Multiplexed Immunohistochemistry to Identify Systematically Classes of Human Colonic Myenteric Neurons
Authors: Brookes Simon Jonathan , Humenick Adam , Chen Bao Nan , Dinning Phil G , Costa Marcello , Wattchow David 
Published: April 2020

ObjectiveThe enteric nervous system (ENS) comprises multiple classes of nerve cells which can be distinguished by combinations of immunohistochemical markers (“chemical coding”) that they express. While many immunohistochemical studies have been carried out, the chemical coding of ENS of human colon has not been characterised systematically.MethodsSurgical specimens of human colon were obtained with prior informed consent (Ethics Approval SAC HREC#207.07), incubated in 83uM colchicine overnight to enhance neuropeptide immunoreactivity, then fixed. Multiplexed indirect immunohistochemistry (5 layers of quadruple labelling) included 13 common neuronal markers; HuC/D, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), 5‐hydroxytryptamie (5‐HT), Substance P/tachykinins (SP), leu‐Enkephalin (ENK), calcitonin gene‐related peptide (CGRP), somatostatin (SOM), Calbindin (Calb), Calretinin (Calret), neurofilament 200 (NF200), vasoactive intestinal polypeptide (VIP) and neuropeptide Y (NPY). Antibodies were eluted between layers. The presence/absence of all 13 markers was recorded for 1303 neurons in human ascending colon (n=6) and 1087 in descending colon (n=5). HuC/D labelled all myenteric nerve cell bodies and was revealed using AMCA as a fluorophore; this staining was resistant to elution and allowed comparison between layers.ResultsChemical codes were surprisingly varied; of 4096 possible codes (212), 108 were present in ascending colon and 112 in descending colon. However, 40 codes accounted for more than 90% of neurons. There was a strong correlation between the rank order of chemical codes in ascending and descending colon (ranked by abundance; Rs=0.884[39]); p<0.001) indicating that there are similar ENS populations in ascending and descending colon. In ascending colon (n=6, 1303 cells) 686 cells (52.6%) were immunoreactive for NOS; 55.5% for ChAT; 9.8% were immunoreactive for both ChAT and NOS and 22 cells (1.7%) lacked both markers. The next most abundant markers were NF200 (50.0%), VIP (31.3%) and calbindin (25.1%). Low frequency markers in myenteric nerve cell bodies included CGRP (0.9%) and SOM (1.3%). A few codes accounted for very large proportions of neurons. For example, putative inhibitory motor neurons, immunoreactive for NOS (with none of the other 11 markers), accounted for 192 of 1303 neurons (14.7%). Dogiel type II neurons (identified by NF200 morphology) were large cells (1795 +/‐ 729 μm2, 25 cells) with NF200, ChAT and SP, with either Calb or Calret (or both) or SOM. It is also possible to identify cell bodies surrounded by dense baskets of varicose axons, which represent strong synaptic inputs. For example, ChAT‐immunoreactivity was present in 55.5% of all myenteric neurons, but made up 93.6% of all myenteric neurons enclosed by dense enkephalin‐immunoreactive inputs.Conclusionsmulti‐layer multiplexed immunoreactivity reveals a wealth of information about the human colonic ENS, making possible systematic identification of classes and identification of some synaptic connectivitySupport or Funding InformationSupported by NIH SPARC grant #1OT2OD24899