vesicular acetylcholine transporter (VAChT). Immunofluorescence was viewed using confocal microscopy. Abundant M2r-immunoreactivity (IR) was present on the surface of circular and longitudinal smooth muscle cells. M2r-IR was present in many but not all nerve fibres in the circular muscle and ganglia. M2r-IR was present in VAChT-IR and cChAT-IR cholinergic nerve fibres and SP-IR nerve fibres in the myenteric ganglia and submucosal ganglia. M2r-IR was present on a few nNOS-IR nerve fibres and around nNOS-IR neurons in the myenteric ganglia. In the circular muscle and deep muscular plexus, M2r-IR was present in many VAChT-IR and SP-IR nerve fibres and in few nNOS-IR nerves. M2rs are not only present on muscle cells in the intestine, but also on nerve fibres. M2rs may mediate cholinergic reflexes via their location on muscle and also via neural transmission. The pre-synaptic location supports pharmacological studies suggesting M2rs mediate neurotransmitter release from nerve fibres. The presence of M2rs on VAChT-IR, SP-IR and nNOS-IR-containing nerve fibres suggests M2rs may regulate ACh, SP and nitric oxide release. Work in this study was funded by the National Health and Medical Research Council (grant numbers: 114215 and 216704; Senior Research Fellowship to B.S.), a Melbourne University Research Scholarship and the Murdoch Children’s Research Institute.     

Neuron-specific enolase and S-100 protein immunohistochemistry for defining the structure and topographical relationship of the different enteric nerve plexuses in the small intestine of the pig

Summary The morphological and topographical features of the intramural enteric nervous system in the small intestine of the pig has been studied on whole mounts by means of neuron-specific enolase (NSE) and S-100 protein immu-nohistochemistry. A clear visualization of the myenteric plexus allows the recognition of its characteristic morphology, including the thin tertiary plexus coursing within the smooth muscle layers. In the tela submucosa two ganglionated plexuses, each with its own specific characteristics, can clearly be demonstrated: (1) the plexus submucosus externus (Schabadasch) located near the inner surface of the circular muscle layer at the abluminal side of the submucosal vascular arcades, and (2) the plexus submucosus internus (Meissner) close to the outer surface of the lamina muscularis mucosae at the luminal side of the submucosal vascular arcades. Due to the possibility to trace clearly the perivascular plexuses of these vascular arcades by use of immunohistochemical techniques with antibodies to NSE and S-100 protein, the two submucosal nerve plexuses can be demonstrated with exceptional clarity. This is the first report of an investigation of the intramural nerve plexuses of the small intestine of the pig using the NSE and S-100 immunostaining methods, which is sufficiently detailed to substantiate the characteristic topography and structure of the two submucosal plexuses and their relation to the smooth muscle layers and perivascular plexuses. The level of NSE immunoreactivity for enteric neurons displays great variation, a substantial proportion of the type-II neurons appearing strongly stained. Although little is known of the specific function of these enzymes, proposals are discussed.     

Distribution of peptide-containing neurons and endocrine cells in the rabbit gastrointestinal tract,with particular reference to the mucosa

Summary The distribution patterns of peptide-containing neurons and endocrine cells were mapped in sections of oesophagus, stomach, small intestine and large intestine of the rabbit, by use of standard immunohistochemical techniques. Whole mounts of separated layers of ileum were similarly examined. Antibodies raised against vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), enkephalins (ENK) and gastrin-releasing peptide (GRP) were used, and for each of these antisera distinct populations of immunoreactive (IR) nerve fibres were observed. Endocrine cells were labelled by the SP, SOM or NPY antisera in some regions.VIP-IR nerve fibres were common in each layer throughout the gastrointestinal tract. With the exception of the oesophagus, GRP-IR nerve fibres also occurred in each layer of the gastrointestinal tract; they formed a particularly rich network in the mucosa of the stomach and small intestine. Fewer nerve fibres containing NPY-IR or SOM-IR were seen in all areas. SOM-IR nerve fibres were very scarce in the circular and longitudinal muscle layers of each area and were absent from the gastric mucosa. The SP-IR innervation of the external musculature and ganglionated plexuses in most regions was rather extensive, whereas the mucosa was only very sparsely innervated. ENK-IR nerve fibres were extremely rare or absent from the mucosa of all areas, although immunoreactive nerve fibres were found in other layers.These studies illustrate the differences in distribution patterns of peptide-containing nerve fibres and endocrine cells along the gastrointestinal tract of the rabbit and also show that there are some marked differences in these patterns, in comparison with other mammalian species.     

