MORPHOMETRIC STUDY OF ISOLATED DOGIEL TYPE II CELLS OF THE INTERMUSCULAR PLEXUS

Aim of the study was to calculate the morphometric parameters of isolated Dogiel type II cells from the intermuscular plexus of the small intestine. Materials and methods: 3D models of the oval-shaped Dogiel type II cells from the intermuscular plexus of a rat (n = 1) were constructed and studied. Neurocytes were identified by the impregnation method on the frontal and horizontal sections of the wall of the small intestine. Results: The results showed that the total number of the nodes in the virtual model was 36534, and the mesh elements — 156595. The resulting 3D model of the cell and nucleus was reduced 900 times to obtain a threedimensional cell and nucleus with absolute dimensions, with a ratio of 1:1 to their true size. The volume of Dogiel type II cell was 2785.11 μm³, the volume of the nucleus was 647.7 μm³ and the volume of its perikaryon was 2785.11 μm³. Conclusion: Dogiel type II cells from the intermuscular plexus of the rat small intestine has an ovoid shaped threedimensional structure. These cells are flattened in transverse direction and elongated in longitudinal direction.

Classification of human enteric neurons

Major advances in our understanding of the functional heterogeneity of enteric neurons are driven by the application of newly developed, innovative methods. In contrast to this progress, both animal and human enteric neurons are usually divided into only two morphological subpopulations, “Dogiel type II” neurons (with several long processes) and “Dogiel type I” neurons (with several short processes). This implies no more than the distinction of intrinsic primary afferent from all other enteric neurons. The well-known chemical and functional diversity of enteric neurons is not reflected by this restrictive dichotomy of morphological data. Recent structural investigations of human enteric neurons were performed by different groups which mainly used two methodical approaches, namely detecting the architecture of their processes and target-specific tracing of their axonal courses. Both methods were combined with multiple immunohistochemistry in order to decipher neurochemical codes. This review integrates these morphological and immunohistological data and presents a classification of human enteric neurons which we believe is not yet complete but provides an essential foundation for the further development of human gastrointestinal neuropathology.

Synaptic activation of putative sensory neurons by hexamethonium-sensitive nerve pathways in mouse colon

The gastrointestinal tract contains its own independent population of sensory neurons within the gut wall. These sensory neurons have been referred to as intrinsic primary afferent neurons (IPANs) and can be identified by immunoreactivity to calcitonin gene-related peptide (CGRP) in mice. A common feature of IPANs is a paucity of fast synaptic inputs observed during sharp microelectrode recordings. Whether this is observed using different recording techniques is of particular interest for understanding the physiology of these neurons and neural circuit modeling. Here, we imaged spontaneous and evoked activation of myenteric neurons in isolated whole preparations of mouse colon and correlated recordings with CGRP and nitric oxide synthase (NOS) immunoreactivity, post hoc. Calcium indicator fluo 4 was used for this purpose. Calcium responses were recorded in nerve cell bodies located 5–10 mm oral to transmural electrical nerve stimuli. A total of 618 recorded neurons were classified for CGRP or NOS immunoreactivity. Aboral electrical stimulation evoked short-latency calcium transients in the majority of myenteric neurons, including ~90% of CGRP-immunoreactive Dogiel type II neurons. Activation of Dogiel type II neurons had a time course consistent with fast synaptic transmission and was always abolished by hexamethonium (300 μM) and by low-calcium Krebs solution. The nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (during synaptic blockade) directly activated Dogiel type II neurons. The present study suggests that murine colonic Dogiel type II neurons receive prominent fast excitatory synaptic inputs from hexamethonium-sensitive neural pathways.NEW & NOTEWORTHY Myenteric neurons in isolated mouse colon were recorded using calcium imaging and then neurochemically defined. Short-latency calcium transients were detected in >90% of calcitonin gene-related peptide-immunoreactive neurons to electrical stimulation of hexamethonium-sensitive pathways. Putative sensory Dogiel type II calcitonin gene-related peptide-immunoreactive myenteric neurons may receive widespread fast synaptic inputs in mouse colon.

