Axon typing of rat muscle nerves using a double staining procedure for cholinesterase and carbonic anhydrase

We developed a method for detecting activity of axonal cholinesterase (CE) and carbonic anhydrase (CA)--markers for motor and sensory nerve fibers (NFs)--in the same histological section. To reach this goal, cross-sections of muscle nerves were sequentially incubated with the standard protocols for CE and CA histochemistry. A modified incubation medium was used for CA in which Co++ is replaced by Ni++. This avoids interference of the two histochemical reactions because Co++ binds unspecifically to the brown copper-ferroferricyanide complex representing CE activity, whereas Ni++ does not. Cross-sections of the trapezius muscle nerve containing efferent and afferent NFs in segregated fascicles showed that CE activity was confined to motor NFs. Axonal CA was detected solely in sensory NFs. The number of labeled motor and sensory NFs determined in serial cross-sections stained with either the new or the conventional technique was not significantly different. Morphometric analysis revealed that small unreactive NFs (diameter less than 5 microns) are afferent, medium-sized ones (5 microns less than d less than 7 microns) are unclassifiable, and large ones (d greater than 7 microns) are efferent. The heterogenous CE activity of thick (alpha) motor NFs is linked to the type of their motor units. "Fast" motor units contain CE reactive NFs; "slow" ones have CE negative neurites.