Characterization of projections of long interneurons in human colon

Study Purpose: The enteric nervous system contains inhibitory and excitatory motor neurons in circular and longitudinal muscle layers which modulate smooth muscle contractility. There are also numerous long interneuronal pathways in the myenteric plexus which have not been systematically investigated in human colon.

Data Collection: We used retrograde tracing ex vivo with DiI, with multiple labeling immu-nohistochemistry, to characterize long interneurons in the myenteric plexus of human colon. The projections of long ascending and descending interneurons have been established and a number of markers specific to polarised pathways (enkephalin, substance P and calretinin and nitric oxide synthase) have been identified.

Primary Conclusion: We confirmed previous studies on the projections of longitudinal and circular muscle motor neurons and showed that descending motor neurons have projections shorter than 18mm and ascending motor neurons have projections shorter than 8mm. Therefore, cells filled by DiI applied to the myenteric plexus, with projections longer than these limits, must be motor neurons and, arguably, long interneurons. We combined DiI tracing with immunohistochemistry for 6 other markers in various combinations. This showed that most ascending interneurons are immunoreactive for enkephalin, with a substantial proportion staining for calbindin or Substance P. They have projections up to 40mm long. Descending interneurons have projections up to 70mm and nearly all are immunoreactive for nitric oxide synthase, calbindin, or calretinin. Calbindin was present in circular muscle motor neurons, ascending interneurons, and descending interneurons and is therefore not a useful discriminatory marker.

Curator's Notes

Experimental Design: Tissue was obtained from 36 patients undergoing elective surgery with prior written informed consent. The serosa was then dissected to remove connective tissue, and the preparation was washed repeatedly in sterile KREBS solution. For fills from the tenia, a small longitudinal incision was made between bundles of tenial muscle, and the DiI‐coated bead was placed within this slit, allowing contact with axons at multiple levels through the tenia. Antibody-stained preparations were viewed on an Olympus IX microscope fit with epifluorescence and appropriate filter sets (Chroma Corporation). Images of labeled neural structures were captured via a CoolSNAP ES digital camera (Roper Scientific, Photometrics) using analySIS 5.0 software (Soft Imaging System).

Completeness: This dataset is a part of a larger study: "Characterisation of human colon longitudinal muscle motor neurons"

Subjects & Samples: 36 human subjects, average age 65 years (std deviation 13 years), 17 female, 19 male

Primary vs derivative data: The primary folder consists of original raw data files showing results of tracing enteric neuronal pathways in the human colon, ex vivo, using the carbocyanine dye DiI in combination with dual-labeling immunohistochemistry for calbindin (Calb) and calretinin (Calret). Each specific folder includes data from a de-identified patient (sub-) with DiI applied to the circular muscle samples. Each filename contains the coordinates of the cell body relative to the DiI application site. All images were taken at 20x magnification giving a calibration of (long axis of micrograph = 465um). Micrographs are arranged in triplets - DiI, Marker 1, and Marker 2. Each sample folder also contains a Microsoft Excel file (.xlsx) with coordinates downloaded from a fluorescence microscope. Derivative folder image data (JPEG2000 and OME-TIFF) was derived from primary images (.tif). Primary images (3 separate channels) were merged and converted with 10:1 compression to JPEG2000 (.jpx) by MBF Bioscience for web streaming and visualization on the SPARC Data Portal. Primary images were also converted with lossless compression to OME-TIFF (.tif) by MBF Bioscience.