Marion Raab

Vesicular glutamate transporter 1 immunoreactivity in extrinsic and intrinsic innervation of the rat esophagus

Encouraged by the recent finding of vesicular glutamate transporter 2 (VGLUT2) immunoreactivity (-ir) in intraganglionic laminar endings (IGLEs) of the rat esophagus, we investigated also the distribution and co-localization patterns of VGLUT1. Confocal imaging revealed substantial co-localization of VGLUT1-ir with selective markers of IGLEs, i.e., calretinin and VGLUT2, indicating that IGLEs contain both VGLUT1 and VGLUT2 within their synaptic vesicles. Besides IGLEs, we found VGLUT1-ir in both cholinergic and nitrergic myenteric neuronal cell bodies, in fibers of the muscularis mucosae, and in esophageal motor endplates. Skeletal neuromuscular junctions, in contrast, showed no VGLUT1-ir. We also tested for probable co-localization of VGLUT1-ir with markers of extrinsic and intrinsic esophageal innervation and glia. Within the myenteric neuropil we found, besides co-localization of VGLUT1 and substance P, no further co-localization of VGLUT1-ir with any of these markers. In the muscularis mucosae some VGLUT1-ir fibers were shown to contain neuronal nitric oxide synthase (nNOS)-ir. VGLUT1-ir in esophageal motor endplates was partly co-localized with vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT)-ir, but VGLUT1-ir was also demonstrated in separately terminating fibers at motor endplates co-localized neither with ChAT/VAChT-ir nor with nNOS-ir, suggesting a hitherto unknown glutamatergic enteric co-innervation. Thus, VGLUT1-ir was found in extrinsic as well as intrinsic innervation of the rat esophagus.

Vesicular glutamate transporter 1 immunoreactivity in motor endplates of striated esophageal but not skeletal muscles in the mouse.

Glutamate, the major excitatory transmitter in the central nervous system, has been speculated for years to influence mammalian motor endplates but trials to identify glutamatergic motor terminals failed because specific markers were not available. Recently, antibodies to vesicular glutamate transporters (VGLUTs) opened new possibilities for further morphological investigations. We detected VGLUT1 immunoreactivity (-ir), but not VGLUT2-ir and VGLUT3-ir, respectively, in many motor nerve terminals in motor endplates of the mouse esophagus as identified by alpha-bungarotoxin or colocalization of VGLUT1 with choline acetyltransferase. These findings suggest that glutamate is co-stored with acetylcholine in esophageal neuromuscular junctions. Surprisingly, we found neither VGLUT1-ir nor VGLUT2-ir or VGLUT3-ir in neuromuscular junctions of somitic and branchiogenic skeletal muscles. This may reflect differences in functional properties and the embryonic origin between skeletal and esophageal striated muscle fibers.

Glutamatergic Functions of Primary Afferent Neurons with Special Emphasis on Vagal Afferents

Glutamate has been identified as the main transmitter of primary afferent neurons. This was established based on biochemical, electrophysiological, and immunohistochemical data from studies on glutamatergic receptors and their agonists/antagonists. The availability of specific antibodies directed against glutamate and, more recently, vesicular glutamate transporters corroborated this and led to significant new discoveries. In particular, peripheral endings of various classes of afferents contain vesicular glutamate transporters, suggesting vesicular storage in and exocytotic release of glutamate from peripheral afferent endings. This suggests that autocrine mechanisms regulate sensory transduction processes. However, glutamate release from peripheral sensory terminals could also enable afferent neurons to influence various cells associated with them. This may be particularly relevant for vagal intraganglionic laminar endings, which could represent glutamatergic sensor-effector components of intramural reflex arcs in the gastrointestinal tract. Thus, morphological analysis of the relationships of putative glutamatergic primary afferents with associated tissues may direct forthcoming studies on their functions.

Reduction of NT-3 or TrkC results in fewer putative vagal mechanoreceptors in the mouse esophagus

Intraganglionic laminar endings (IGLEs) represent major vagal afferent structures throughout the gastrointestinal tract. Both morphological and functional data suggested a mechanosensory role. Elucidation of their functional significance in a particular organ would be facilitated by the availability of animal models with significantly altered numbers of IGLEs. The present study was aimed at searching for mouse strains fulfilling this criterion in the esophagus. Anterograde wheat germ agglutinin-horseradish peroxidase tracing (WGA-HRP) from nodose ganglion was used in order to label esophageal IGLEs in mice deficient for neurotrophin-3 (NT-3) or tyrosine kinase C-receptor (TrkC) and in control littermates. This approach was feasible only in heterozygous mutants which are viable. IGLEs were counted in tetramethylbenzidine (TMB) processed wholemounts using a standardised protocol. Quantification of myenteric neurons was done in cuprolinic blue-stained specimens. Nodose neuron counts were performed in cryostat sections stained with cresyl violet. Numbers of IGLEs in the esophagus were significantly reduced in both heterozygous NT-3 (NT-3+/-) and heterozygous TrkC (TrkC+/-) mutants (65% and 40% reduction, respectively). Numbers of nodose neurons were also significantly reduced in NT-3+/- mice (48% reduction), while their reduction in TrkC+/- mutants was insignificant (11% reduction). There was no reduction of myenteric neurons in the esophagus of either mutant strain. The numeric deficiency of IGLEs was unlikely to be secondary to reduction of myenteric neurons. Although only heterozygous mutants could be studied, these results suggest that esophageal IGLEs share neurotrophin dependence on NT-3/TrkC with spinal proprioceptors and some cutaneous mechanosensors. This concurs with their proposed function as vagal mechanosensors crucial for reflex peristalsis.

