Axon Biology and Ultrastructure

Introduction

The axon is the output process of the neuron. Each neuron typically gives rise to a single axon that carries action potentials away from the soma and delivers signals to target cells through synaptic transmission. Axons are structurally and molecularly distinct from dendrites, and understanding their ultrastructure is critical for annotators working in EM volumes. This script covers the axon from its origin at the axon initial segment through its unmyelinated and myelinated lengths to its synaptic terminals.

1. Axon Overview

Key distinguishing properties of axons compared to dendrites:

Single per neuron: In most cases, each neuron has exactly one axon (though it may branch extensively).
Uniform caliber: Unlike dendrites, axons maintain a relatively constant diameter along their length (except at branch points and boutons).
Extended length: Axons range from less than a millimeter (local interneurons) to over a meter (corticospinal tract neurons, peripheral motor neurons).
No ribosomes: Mature vertebrate axons lack ribosomes and rough ER under normal conditions. All axonal proteins must be synthesized in the soma and transported.
Uniform microtubule polarity: In vertebrates, axonal microtubules are oriented uniformly with plus-ends pointing distally (away from the soma). This contrasts with the mixed polarity in dendrites.
Synaptic vesicles: Axons contain clusters of synaptic vesicles at their boutons, a feature never found in dendrites.

2. The Axon Initial Segment (AIS)

The axon initial segment is the specialized proximal region of the axon where action potential initiation occurs. It spans roughly 20-60 micrometers from the soma and has a unique ultrastructure that makes it identifiable in EM (Leterrier, 2018).

Structural Features Visible in EM

Dense undercoat: The most conspicuous AIS feature is a thick, electron-dense coating on the cytoplasmic face of the plasma membrane, typically 5-10 nm thick. This undercoat is composed of ankyrin-G, betaIV-spectrin, and associated proteins that anchor voltage-gated sodium channels (Nav1.6, Nav1.2) and potassium channels (Kv7, Kv1).
Fasciculated microtubules: Unlike the loosely arranged microtubules in dendrites and the soma, AIS microtubules are bundled into tight fascicles of 3-10 microtubules cross-linked by TRIM46 protein. These fascicles run parallel to the long axis of the AIS and are a reliable identification cue.
Absence of ribosomes: The AIS marks the boundary beyond which ribosomes and rough ER are excluded. The “Nissl cap” observed at light microscopy corresponds to the end of ribosome-containing cytoplasm at the axon hillock.
Sparse organelles: Compared to proximal dendrites of similar caliber, the AIS has fewer mitochondria, no Golgi outposts, and no rough ER.
Occasional synapses: The AIS receives GABAergic synapses, often from chandelier cells (axo-axonic cells). These Type II symmetric synapses on the AIS are a distinctive feature and can help confirm AIS identity.

Functional Significance

The AIS is the site where the neuron integrates all incoming synaptic input into a binary decision: to fire or not. The high density of voltage-gated sodium channels (estimated at 40-100 times higher than on the soma) creates the lowest threshold for action potential generation. The dense undercoat provides the scaffold that maintains this channel enrichment.

3. Unmyelinated Axons

Many axons in the central nervous system — particularly those of local-circuit interneurons and some long-range projection neurons — are unmyelinated. In EM:

Diameter: Typically 0.1-1.0 micrometers. The smallest unmyelinated axons can be as thin as 0.08 micrometers.
Cytoskeletal contents: Neurofilaments (10 nm intermediate filaments) and microtubules (25 nm outer diameter) run longitudinally. The ratio of neurofilaments to microtubules increases with axon diameter.
Smooth ER: A single smooth ER tubule often runs the length of the axon, serving as a calcium store and membrane reservoir.
Mitochondria: Sparse, small (0.5-2 micrometers), and elongated. They are distributed along the axon at intervals, more densely near branch points and boutons.
No ribosomes: The complete absence of ribosomes and rough ER is a defining negative feature.
Ensheathing glia: In the CNS, unmyelinated axons may be partially wrapped by astrocyte processes but lack the compact myelin of oligodendrocytes. In the PNS, Remak bundles group multiple unmyelinated axons within a single Schwann cell.

Annotation Challenges

Unmyelinated axons are among the most difficult structures to trace in EM volumes because:

Their small diameter means they occupy only a few pixels in lower-resolution datasets.
They can be confused with thin dendritic branches, glial processes, or even imaging artifacts.
They run in dense bundles where individual axons are hard to separate.

4. Axon Terminals: Boutons

Axon terminals are the synaptic output sites where action potentials trigger neurotransmitter release. Two major types exist.