Critical role of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in the initiation, generation, and propagation of the murine colonic migrating motor complex

The colonic migrating motor complex (CMMC) is necessary for fecal pellet propulsion in the murine colon. We have previously shown that 5-hydroxytryptamine (5-HT) released from enterochromaffin cells activates 5-HT3 receptors on the mucosal processes of myenteric Dogiel type II neurons to initiate the events underlying the CMMC. Our aims were to further investigate the roles of 5-HT1A, 5-HT3, and 5-HT7 receptor subtypes in generating and propagating the CMMC using intracellular microelectrodes or tension recordings from the circular muscle (CM) in preparations with and without the mucosa. Spontaneous CMMCs were recorded from the CM in isolated murine colons but not in preparations without the mucosa. In mucosaless preparations, ondansetron (3 μM; 5-HT3 antagonist) plus hexamethonium (100 μM) completely blocked spontaneous inhibitory junction potentials, depolarized the CM. Ondansetron blocked the preceding hyperpolarization associated with a CMMC. Spontaneous CMMCs and CMMCs evoked by spritzing 5-HT (10 and 100 μM) or nerve stimulation in preparations without the mucosa were blocked by SB 258719 or SB 269970 (1–5 μM; 5-HT7 antagonists). Both NAN-190 and (S)-WAY100135 (1–5 μM; 5-HT1A antagonists) blocked spontaneous CMMCs and neurally evoked CMMCs in preparations without the mucosa. Both NAN-190 and (S)-WAY100135 caused an atropine-sensitive depolarization of the CM. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP) (10 μM), and 5-carboxamidotryptamine (5-CT) (5 μM; 5-HT1/5/7 agonist) increased the frequency of spontaneous CMMCs. 5-HTP and 5-CT also induced CMMCs in preparations with and without the mucosa, which were blocked by SB 258719. 5-HT1A, 5-HT3, and 5-HT7 receptors, most likely on Dogiel Type II/AH neurons, are important in initiating, generating, and propagating the CMMC. Tonic inhibition of the CM appears to be driven by ongoing activity in descending serotonergic interneurons; by activating 5-HT7 receptors on AH neurons these interneurons also contribute to the generation of the CMMC.

Characterization of Myenteric Sensory Neurons in the Mouse Small Intestine

We recorded from myenteric AH/Dogiel type II cells, demonstrated mechanosensitive responses, and characterized their basic properties. Recordings were obtained using the mouse longitudinal muscle myenteric plexus preparation with patch-clamp and sharp intracellular electrodes. The neurons had an action potential hump and a slow afterhyperpolarization (AHP) current. The slow AHP was carried by intermediate conductance Ca2+-dependent K+-channel currents sensitive to charybdotoxin and clotrimazole. All possessed a hyperpolarization-activated current that was blocked by extracellular cesium. They also expressed a TTX-resistant Na+ current with an onset near the resting potential. Pressing on the ganglion containing the patched neuron evoked depolarizing potentials in 17/18 cells. The potentials persisted after synaptic transmission was blocked. Volleys of presynaptic electrical stimuli evoked slow excitatory postsynaptic potentials (EPSPs) in 9/11 sensory neurons, but 0/29 cells received fast EPSP input. The slow EPSP was generated by removal of a voltage-insensitive K+ current. Patch-clamp recording with a KMeSO4-containing, but not a conventional KCl-rich, intracellular solution reproduced the single-spike slow AHPs and low input resistances seen with sharp intracellular recording. Cell-attached recording of intermediate conductance potassium channels supported the conclusion that the single-spike slow AHP is an intrinsic property of intestinal AH/sensory neurons. Unitary current recordings also suggested that the slow AHP current probably does not contribute significantly to the high resting background conductance seen in these cells. The characterization of mouse myenteric sensory neurons opens the way for the study of their roles in normal and pathological physiology.