A Novel Approach for Studying the Physiology and Pathophysiology of Myelinated and Non-Myelinated Axons in the CNS White Matter

Abstract

Advances in brain connectomics set the need for detailed knowledge of functional properties of myelinated and non-myelinated (if present) axons in specific white matter pathways. The corpus callosum (CC), a major white matter structure interconnecting brain hemispheres, is extensively used for studying CNS axonal function. Unlike another widely used CNS white matter preparation, the optic nerve where all axons are myelinated, the CC contains also a large population of non-myelinated axons, making it particularly useful for studying both types of axons. Electrophysiological studies of optic nerve use suction electrodes on nerve ends to stimulate and record compound action potentials (CAPs) that adequately represent its axonal population, whereas CC studies use microelectrodes (MEs), recording from a limited area within the CC. Here we introduce a novel robust isolated "whole" CC preparation comparable to optic nerve. Unlike ME recordings where the CC CAP peaks representing myelinated and non-myelinated axons vary broadly in size, "whole" CC CAPs show stable reproducible ratios of these two main peaks, and also reveal a third peak, suggesting a distinct group of smaller caliber non-myelinated axons. We provide detailed characterization of "whole" CC CAPs and conduction velocities of myelinated and non-myelinated axons along the rostro-caudal axis of CC body and show advantages of this preparation for comparing axonal function in wild type and dysmyelinated shiverer mice, studying the effects of temperature dependence, bath-applied drugs and ischemia modeled by oxygen-glucose deprivation. Due to the isolation from gray matter, our approach allows for studying CC axonal function without possible "contamination" by reverberating signals from gray matter. Our analysis of "whole" CC CAPs revealed higher complexity of myelinated and non-myelinated axonal populations, not noticed earlier. This preparation may have a broad range of applications as a robust model for studying myelinated and non-myelinated axons of the CNS in various experimental models.

Distinctive functional deficiencies in axonal conduction associated with two forms of cerebral white matter injury

“…Parameters of the N1 (myelinated axons) and N2 (unmyelinated axons) components of the CAPs were contrasted in the anterior CC after cuprizone and L‐NIO induced WM injury mouse models. Brain slices from the anterior CC were chosen for this comparative analysis, similar as a previous study, our data presented in the current study have showed that the anterior CC contains a higher proportion of myelinated axons than the posterior CC. The results indicated that the parameters beyond the amplitude and the area of the N1 component of the CAPs might be used to distinguish between either pure demyelination or demyelination concurrent with axonal injury.…”

“…The early peak (N1) signifies the conduction of myelinated axons, while the later phase peak (N2) represents unmyelinated axons. Previous research has observed heterogeneity in the CAPs response within the rostral‐caudal extent of the CC that appears to be linked to variations in the proportion of myelinated and unmyelinated axons . We therefore surmised that demyelination and axonal damage might give different CAPs profiles relative to the normal CAPs response.…”

“…Thus, WM injury can be characterized as demyelination and/or axonal damage. As myelinated and unmyelinated axons display different electrophysiological properties, functional studies employing electrophysiological techniques capitalizing on these differences may allow researchers to discern the nature and severity of any underlying WM damage. To this end, the corpus callosum (CC), the largest tract of WM in the brain, has been used extensively in research to study WM function under normal and pathological conditions (including axonal injury and remyelination) .…”

Structural adaption of axons during de‐ and remyelination in the Cuprizone mouse model

“…This finding is consistent with the functional readout applied, the measuring of compound action potentials. While it is generally assumed that the two peaks that are usually measured when recording CAPs in fibers of the corpus callosum belong to myelinated and unmyelinated fibers , the ratio between these two peaks is highly depending on positioning of the electrodes [summarized in ]. After complete demyelination in the corpus callosum, the first of the two peaks is lost .…”

Splice‐dependent trans‐synaptic PTP δ– IL 1 RAPL 1 interaction regulates synapse formation and non‐ REM sleep

“…Corpus callosum samples were prepared as described previously (Li et al , ). Briefly, coronal brain sections (400 μm) containing the corpus callosum were sliced in aCSF using a vibratome (VT1200s, Leica).…”