4.1 En Passant Boutons

Definition: Swellings along the axon shaft that form synapses without terminating the axon. The axon continues beyond the bouton to form additional synapses.
Morphology: The axon locally expands to 0.5-2.0 micrometers diameter (compared to 0.1-0.3 micrometers for the inter-bouton axon).
Vesicle content: Clusters of synaptic vesicles congregate at the active zone face of the bouton. Mitochondria are frequently present.
Prevalence: Extremely common. A single cortical axon may form dozens to hundreds of en passant boutons along its length.
EM identification: Look for a local swelling in an axon profile containing vesicle clusters apposed to a postsynaptic target with visible PSD.

4.2 Terminal Boutons

Definition: Boutons at the end of an axonal branch, where the axon terminates.
Morphology: Often larger than en passant boutons (1-5 micrometers), with more extensive vesicle pools.
Classic examples: Neuromuscular junction terminals, calyx of Held, mossy fiber terminals in hippocampus (which can be very large, 3-5 micrometers).
EM identification: A large vesicle-filled profile at the end of an axon branch, often contacting multiple postsynaptic targets.

5. Synaptic Vesicle Pools

Within each bouton, synaptic vesicles are organized into functionally distinct pools (Rizzoli & Betz, 2005). While these pools are defined physiologically, they have ultrastructural correlates:

5.1 Readily Releasable Pool (RRP)

Size: Approximately 1-2% of total vesicles (roughly 5-10 vesicles per active zone).
Location: Docked at the active zone membrane, in direct contact with or within nanometers of the presynaptic membrane.
EM correlate: Vesicles directly touching the presynaptic membrane at the active zone. These “docked vesicles” are visible in well-preserved tissue.
Function: Released first upon action potential arrival. Determines initial release probability.

5.2 Recycling Pool

Size: Approximately 10-20% of total vesicles.
Location: Near the active zone but not docked. Within roughly 100-200 nm of the membrane.
EM correlate: The cluster of vesicles just behind the docked vesicles, within the active zone vicinity.
Function: Replenishes the RRP during moderate, sustained activity. Vesicles cycle between release, endocytosis, refilling, and re-docking.

5.3 Reserve Pool

Size: Approximately 80-90% of total vesicles — the vast majority.
Location: Distributed throughout the bouton, often tethered to the cytoskeleton by synapsin proteins.
EM correlate: The large cloud of vesicles filling the bouton away from the active zone.
Function: Mobilized only during intense, prolonged stimulation. Synapsin phosphorylation releases vesicles from cytoskeletal tethers.

Practical Note for Annotators

The total number of vesicles per bouton varies enormously: small en passant boutons in cortex may contain 100-300 vesicles, while large terminals like the calyx of Held contain over 70,000. Vesicle counts from serial-section EM have been fundamental to quantifying these pools (Shepherd & Harris, 1998).

6. Active Zones

Active zones are the specialized presynaptic membrane domains where vesicle fusion occurs. In EM:

Electron-dense material: A fuzzy, electron-dense coating on the cytoplasmic face of the presynaptic membrane, directly opposite the postsynaptic PSD.
Vesicle docking: Vesicles are clustered at and docked to the active zone membrane.
Size: Typically 200-500 nm in diameter (en face), matching or slightly smaller than the opposing PSD.
Molecular composition: RIM, Munc13, RIM-BP, ELKS, and liprin-alpha proteins form the active zone scaffold (not directly visible in conventional EM but demonstrated by immuno-EM).
Number per bouton: Small cortical boutons typically have one active zone; large terminals may have multiple (the calyx of Held has approximately 600).

7. Dense-Core Vesicles

In addition to the small, clear synaptic vesicles that contain classical neurotransmitters (glutamate, GABA), some boutons contain dense-core vesicles (DCVs):

Size: 80-120 nm diameter, distinctly larger than clear vesicles (35-50 nm).
Appearance: A dark, electron-dense core surrounded by a clear halo and a vesicle membrane. The dense core contains the packaged neuropeptide or monoamine.
Contents: Neuropeptides (substance P, neuropeptide Y, enkephalins, BDNF) or monoamines (dopamine, norepinephrine, serotonin).
Distribution: Not concentrated at active zones like clear vesicles. DCVs are often found scattered throughout the bouton and may be released extrasynaptically through volume transmission.
Neuron-type specificity: Particularly abundant in monoaminergic neurons (locus coeruleus, raphe nuclei, ventral tegmental area) and peptidergic interneurons. Relatively rare in glutamatergic pyramidal neurons.
Annotation note: The presence of numerous DCVs in a bouton can help identify the presynaptic neuron type. A bouton with exclusively clear, round vesicles is likely glutamatergic. A bouton with pleomorphic vesicles and scattered DCVs may be from a peptide-co-releasing interneuron.

