Myelin Sheath and Nodes of Ranvier

Introduction

Myelin is one of the most visually striking and functionally important structures in the nervous system. The spirally wrapped membrane sheath produced by glial cells dramatically increases the speed of action potential conduction and reduces the metabolic cost of signaling. For EM annotators, myelin serves as one of the most reliable markers of axonal identity, and its features — periodicity, wrap number, nodal domains, and associated specializations — provide a wealth of information about axon caliber, maturation, and health.

This script covers myelin ultrastructure from the molecular level of membrane periodicity to the macroscopic level of nodal architecture, with practical guidance for identification in EM datasets.

1. Origin of the Myelin Sheath

Central Nervous System (CNS)

In the CNS, myelin is produced by oligodendrocytes. Each oligodendrocyte extends multiple processes (typically 20-60 in rodents, fewer in humans), each of which wraps a segment of a different axon. A single oligodendrocyte therefore myelinates multiple axons simultaneously. The oligodendrocyte cell body is typically located some distance from the myelinated segments, connected by thin cytoplasmic processes.

Peripheral Nervous System (PNS)

In the PNS, myelin is produced by Schwann cells. Each Schwann cell wraps a single internode of a single axon — a one-to-one relationship. The Schwann cell body sits adjacent to the myelin sheath, and its nucleus is often visible alongside the outer myelin surface in longitudinal sections.

The Wrapping Process

Myelin formation begins when a glial process contacts an axon and begins to spiral around it. The cytoplasm is progressively squeezed out as the membrane wraps tighten, producing compact myelin. The innermost wrap (inner tongue) and outermost wrap (outer tongue) retain cytoplasm, as do occasional channels through the compact myelin (Schmidt-Lanterman incisures in PNS).

2. Compact Myelin Ultrastructure

Compact myelin has a highly regular, periodic structure that is one of the most distinctive features in EM.

2.1 The Major Dense Line

Formation: When the cytoplasmic faces of the glial cell membrane come together (as the wrapping process squeezes out cytoplasm), the intracellular surfaces fuse to form the major dense line.
Appearance: A dark, electron-dense line approximately 3 nm thick.
Composition: Primarily myelin basic protein (MBP) in the CNS, which is essential for holding the cytoplasmic faces together. In the PNS, both MBP and P0 protein contribute.

2.2 The Intraperiod Line

Formation: Where the extracellular faces of adjacent membrane wraps appose each other.
Appearance: A slightly less dense line than the major dense line, sometimes appearing as a double line at high resolution (because the extracellular surfaces do not fully fuse).
Composition: Proteolipid protein (PLP) and P0 protein span the membrane and contribute to extracellular adhesion.

2.3 Myelin Periodicity

The repeat distance from one major dense line to the next — encompassing two lipid bilayers, one major dense line, and one intraperiod line — is the myelin period:

CNS myelin: Approximately 12 nm period (some sources cite 11-13 nm depending on species and fixation).
PNS myelin: Approximately 13-14 nm period, slightly larger due to the more prominent intraperiod line.
EM appearance: In high-resolution cross-sections, compact myelin appears as a series of alternating dark (major dense) and lighter (intraperiod) lines, creating a characteristic laminated or banded pattern. At lower magnification, the myelin sheath appears as a dark, homogeneous ring around the axon.

2.4 Visualization Considerations

High magnification (>30,000x): Individual lamellae are resolved. The alternating dark/light periodicity is clearly visible. Annotators can count individual wraps.
Medium magnification (10,000-30,000x): The myelin sheath appears as a thick dark ring. Individual lamellae may be partially resolved.
Low magnification (<10,000x): Myelin appears as a dark annulus around a lighter axonal profile. The number of wraps is not discernible, but myelinated axons are easily distinguished from unmyelinated ones.

3. Number of Myelin Wraps

The thickness of the myelin sheath is not uniform but scales with axon diameter:

Small axons (0.5-1 micrometer diameter): Typically 10-30 lamellae.
Medium axons (1-3 micrometers): Typically 30-80 lamellae.
Large axons (3-10+ micrometers, e.g., motor axons in PNS): Can have 80-150+ lamellae.

