Quantification of CGRP-immunoreactive myenteric neurons in mouse colon

Area of Science:

• Neuroscience
• Gastroenterology
• Cell Biology

Background:

• Quantitative data are crucial for understanding biological systems, including pathology and experimental perturbations.
• Calcitonin gene-related peptide (CGRP) is expressed in mouse colon myenteric neurons with Dogiel type II morphology, characteristic of intrinsic primary afferent neurons (IPANs).
• IPANs represent 5-35% of myenteric neurons across species and gut regions.

Purpose of the Study:

• To quantify the proportion of CGRP-immunopositive (CGRP+) myenteric neurons in the mouse colon.
• To determine if CGRP+ neurons exhibit characteristics of IPANs.

Main Methods:

• Whole-mount preparations of mouse colon (proximal, mid, distal) were treated with colchicine and labeled for neuronal markers HuC/D, CGRP, nitric oxide synthase (NOS), and peripherin (Per).
• Pan-neuronal markers (Hu+/Per+) identified 94% of neurons; quantification focused on Hu+ myenteric neurons (n=7, total 8576).
• Immunoreactivity for CGRP, NOS, and peripherin was analyzed, with CGRP+ neuron cell body size compared to the average Hu+ neuron size.

Main Results:

• CGRP+ myenteric neurons constituted 19% ± 3% of total myenteric neurons, with no significant regional variation.
• CGRP+ neuron cell bodies were significantly larger (329 ± 13 μm²) than the average Hu+ neuron (261 ± 12 μm²).
• Nitric oxide synthase (NOS)+ neurons comprised 42% ± 2% of myenteric neurons, with a lower proportion in the proximal colon.
• All 118 peripherin-positive (Per+) multiaxonal neurons, identified as Dogiel type II/IPANs, were also CGRP+.

Conclusions:

• CGRP+ myenteric neurons in the mouse colon were quantified, with their proportion falling within the expected range for a putative IPAN marker.
• The finding that all Per+ multiaxonal neurons are CGRP+ strongly supports CGRP as a reliable marker for IPANs in the mouse colon.