Distribution of pituitary adenylyl cyclase activating peptide (PACAP) immunoreactivity in neurons of the guinea-pig digestive tract and their projections in the ileum and colon

Pituitary adenylyl cyclase activating peptide (PACAP) is a novel hypothalamic peptide that is widely distributed in neurons, including those of the gastrointestinal tract. In this study, a polyclonal antiserum directed against PACAP-27 was used to investigate the localisation of PACAP throughout the gut and to determine the projections of PACAP-immunoreactive (IR) neurons in the guinea-pig small and large intestines. PACAP-IR fibres were seen in the myenteric and submucous plexuses, in the longitudinal and circular muscle layers and around blood vessels of the submucosa throughout the gut. In both the small and large intestine, PACAP-IR cell bodies, most with Dogiel type-I morphology, were seen in the myenteric ganglia following colchicine treatment. Lesion studies (myotomy and myectomy operations) revealed that PACAP-IR interneurons projected anally in the ileum and colon. Myectomy operations resulted in a loss of PACAP-IR fibres in the circular muscle under the operation, whereas PACAP-IR fibres remained in the submucosa and around blood vessels. Following extrinsic denervation of the ileum, the number of PACAP-IR fibres in the submucosal ganglia and around blood vessels decreased. This suggests that a portion of PACAP-IR fibres supplying the submucosal ganglia and blood vessels have an extrinsic source. To investigate this, immunohistochemical studies were performed on sympathetic and dorsal root ganglia. Numerous reactive cells were seen in the dorsal root ganglia, but none was seen in sympathetic pre- or paravertebral ganglia.     

High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

Previous studies have demonstrated that neurofilament proteins are expressed by type II neurons in the enteric plexuses of a range of species from mouse to human. However, two previous studies have failed to reveal this association in the guinea-pig. Furthermore, immunohistochemistry for neurofilaments has revealed neurons with a single axon and spiny dendrites in human and pig but this morphology has not been described in the guinea-pig or other species. We have used antibodies against high- and medium-weight neurofilament proteins (NF-H and NF-M) to re-examine enteric neurons in the guinea-pig. NF-H immunoreactivity occurred in all type II neurons (identified by their IB4 binding) but these neurons were never NF-M-immunoreactive. On the other hand, 17% of myenteric neurons expressed NF-M. Many of these were uni-axonal neurons with spiny dendrites and nitric oxide synthase (NOS) immunoreactivity. NOS immunoreactivity occurred in surface expansions of the cytoplasm that did not contain neurofilament immunoreactivity. Thus, because of their NOS immunoreactivity, spiny neurons had the appearance of type I neurons. This indicates that the apparent morphologies and the morphological classifications of these neurons are dependent on the methods used to reveal them. We conclude that spiny type I NOS-immunoreactive neurons have similar morphologies in human and guinea-pig and that many of these are inhibitory motor neurons. Both type II and neuropeptide-Y-immunoreactive neurons in the submucosal ganglia exhibit NF-H immunoreactivity. NF-M has been observed in nerve fibres, but not in nerve cell bodies, in the submucosa. This work was supported by a grant from the National Health and Medical Council of Australia (grant number 400020).     