The g-ratio

The ratio of inner axon diameter to total outer diameter (including myelin) is called the g-ratio:

Optimal g-ratio: Approximately 0.6-0.7 for CNS axons, meaning the myelin sheath occupies roughly 30-40% of the total fiber diameter.
Significance: The g-ratio is optimized for maximal conduction velocity. Axons with too little myelin (high g-ratio) conduct slowly. Axons with excessive myelin (low g-ratio) waste resources and space.
Annotation use: Measuring the g-ratio in EM volumes can identify axons with abnormally thin myelin (possible demyelination or remyelination) or abnormally thick myelin (possible tomaculous neuropathy).

4. Nodes of Ranvier

Nodes of Ranvier are the regularly spaced gaps in the myelin sheath where the axon membrane is exposed to the extracellular space. They are the sites of saltatory conduction — action potentials “jump” from node to node.

4.1 Nodal Architecture

Length: The bare nodal gap is typically 1-2 micrometers long.
Internode distance: The myelinated segment between nodes (the internode) ranges from approximately 100 micrometers to over 1 millimeter, roughly proportional to axon diameter.
Exposed axolemma: The nodal membrane is not truly bare — it is covered by a specialized extracellular matrix and contacted by glial processes.

4.2 Ultrastructural Features in EM

Dense undercoat: The nodal axolemma has a thick, electron-dense coating on its cytoplasmic face, similar to (and molecularly related to) the AIS undercoat. This undercoat is composed of ankyrin-G and betaIV-spectrin, which anchor voltage-gated sodium channels (Nav1.6 is the predominant subtype at mature nodes).
Sodium channel clustering: Although individual channels are not visible in conventional EM, their functional presence is inferred from the dense undercoat. Immuno-EM confirms the extreme concentration of Nav channels at nodes.
Nodal microvilli (PNS): In peripheral nerves, Schwann cell microvilli project from the terminal ends of the myelin sheath to contact the nodal axolemma. These appear as small finger-like projections surrounding the node.
Astrocyte contacts (CNS): In the central nervous system, perinodal astrocyte processes fill the space around the node instead of Schwann cell microvilli. These pale, glycogen-containing processes can be identified by their characteristic astrocytic features.
Extracellular matrix: The nodal gap is filled with a proteoglycan-rich extracellular matrix that helps maintain the nodal domain. This material may appear as a faint, granular density in the extracellular space.

4.3 Paranodal Regions

Flanking each node are the paranodal regions, where the myelin sheath terminates:

Paranodal loops: The terminal turns of the myelin spiral do not end abruptly. Instead, each lamella forms a cytoplasm-containing loop that contacts the axon membrane. These loops stack in a regular array along the paranode, creating a distinctive “staircase” pattern in longitudinal sections.
Septate-like junctions: Each paranodal loop forms a specialized junction with the axolemma, mediated by the Caspr/contactin/neurofascin-155 complex. In EM, these junctions appear as periodic densities (septae) bridging the 3-5 nm gap between the loop membrane and the axon membrane.
Function: Paranodal junctions serve as a diffusion barrier, preventing lateral movement of nodal sodium channels and juxtaparanodal potassium channels. They effectively define and maintain the molecular domains of the node.
Length: The paranodal region is typically 3-10 micrometers long on each side of the node.

4.4 Juxtaparanodal Region

Just beyond the paranode, beneath the compact myelin:

Potassium channels: Kv1.1 and Kv1.2 channels cluster in the juxtaparanodal axolemma. They are not visible in conventional EM but are important for understanding nodal physiology.
EM appearance: The juxtaparanodal axolemma may show a modest undercoat density, less prominent than the nodal undercoat.

5. Schmidt-Lanterman Incisures

Schmidt-Lanterman incisures (also called Schmidt-Lanterman clefts) are cytoplasmic channels that spiral through the compact myelin sheath:

Location: Primarily found in PNS myelin; rare or absent in CNS myelin.
Structure: Regions where the major dense line opens to create a narrow channel of Schwann cell cytoplasm connecting the inner and outer tongues through the myelin sheath. The cytoplasmic channel spirals through the myelin, following the original wrapping trajectory.
EM appearance:
- Longitudinal section: Lighter streaks or bands running obliquely through the dark myelin sheath, creating a characteristic “herringbone” pattern.
- Cross-section: Appear as interruptions in the compact myelin lamellae where a small pocket of cytoplasm separates adjacent lamellae.
Function: Provide a cytoplasmic pathway for transport of metabolites, signaling molecules, and organelles between the Schwann cell body (outer surface) and the inner tongue (nearest the axon). Without incisures, the compact myelin would completely isolate the inner tongue from the cell body.
Contents: The cytoplasm within incisures contains microtubules, actin filaments, and occasionally small organelles (mitochondria, ER tubules).

