Enteric neuroanatomy and smooth muscle activity in the western diamondback rattlesnake (Crotalus atrox).

BACKGROUND: Gastrointestinal (GI) functions are controlled by the enteric nervous system (ENS) in vertebrates, but data on snakes are scarce, as most studies were done in mammals. However, the feeding of many snakes, including Crotalus atrox, is in strong contrast with mammals, as it consumes an immense, intact prey that is forwarded, stored, and processed by the GI tract. We performed immunohistochemistry in different regions of the GI tract to assess the neuronal density and to quantify cholinergic, nitrergic, and VIPergic enteric neurons. We recorded motility patterns and determined the role of different neurotransmitters in the control of motility. Neuroimaging experiments complemented motility findings. RESULTS: A well-developed ganglionated myenteric plexus (MP) was found in the oesophagus, stomach, and small and large intestines. In the submucous plexus (SMP) most neurons were scattered individually without forming ganglia. The lowest number of neurons was present in the SMP of the proximal colon, while the highest was in the MP of the oesophagus. The total number of neurons in the ENS was estimated to be approx. 1.5 million. In all regions of the SMP except for the oesophagus more nitric oxide synthase+ than choline-acetyltransferase (ChAT)+ neurons were counted, while in the MP ChAT+ neurons dominated. In the SMP most nerve cells were VIP+, contrary to the MP, where numerous VIP+ nerve fibers but hardly any VIP+ neuronal cell bodies were seen. Regular contractions were observed in muscle strips from the distal stomach, but not from the proximal stomach or the colon. We identified acetylcholine as the main excitatory and nitric oxide as the main inhibitory neurotransmitter. Furthermore, 5-HT and dopamine stimulated, while VIP and the ß-receptor-agonist isoproterenol inhibited motility. ATP had only a minor inhibitory effect. Nerve-evoked contractile responses were sodium-dependent, insensitive to tetrodotoxin (TTX), but sensitive to lidocaine, supported by neuroimaging experiments. CONCLUSIONS: The structure of the ENS, and patterns of gastric and colonic contractile activity of Crotalus atrox are strikingly different from mammalian models. However, the main excitatory and inhibitory pathways appear to be conserved. Future studies have to explore how the observed differences are an adaptation to the particular feeding strategy of the snake.

Immunofluorescence Staining of P2X7 Receptors in Whole-Mount Myenteric Plexus Preparations.

P2X7 receptors play an important role in cytokine release and immune cell regulation. Their upregulation has been described in inflammatory and degenerative processes and P2X7 blockade or deletion has been shown to reduce tissue damage and severity of symptoms in animal models of inflammatory bowel disease (IBD). Several studies have found that P2X7 receptors are present on enteric neurons and glia and it was proposed that they mediate neuronal death during IBD. However, the cell type-specific localization of P2X7 receptors has been a matter of debate, since some antibodies have been found to be unspecific. Here we describe the preparation of whole-mount myenteric plexus from the colon of BAC transgenic P2X7-EGFP reporter mice and subsequent immunofluorescence staining of P2X7 receptors together with cell type-specific marker proteins.

How big is the little brain in the gut? Neuronal numbers in the enteric nervous system of mice, Guinea pig, and human.

BACKGROUND: Despite numerous studies on the enteric nervous system (ENS), we lack fundamental knowledge on neuronal densities or total neuron numbers in different species. There are more anecdotal than actual figures on nerve counts. METHODS: We used standardized preparation techniques and immunohistochemistry with validated panneuronal markers (human or mouse anti-HuD/C) to determine neuronal densities in specimen from the entire gastrointestinal tract of mice, guinea pig, and humans. In parallel, we measured the dimensions of the gastrointestinal regions in mouse and guinea pig. For humans, we had to rely on literature data. KEY RESULTS: The average neuronal densities along the gastrointestinal tract were 35,011 ± 25,017 1/cm2 for the myenteric and 16,685 ± 9098 1/cm2 for the submucous plexus in mice, 24,315 ± 16,627 and 11,850 ± 6122 1/cm2 for guinea pig myenteric and submucous plexus, respectively, and 21,698 ± 9492 and 16,367 ± 5655 1/cm2 for human myenteric and submucous plexus, respectively. The total number of neurons in the ENS was 2.6 million for mice, 14.6 million for guinea pig, and 168 million for human. CONCLUSIONS & INFERENCES: This study reports the first comprehensive nerve cell count in mice, guinea pig, and human ENS. Neuronal densities were comparable between the three species and the differences in the total numbers of enteric neurons are likely due to body size and intestinal length. The number of enteric neurons is comparable to the number of neurons in the spinal cord for all three species.

Therapy-refractory gastrointestinal motility disorder in a child with c-kit mutations.

Constipation and fecal impaction are frequent and distressing complaints in pediatric gastroenterology. Especially in neurologically handicapped children, treatment of severe forms of slow-transit constipation (STC) can be difficult. In the majority of cases, STC is of unknown etiology. However, in recent years, there is growing evidence that interstitial cells of Cajal (ICCs), which serve as electrical pacemakers and generate spontaneous electrical slow waves in the gastrointestinal tract, might play an important role in the pathophysiology of STC. It remains unclear whether morphological ICC alterations seen in affected patients are based on congenital developmental anomalies, or whether they are a consequence of long-term constipation with secondary damage of the gastrointestinal nervous system. To the best of our knowledge, we present the first case of a patient with histological alterations in ICC morphology who displayed multiple alterations of c-kit at the level of mRNA. The protein encoded by c-kit is the receptor tyrosine kinase Kit (CD117), which is crucial for development and function of ICCs. Therefore, these findings provide a new explanation for congenital alterations of ICC development that result in gastrointestinal motility disorders.