Quantification of CGRP‐immunoreactive myenteric neurons in mouse colon

Abstract Quantitative data of biological systems provide valuable baseline information for understanding pathology, experimental perturbations, and computational modeling. In mouse colon, calcitonin gene‐related peptide (CGRP) is expressed by myenteric neurons with multiaxonal (Dogiel type II) morphology, characteristic of intrinsic primary afferent neurons (IPANs). Analogous neurons in other species and gut regions represent 5–35% of myenteric neurons. We aimed to quantify proportions of CGRP‐immunopositive (CGRP+) myenteric neurons. Colchicine‐treated wholemount preparations of proximal, mid, and distal colon were labeled for HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per). The pan‐neuronal markers (Hu+/Per+) co‐labeled 94% of neurons. Hu+/Per– neurons comprised ∼6%, but Hu‐/Per+ cells were rare. Thus, quantification was based on Hu+ myenteric neurons (8576 total; 1225 ± 239 per animal, n = 7). CGRP+ cell bodies were significantly larger than the average of all Hu+ neurons (329 ± 13 vs. 261 ± 12 μm 2 , p < .0001). CGRP+ neurons comprised 19% ± 3% of myenteric neurons without significant regional variation. NOS+ neurons comprised 42% ± 2% of myenteric neurons overall, representing a lower proportion in proximal colon, compared to mid and distal colon (38% ± 2%, 44% ± 2%, and 44% ± 3%, respectively). Peripherin immunolabeling revealed cell body and axonal morphology in some myenteric neurons. Whether all CGRP+ neurons were multiaxonal could not be addressed using peripherin immunolabeling. However, of 118 putatively multiaxonal neurons first identified based on peripherin immunoreactivity, all were CGRP+ ( n = 4). In conclusion, CGRP+ myenteric neurons in mouse colon were comprehensively quantified, occurring within a range expected of a putative IPAN marker. All Per+ multiaxonal neurons, characteristic of Dogiel type II/IPAN morphology, were CGRP+.