6. Inner and Outer Tongues

The innermost and outermost wraps of the myelin sheath retain cytoplasm and serve as the interface between compact myelin and the cell:

6.1 Inner Tongue (Innermost Loop)

Location: The cytoplasm-containing layer directly apposed to the axon membrane.
Appearance: A thin crescent of pale cytoplasm between the innermost myelin lamella and the axolemma.
Function: Site of membrane growth during myelination. Contains the machinery for membrane addition and lipid synthesis. The inner tongue is where new myelin membrane is added during sheath growth.
Width: Varies; typically very thin (50-200 nm) in mature myelin but can be thicker during active myelination.

6.2 Outer Tongue (Outermost Loop)

Location: The outermost wrap of the myelin sheath, connected to the glial cell body (via the process in CNS, directly in PNS).
Appearance: A thin crescent of cytoplasm on the external surface of the myelin sheath.
Function: Continuous with the cell body cytoplasm (in Schwann cells) or the oligodendrocyte process (in CNS). Serves as the route for transport of newly synthesized myelin components.

7. Annotation Implications of Myelin

7.1 Myelin as an Axon Identifier

Strong positive cue: The presence of a myelin sheath is strong evidence that the enclosed process is an axon. In the CNS, only axons are myelinated (dendrites and somata are not).
Caveat — unmyelinated axons: Many CNS axons are unmyelinated, especially those of local interneurons and thin-caliber projection axons. The absence of myelin does not exclude axon identity.
Caveat — partial myelination: Some axons are myelinated along part of their length but unmyelinated at their terminals and sometimes along intermediate segments. An axon may therefore transition from myelinated to unmyelinated within a single EM volume.

7.2 Node Identification for Tracing

Nodes of Ranvier can be challenging for automated segmentation and manual tracing because:

The axon caliber often narrows at the node (by approximately 20-30%), changing the profile size.
The myelin sheath is absent, so the characteristic dark ring that aids tracing disappears.
Paranodal loops and glial contacts create a complex local environment that can confuse segmentation algorithms.

Strategy: When a myelinated axon seems to “disappear” between sections, check for a node. Look for the characteristic paranodal loop pattern, the dense undercoat on a bare segment of axolemma, and the re-emergence of myelin on the far side.

7.3 Myelin Pathology Indicators

Thin myelin relative to axon diameter (high g-ratio): May indicate remyelination (new myelin sheaths are thinner than original ones) or incomplete myelination.
Split or vacuolated myelin: Separation of lamellae with fluid-filled spaces, seen in edema or early demyelination.
Myelin debris (ovoid bodies): Fragments of myelin in the neuropil, indicating active demyelination. Often phagocytosed by macrophages or microglia.
Redundant myelin (outfolding): Excessive myelin forming loops that extend away from the axon. Can be a fixation artifact or a genuine pathological finding (tomaculous neuropathy).

8. Worked Example: Recognizing a Myelinated Axon in Cross-Section

Scenario: In a cross-section through cortical white matter, you see a circular profile surrounded by a thick dark ring.

Step-by-step identification:

Dark ring assessment: The ring shows a laminated structure at high magnification — alternating dark and lighter lines with approximately 12 nm periodicity. This is compact myelin.
Wrap count: Approximately 40 lamellae are visible, consistent with a medium-caliber axon.
Axon contents: The enclosed profile contains a few small mitochondria (approximately 0.5 micrometers), longitudinally cut neurofilaments and microtubules, and a single SER tubule. No ribosomes are visible.
g-ratio estimation: The inner axon diameter is approximately 1.5 micrometers; the total outer diameter (with myelin) is approximately 2.3 micrometers. The g-ratio is approximately 0.65 — within the normal range.
Inner tongue: A thin crescent of pale cytoplasm is visible between the innermost lamella and the axon membrane.
Conclusion: Compact myelin + appropriate contents + normal g-ratio = healthy myelinated axon.

9. Worked Example: Identifying a Node of Ranvier

Scenario: While tracing a myelinated axon through serial sections, the myelin sheath suddenly terminates on both sides, leaving a short bare segment.