Distribution of vesicular glutamate transporter 1 (VGLUT1) in the mouse esophagus

In rat and mouse esophagus, vesicular glutamate transporter 2 (VGLUT2) has been demonstrated to identify vagal intraganglionic laminar endings (IGLEs); this has recently also been shown for VGLUT1 in rat esophagus. In this study, we have investigated the distribution of VGLUT1 in the mouse esophagus and compared these results with the recently published data from the rat esophagus. Unexpectedly, we have discovered that VGLUT1 mostly fails to identify IGLEs in the mouse esophagus. This is surprising, since the distribution of VGLUT2 shows comparable results in both species. Confocal imaging has revealed substantial colocalization of VGLUT1 immunoreactivity (-ir) with cholinergic and nitrergic/peptidergic markers within the myenteric neuropil and in both cholinergic and nitrergic myenteric neuronal cell bodies. VGLUT1 and cholinergic markers have also been colocalized in fibers of the muscularis mucosae, whereas VGLUT1 and nitrergic markers have never been colocalized in fibers of the muscularis mucosae, although this does occur in fibers of the muscularis running to motor endplates. Thus, VGLUT1 is contained in the nitrergic innervation of mouse esophageal motor endplates, another difference from the rat esophagus. VGLUT1-ir is therefore present in extrinsic and intrinsic innervation of the mouse esophagus, but the significant differences from the rat indicate species variations concerning the distribution of VGLUTs in the peripheral nervous system.

Quantitative evaluation of neurons in the mucosal plexus of adult human intestines

The consequence of presence versus absence of mucosal neurons is not consistently assessed. Here, we addressed two questions. First, based on resected gut specimens of 65 patients/body donors suffering from different diseases, counts of mucosal neurons per mm2 were analysed with respect to age, gender and region. Second, we evaluated resected megacolonic specimens of four patients suffering from chronic Chagas’ disease. Mucosal wholemounts were triple-stained for calretinin (CALR), peripherin (PER) and human neuronal protein Hu C/D (HU). Counts revealed no clear correlation between the presence of mucosal neurons and age, gender or region. Mucosal neurons were present in 30 of 36 specimens derived from males (83%) and in 20 of 29 from females (69%). The numbers per mm2 increased from duodenum to ileum (1.7–10.8) and from ascending to sigmoid colon (3.2–9.9). Out of 149 small intestinal mucosal neurons, 47% were co-reactive for CALR, PER and HU (large intestine: 76% of 300 neurons) and 48% for PER and HU only (large intestine: 23%). In 12 megacolonic specimens (each 3 from 4 patients), all 23 mucosal neurons found (1.9 per mm2) displayed co-reactivity for CALR, PER and HU. We suggest that the presence or the absence of mucosal neurons is variable, ongoing studies will address our assumption that they correspond in their morphochemical characteristics to submucosal neurons. Furthermore, both the architecture and neuron number of the megacolonic mucosal plexus displayed no dramatic changes indicating that mucosal nerves might be less involved in chagasic/megacolonic neurodegeneration as known from the myenteric plexus.

Pitfalls using tyramide signal amplification (TSA) in the mouse gastrointestinal tract: endogenous streptavidin-binding sites lead to false positive staining.

Highly sensitive immunohistochemical detection systems such as tyramide signal amplification (TSA) are widely used, since they allow using two primary antibodies raised in the same species. Most of them are based on the streptavidin-biotin-peroxidase system and include streptavidin-coupled secondary antibodies. Using TSA in cryostat-sectioned tissues of mouse esophagus, we were puzzled by negative controls with unexpected staining mostly in the ganglionic areas. This prompted us to search for the causing agent and to include also other parts of the mouse gastrointestinal tract for comparison. Streptavidin-coupled antibodies bound to endogenous binding sites yet to be characterized, which are present throughout the mouse intestines. Staining was mainly localized around neuronal cell bodies of enteric ganglia. Thus, caution is warranted when applying streptavidin-coupled antibodies in the mouse gastrointestinal tract. The use of endogenous biotin-blocking kits combined with a prolonged post-fixation time could significantly reduce unintentional staining.

Neurovaskuläre Mobilisation des Plexus hypogastricus inferior

Zusammenfassung Haufig fuhren Operationen im kleinen Becken oder Verletzungen des Beckens, insbesondere Sturze auf das Sakrum, aber auch unterschiedliche Traumata der unteren Extremitat durch Zugkrafte zu einer Irritation der Beckengeflechte, aus der zahlreiche Beschwerden resultieren konnen. Der Beitrag beschreibt Tests und Techniken, um Restriktionen des Plexus hypogastricus inferior festzustellen und zu behandeln. Zudem werden Hypothesen diskutiert, die einen Zusammenhang der Irritation/Mindermobilisation der Beckengeflechte mit der scheinbar bunten Symptomatik aufzeigen sollen.

Funktionsstörungen des Ösophagus und des gastro-ösophagealen Übergangs: Anatomie und Pathomechanismus

Zusammenfassung Haufig fuhrt ein Schleudertrauma zu einer Mindermobilitat im Thorax, im Umgebungsbereich der Speiserohre und damit zu einer Zugbelastung im Bereich des Hiatus oesophageus, aus der zahlreiche Beschwerden resultieren konnen. Der Beitrag beschreibt die anatomischen Grundlagen der Region, die Anatomie des gastro-osophagealen Ubergangs, den Verschlussmechanismus des Osophagus und die Innervation des unteren Verschlusssegments.

Funktionsstörungen des Ösophagus und des gastro-ösophagealen Übergangs: Techniken zur Behandlung

Zusammenfassung Der Artikel beschreibt eine Reihe von Tests und Techniken, um Restriktionen des thorakalen Osophagus und Funktionsstorungen im Bereich des gastro-osophagealen Ubergangs festzustellen und zu behandeln.