Shapes of nerve cells in the myenteric plexus of the guinea-pig small intestine revealed by the intracellular injection of dye

Summary The shapes of myenteric neurons in the guineapig small intestine were determined after injecting living neurons with the dye Lucifer yellow via a microelectrode. The cells were fixed and the distribution of Lucifer yellow rendered permanent by an immunohistochemical method. Each of 204 nerve cells was examined in whole-mount preparations of the myenteric plexus and drawn using a camera lucida at 1250 x magnification. Four cell shapes were distinguished: (1) neurons with several long processes corresponding to type II of Dogiel; (2) neurons with a single long process and lamellar dendrites corresponding to type I of Dogiel; (3) neurons with numerous filamentous dendrites; and (4) small neurons with few processes. About 15% of the neurons could not be placed into these classes or into any single class. The type II neurons (39% of the sample) had generally smooth somata and up to 7 (average 3.3) long processes, most of which ran circumferentially. Dogiel type I neurons (34% of sampled neurons) had characteristic lamellar dendrites, i.e., broad dendrites that were flattened in the plane of the plexus. The filamentous neurons (7% of the sample), had, on average, 14 fine processes up to about 50 m in length. Small neurons with smooth outlines and a few fine processes made up 5% of the neurons encountered. We conclude that myenteric neurons that have been injected with dye can be separated into morphologically distinct classes and that the different morphological classes probably correspond to different functional groupings of neurons.     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.     

Immunohistochemical analysis of neuron types in the mouse small intestine

The definition of the nerve cell types of the myenteric plexus of the mouse small intestine has become important, as more researchers turn to the use of mice with genetic mutations to analyze roles of specific genes and their products in enteric nervous system function and to investigate animal models of disease. We have used a suite of antibodies to define neurons by their shapes, sizes, and neurochemistry in the myenteric plexus. Anti-Hu antibodies were used to reveal all nerve cells, and the major subpopulations were defined in relation to the Hu-positive neurons. Morphological Type II neurons, revealed by anti-neurofilament and anti-calcitonin gene-related peptide antibodies, represented 26% of neurons. The axons of the Type II neurons projected through the circular muscle and submucosa to the mucosa. The cell bodies were immunoreactive for choline acetyltransferase (ChAT), and their terminals were immunoreactive for vesicular acetylcholine transporter (VAChT). Nitric oxide synthase (NOS) occurred in 29% of nerve cells. Most were also immunoreactive for vasoactive intestinal peptide, but they were not tachykinin (TK)-immunoreactive, and only 10% were ChAT-immunoreactive. Numerous NOS terminals occurred in the circular muscle. We deduced that 90% of NOS neurons were inhibitory motor neurons to the muscle (26% of all neurons) and 10% (3% of all neurons) were interneurons. Calretinin immunoreactivity was found in a high proportion of neurons (52%). Many of these had TK immunoreactivity. Small calretinin neurons were identified as excitatory neurons to the longitudinal muscle (about 20% of neurons, with ChAT/calretinin/± TK chemical coding). Excitatory neurons to the circular muscle (about 10% of neurons) had the same coding. Calretinin immunoreactivity also occurred in a proportion of Type II neurons. Thus, over 90% of neurons in the myenteric plexus of the mouse small intestine can be currently identified by their neurochemistry and shape.     

Distinct distribution of CGRP-, enkephalin-, galanin-, neuromedin U-, neuropeptide Y-, somatostatin-, substance P-, VIP- and serotonin-containing neurons in the two submucosal ganglionic neural networks of the porcine small intestine

Summary In addition to differences between the two submucosal ganglionic neural networks, i.e., the plexus submucosus externus (Schabadasch) and the plexus submucosus internus (Meissner), with respect to the occurrence and distribution of serotonin as neurotransmitter, immunocytochemistry also revealed a distinct distribution for various neuropeptides in these two plexuses. Immunoreactivity for galanin, vasoactive intestinal polypeptide, calcitonin gene-related peptide, substance P, neuromedin U, enkephalin, somatostatin and neuropeptide Y was found in varicose and non-varicose nerve fibres of both submucosal ganglionic plexuses, albeit with a distinct distributional pattern. The difference in neurotransmitter and/or neuromodulator content between both neural networks became even more obvious when attention was focussed on the immunoreactivity of the nerve cell bodies for these substances. Indeed, neuropeptide Y, enkephalin-and somatostatin-immunoreactive neuronal perikarya as well as serotonergic neuronal cell bodies appear solely in the plexus submucosus externus. Neuromedin U-immunoreactive perikarya, mostly coexisting with substance P, are observed in large numbers in the plexus submucosus internus, whilst they are rare in the plexus submucosus externus. Double-labelling immunostaining for substance P with CGRP and galanin revealed a different coexistence pattern for the two submucosal ganglionic plexuses. The differing chemical content of the neuronal populations supports the hypothesis that the existence of the two submucosal ganglionic plexuses, present in most large mammals including man, not only reflects a morphological difference but also points to differentiated functions.     