Step-by-step identification:

Myelin termination pattern: On each side of the bare segment, the myelin lamellae terminate in a series of cytoplasm-filled loops (paranodal loops) that stack against the axon membrane. The characteristic “staircase” pattern is visible.
Nodal gap length: The bare axon segment is approximately 1.5 micrometers long — consistent with a node of Ranvier.
Dense undercoat: The axolemma in the bare segment shows a dark electron-dense coating on the cytoplasmic face. This is the ankyrin-G-based undercoat enriched in sodium channels.
Glial contacts: Pale astrocyte processes (containing glycogen granules) contact the nodal axolemma. No Schwann cell microvilli are present (confirming CNS location).
Axon caliber: The axon diameter narrows slightly at the node (approximately 20% reduction compared to the internodal diameter). This is normal.
Continuity confirmation: The myelin resumes on the far side with similar periodicity and wrap number, confirming this is the same axon.
Conclusion: Paranodal loops + bare axolemma with dense undercoat + astrocyte contacts + transient caliber reduction = node of Ranvier (CNS).

10. Worked Example: Distinguishing Compact Myelin from Artifact

Scenario: A dark, laminated ring surrounds a small profile, but something looks unusual.

Potential artifacts that mimic or distort myelin:

Fixation-induced myelin splitting: If alternating lamellae are separated by clear spaces, the “myelin” may be artifactually swollen. Genuine compact myelin has tightly apposed lamellae with no visible gaps at the light-microscopic level. Mild splitting at the intraperiod line can occur during fixation and is a common processing artifact, not necessarily pathology.
Oblique sectioning: If the section cuts through myelin at an angle rather than perpendicular to the axon, the myelin ring may appear asymmetric — thicker on one side, thinner on the other. This is a geometric artifact, not a biological feature.
Membrane whorls: Occasionally, membrane debris in the neuropil forms concentric lamellated structures that superficially resemble myelin. To distinguish: (a) check for an enclosed axon with appropriate cytoskeletal contents, (b) check for inner and outer tongues, (c) verify that the periodicity matches known myelin values. Membrane whorls often have irregular periodicity and lack a central axon.
Resin artifacts: Folds or wrinkles in the section can create dark bands that mimic lamellae. These typically extend linearly across multiple structures rather than forming a closed ring.

11. Common Misconceptions

Misconception	Reality
“All axons in the CNS are myelinated.”	Many CNS axons are unmyelinated, especially those of local interneurons. In cortical gray matter, unmyelinated axons vastly outnumber myelinated ones.
“Myelin is made of fat.”	Myelin is made of tightly compacted glial cell membrane, which is lipid-rich (approximately 70% lipid by dry weight) but also contains critical structural proteins (MBP, PLP, P0).
“The node of Ranvier is a gap in insulation.”	While the node is a gap in myelin, it is a highly organized functional domain with clustered ion channels, specialized extracellular matrix, and glial contacts — not simply a bare patch.
“CNS and PNS myelin are the same.”	CNS myelin (oligodendrocyte) and PNS myelin (Schwann cell) differ in protein composition (PLP vs. P0), periodicity, one-to-many vs. one-to-one wrapping, and presence of Schmidt-Lanterman incisures.
“Thicker myelin always means faster conduction.”	Conduction velocity is optimized at a specific g-ratio. Excessively thick myelin (very low g-ratio) actually reduces conduction velocity because it increases fiber diameter without proportionally increasing axon diameter.
“Paranodal loops are just myelin endings.”	Paranodal loops form critical septate-like junctions that serve as molecular fences, maintaining the distinct ion channel domains of the node and juxtaparanode.

References

Peters A, Palay SL, Webster HdeF (1991) The Fine Structure of the Nervous System, 3rd edition. Oxford University Press.
Hildebrand C, Remahl S, Persson H, Bjartmar C (1993) “Myelinated nerve fibres in the CNS.” Progress in Neurobiology 40:319-384.
Stassart RM, Mobius W, Nave KA, Edgar JM (2018) “The axon-myelin unit in development and degenerative disease.” Frontiers in Neuroscience 12:467.
Salzer JL (2003) “Polarized domains of myelinated axons.” Neuron 40:297-318.
Rasband MN, Peles E (2021) “Mechanisms of node of Ranvier assembly.” Nature Reviews Neuroscience 22:7-20.
Nave KA, Werner HB (2014) “Myelination of the nervous system: mechanisms and functions.” Annual Review of Cell and Developmental Biology 30:503-533.
Waxman SG, Ritchie JM (1993) “Molecular dissection of the myelinated axon.” Annals of Neurology 33:121-136.

This document is part of the NeuroTrailblazers Content Library. It is intended as an instructor reference and annotator training script. Last updated: 2026.