8. Worked Example: Identifying the Axon Initial Segment

Scenario: Tracing outward from a pyramidal neuron soma, you encounter two major processes. One tapers and contains Nissl substance; the other maintains uniform caliber and has a distinctive membrane undercoat.

Step-by-step identification:

Check for ribosomes: The tapering process contains scattered polyribosomes — it is a dendrite. The uniform-caliber process lacks ribosomes — candidate axon.
Look for dense undercoat: The candidate axon has a conspicuous electron-dense lining along the inner membrane, extending for approximately 40 micrometers from the soma. This is the AIS dense undercoat.
Check microtubule organization: Within the process, microtubules are bundled into tight fascicles rather than loosely distributed. This is characteristic of the AIS.
Look for axo-axonic synapses: Two symmetric synapses with pleomorphic vesicles are present on the process — consistent with chandelier cell inputs to the AIS.
Confirm: Dense undercoat + fasciculated microtubules + no ribosomes + axo-axonic synapses = axon initial segment.

9. Worked Example: Distinguishing an En Passant Bouton from a Dendritic Spine

Scenario: You see a small swelling (approximately 0.8 micrometers) associated with a synapse. Is it presynaptic (bouton) or postsynaptic (spine)?

Feature	En Passant Bouton	Dendritic Spine
Contains vesicle cluster	Yes — clustered at active zone	No vesicles (or very rare)
Contains PSD	No (the density is on the other side of the cleft)	Yes — thick electron-dense band on cytoplasmic face
Ribosomes	Absent	May have polyribosomes at base
Continuous with	An axon of uniform caliber	A tapering dendrite shaft
Mitochondria	Often present	Occasionally present in larger spines
Spine apparatus	Never	Sometimes (smooth ER stacks)

Decision process:

Which side has the vesicles? The vesicle-containing side is presynaptic (bouton).
Which side has the thick PSD? The PSD side is postsynaptic (spine or shaft).
Trace connections in adjacent sections to confirm continuity with parent processes.

10. Common Misconceptions

Misconception	Reality
“Axons are always thinner than dendrites.”	Myelinated axons can be several micrometers in diameter, much thicker than distal dendrites. The caliber comparison is not a reliable identification rule.
“All axons are myelinated.”	Many CNS axons are unmyelinated, especially those of local interneurons. In cortex, unmyelinated axons vastly outnumber myelinated ones.
“One bouton contacts one target.”	Multi-synapse boutons are common in cortex: a single bouton may form synapses with 2-3 different postsynaptic targets (Shepherd & Harris, 1998).
“Vesicles are randomly distributed in the bouton.”	Vesicles are organized into functionally distinct pools with specific spatial relationships to the active zone.
“Dense-core vesicles are released at active zones.”	DCVs are often released at non-active-zone sites on the bouton membrane, contributing to volume transmission rather than point-to-point synaptic signaling.
“The AIS is just a bare patch of membrane.”	The AIS has a highly organized molecular architecture — dense undercoat, fasciculated microtubules, clustered ion channels — that is visible in EM.
“Axons never have mitochondria.”	Axons contain mitochondria, though they are smaller and sparser than in somata. Boutons frequently contain mitochondria to support the energy demands of vesicle cycling.

References

Peters A, Palay SL, Webster HdeF (1991) The Fine Structure of the Nervous System, 3rd edition. Oxford University Press.
Rizzoli SO, Betz WJ (2005) “Synaptic vesicle pools.” Nature Reviews Neuroscience 6:57-69.
Leterrier C (2018) “The axon initial segment: an updated viewpoint.” Journal of Neuroscience 38:2135-2145.
Shepherd GMG, Harris KM (1998) “Three-dimensional structure and composition of CA3-CA1 axons in rat hippocampal slices.” Journal of Neuroscience 18:8300-8310.
Rasband MN (2010) “The axon initial segment and the maintenance of neuronal polarity.” Nature Reviews Neuroscience 11:552-562.
Kole MHP, Stuart GJ (2012) “Signal processing in the axon initial segment.” Neuron 73:235-247.
Harris KM, Weinberg RJ (2012) “Ultrastructure of synapses in the mammalian brain.” Cold Spring Harbor Perspectives in Biology 4:a005587.

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