Quadruple colocalization of calretinin,calcitonin gene-related peptide,vasoactive intestinal peptide,and substance P in fibers within the villi of the rat intestine

Double-labeling immunofluoresenct histochemistry demonstrates that calretinin, a calcium-binding protein, coexists with calcitonin gene-related peptide, vasoactive intestinal peptide, and substance P in the fibers innervating the lamina propria of the rat intestinal villi. An acetylcholinesterase histochemical stain revealed that the majority of calretinin-containing cells in the myenteric ganglia were cholinergic and that about one half of the submucosal calretinin-containing cells colocalized with acetylcholinesterase. In situ hybridization studies confirmed the presence of calretinin mRNA in the dorsal root ganglia, and a ribonuclease protection assay verified the presence of calretinin message in the intestine. The coexistence of calretinin in calcitonin-gene-related-peptide-containing cells that also contained substance P and vasoactive intestinal polypeptide in the dorsal root ganglia suggest that these ganglia are the source of the quadruple colocalization within the sensory fibers of the villi. Although the function of calretinin in these nerves is unknown, it is hypothesized that the coexistence of three potent vasodilatory peptides influences the uptake of metabolized food products within the vasculature of the villi.     

Evidence that myenteric neurons of the gastric corpus project to both the mucosa and the external muscle: myectomy operations on the canine stomach

Summary The distribution of nerve cell bodies and fibres in the canine stomach was investigated using antibodies to the general neuronal marker, neuron-specific enolase. Prominent ganglia containing many reactive nerve cells were found in the myenteric plexus of the gastric corpus and antrum. Nerve cells were absent from the submucosa of the corpus and were extremely rare in the antrum. Renoval of areas of longitudinal muscle and myenteric plexus from the corpus (myectomy), with 7 days allowed for axon degeneration, resulted in the loss of fibres reactive for galanin, gastrin-releasing peptide, substance P and vasoactive intestinal peptide from both the circular muscle and mucosa in the area covered by the lesion. Combined vagotomy and sympathetic denervation did not significantly affect these fibres, but did cause fibres reactive for calcitonin gene-related peptide to degenerate. It is concluded that the myenteric plexus of the gastric corpus, like the myenteric plexus of the small intestine and colon, is the source of nerve fibres innervating the circular muscle, but, in contrast to other regions of the gastrointestinal tract, myenteric ganglia, not submucous ganglia, are the major, or sole, source of the intrinsic innervation of the mucosa.     

Occurrence,distribution and neurochemical features of small intestinal neurons projecting to the cranial mesenteric ganglion in the pig

The small intestine of the pig has been investigated for its topographical distribution of enteric neurons projecting to the cranial mesenteric ganglion, by using Fast Blue or Fluorogold as a retrogradely transported neuronal tracer. Contrary to the situation in small laboratory animals such as rat and guinea-pig, the intestinofugally projecting neurons in the porcine small intestine were not restricted to the myenteric plexus, but were observed in greater numbers in ganglia of the outer submucous plexus. The inner submucous plexus was devoid of labelled neurons. Retrogradely labelled neurons were mostly found, either singly or in small aggregates, in ganglia located within a narrow border on either side of the mesenteric attachment. For both nerve networks, their number increased from duodenum to ileum. All the retrogradely labelled neurons exhibited a multidendritic uniaxonal appearance. Some of them displayed type-III morphology and stained for serotonin. This study indicates that, in the pig, not only the myenteric plexus but also one submucous nerve network is involved in the afferent component of intestino-sympathico-intestinal reflex pathways. The finding that some of the morphologically defined type-III neurons participate in these reflexes is in accord with the earlier proposal that type-III neurons are supposed to fulfill an interneuronal role, whether intra- or extramurally.     

Extrinsic and intrinsic sources of calcitonin gene-related peptide immunoreactivity in the lamb ileum: a morphometric and neurochemical investigation

To investigate extrinsic origins of calcitonin gene-related peptide immunoreactive (CGRP-IR) nerve fibres in the sheep ileum, the retrograde fluorescent tracer Fast Blue (FB) was injected into the ileum wall. Sections of thoraco-lumbar dorsal root ganglia (DRG) and distal (nodose) vagal ganglia showing FB-labelled neurons were processed for CGRP immunohistochemistry. The distribution of CGRP-IR in fibres and nerve cell bodies in the ileum was also studied. CGRP-IR enteric neurons were morphometrically analysed in myenteric (MP) and submucosal plexuses (SMP) of lambs (2–4 months). Sensory neurons retrogradely labelled with FB were scattered in T5-L4 DRG but most were located at the upper lumbar levels (L1-L3); only a minor component of the extrinsic afferent innervation of the ileum was derived from nodose ganglia. In the DRG, 57% of retrogradely labelled neurons were also CGRP-IR. In cryostat sections, a dense network of CGRP-IR fibres was observed in the lamina propria beneath the epithelium, around the lacteals and lymphatic follicles (Peyer's platches), and along and around enteric blood vessels. Rare CGRP-IR fibres were also present in both muscle layers. Dense pericellular baskets of CGRP-IR fibres were observed around CGRP-negative somata. The only CGRP-IR nerve cells were well-defined Dogiel type II neurons localised in the MP and in the external and internal components of the SMP. CGRP-IR neurons in the myenteric ganglia were significantly larger than those in the submucosal ganglia (mean profile areas: about 1,400 μm² for myenteric neurons, 750 μm² for submucosal neurons). About 6% of myenteric neurons and 25% of submucosal neurons were CGRP-IR Dogiel type II neurons. The percentages of CGRP-IR neurons that were also tachykinin-IR were about 9% (MP) and 42% (SMP), whereas no CGRP-IR neurons exhibited immunoreactivity for vasoactive intestinal peptide, nitric oxide synthase or tyrosine hydroxylase in either plexus. Thus, CGRP immunoreactivity occurs in the enteric nervous system of the sheep ileum (as in human small intestine and MP of pig ileum) in only one morphologically defined type of neuron, Dogiel type II cells. These are probably intrinsic primary afferent neurons. This work was supported by grants from the Ricerca Fondamentale Orientata (RFO) and Fondazione Del Monte di Bo e Ra.     

Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development

 The tactile bristles of the fly comprise four cells that originate from a single precursor cell through a fixed lineage. The gene tramtrack (ttk) plays a crucial role in defining the fates of these cells. Here we analyse the normal pattern of expression of ttk, as well as the effect of ttk overexpression at different steps of the lineage. We show that ttk is never expressed in cells having a neural potential, and that in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. Furthermore, this expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. Our results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice. Received: 10 October 1996 / Accepted: 11 February 1997     

Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Single-stranded DNA in the genetic transformation of wheat (Triticum aestivum L.): transformation frequency and integration pattern

Two non-linked marker genes (gus and bar) were co-introduced by microprojectile bombardment into wheat cells. Four different DNA structures were compared with respect to ability to integrate into the wheat genome: circular or linear (l) DNA as a single- or double-stranded plasmid (ss and ds, respectively). In eight independent experiments, linearized DNA integrated in the ds or ss form with a high efficiency of up to 14% for l-ssDNA. Molecular analyses by Southern blotting showed that all DNA forms gave a similar complicated integration pattern of the bar gene. Received: 20 July 1998 / Accepted: 30 January 1999