One Axon, Many Dendrites: Neuronal Polarity

Cut a neuron’s axon close to the cell body, and one of its dendrites can grow out as a new axon. Cut it farther away, and the old axon simply regrows.

That result tells you that “axon” is not a permanent label stamped on a process when it first sprouts. It is a state the cell keeps, carried by the axon itself, and it can be handed to a different process if enough of the axon is lost. This lesson asks how axons are formed, and then a second question: once formed, how does an axon stay an axon?

Special cell biology of the neuron: one cell, two kinds of process

Most neurons in a vertebrate brain have two kinds of process. Dendrites are usually several, short, branched and tapering; they receive input from other cells. The axon is usually single, of even width and often very long; it carries the neuron’s output, the action potential, to other cells. Polarity is the word for a cell having two distinct ends or domains that differ in structure and molecules. Some neurons break the pattern. Anaxonic neurons, such as some amacrine cells of the retina, have no clear axon, and many sensory neurons have a single process that splits into two axon branches.

The difference is molecular as well as visible. Dendrites contain the microtubule-associated protein MAP2, receptors for neurotransmitters, and, in many neurons, spines where synapses form. The axon contains other proteins, such as the microtubule-associated protein tau, and its own channels and vesicle machinery. A neuron must sort thousands of proteins to the right side and keep them there for decades. That is what makes the cell biology special.

Establishing polarity during development: a race among equal neurites

The clearest view of how a neuron polarizes comes from a dish. In 1988 Carlos Dotti, C. A. Sullivan and Gary Banker followed single neurons taken from the embryonic rat hippocampus and grown at low density. They described five stages.

1. Lamellipodia. Within hours of plating, the round cell spreads thin, flat veils of membrane.
2. Minor neurites. The veils condense into several short neurites, all alike.
3. The axon. After a day or two, one neurite starts to grow much faster than the rest. It becomes the axon.
4. Dendrites. Over the next few days the other neurites grow, taper and branch as dendrites.
5. Maturation. In the second week and later, dendritic spines and synapses form. (Synapses get their own lesson later: that lesson.)

Notice the logic of stage 2. The neurites look equal, and Dotti and Banker had shown in 1987 that cutting the axon of a young neuron within a day of its appearance can let a different neurite become the axon. Goslin and Banker later reported that a neurite tends to become the axon once it is roughly 10 micrometres longer than the others. The choice behaves like a race.

A race with one winner needs two kinds of feedback. Positive feedback in the leading neurite makes it grow faster the more it grows. Negative feedback holds the others back. The players reported in cultured neurons are these:

• PI3-kinase and PIP3. PI3-kinase makes the lipid PIP3 in the membrane, which proteins with PH domains read as an address (see that lesson and that lesson). PIP3 builds up at the tip of the future axon, and blocking PI3-kinase is reported to stop any neurite from becoming an axon.
• The Par3/Par6/aPKC complex. The same polarity complex that marks the top of an epithelial cell gathers at the tip of the future axon.
• Rho-family GTPases. Cdc42 and Rac, molecular switches on when bound to GTP (that lesson), reorganize actin at the tip, while RhoA tends to oppose neurite growth (that lesson).
• Actin instability and microtubule stabilization. Frank Bradke and Dotti reported in 1999 that the growth cone of the future axon has a looser, more dynamic actin network. A growth cone is the motile tip of a growing neurite (that lesson). Loosened actin is proposed to let microtubules push in, and microtubules in the future axon are more stable than in its siblings. Locally loosening actin with cytochalasin D, or locally stabilizing microtubules with taxol, can make a chosen neurite the axon.

Part of the loop is thought to be physical: a neurite that grows faster draws more building material and more transport, and then grows faster still. How the losers are held back is less settled. Proposals include competition for a limited supply of material and a spreading inhibitory signal.

One caution. These five stages describe dissociated neurons in culture, which have lost their neighbours and their history. In the brain many neurons arrive with polarity already set by their migration. In the developing cortex, for example, the trailing process of a migrating neuron often becomes its axon. The molecules overlap, but the starting point differs.

Development of microtubule polarity: the tracks point different ways

A microtubule has a fast-growing plus end and a slower minus end. Motor proteins read that direction. Most kinesins walk toward plus ends; dynein walks toward minus ends. So the orientation of microtubules in a process decides which motors can carry cargo into it.

In 1988 Peter Baas and colleagues used hook decoration to score microtubule orientation in cultured rat hippocampal neurons. In the axon, nearly all microtubules point their plus ends away from the cell body. In dendrites the orientation is mixed: a large fraction point each way. In fly neurons, dendrites are instead mostly minus-end-out, so the details depend on the animal. In both, axon and dendrites offer different tracks.

The difference gives the neuron a sorting rule. Cargo carried by a kinesin that prefers stable plus-end-out microtubules can be steered into the axon. Cargo carried by dynein can travel outward only on minus-end-out tracks, which vertebrate dendrites have and the axon lacks. Mixed tracks in dendrites appear as the dendrites mature, so this rule is set up after the axon is chosen and helps keep it.

Setting up the axon initial segment: a gate that holds identity

The first few tens of micrometres of the axon form a specialized zone, the axon initial segment (AIS). Under its membrane sits a dense coat built on the scaffold ankyrin G. Ankyrin G binds beta-IV spectrin, which links it to rings of actin under the membrane. Through its ANK repeats it binds voltage-gated sodium channels, KCNQ2/3 potassium channels, and the adhesion molecules neurofascin-186 and NrCAM. Kv1 potassium channels gather there too.

The AIS has two jobs. The first is electrical. Its high density of sodium channels gives it a low threshold, so in many neurons it is where inputs summed from the dendrites and cell body start the action potential. The second is to hold polarity.

The AIS may also be old. Comparative sequence work has been read as suggesting that the ankyrin-G binding motif on sodium channels arose in early chordates, and that the motif on KCNQ2/3 channels arose later, around the time myelin appeared, though that dating rests on which animals have been sequenced.

How does the AIS hold polarity? In 1999 Bettina Winckler, Paul Forscher and Ira Mellman dragged membrane proteins with a laser trap and found that they moved freely in the cell body and dendrites but were held back at the initial segment, and that the hold depended on actin. Several models of the barrier are proposed and may all contribute: a fence of anchored membrane proteins that slows the diffusion of lipids and proteins; an actin-based filter that blocks vesicles carrying dendritic cargo; and sorting by motors that read microtubule orientation. Which matters most is still argued.

Ankyrin G is required to maintain neuronal polarity. In 2008 Kristian Hedstrom, Yasuhiro Ogawa and Matthew Rasband lowered ankyrin G in cultured hippocampal neurons after the AIS had formed. The AIS fell apart, and MAP2, normally excluded, spread into the axon. The axon began to look like a dendrite, with dendritic spines. The axon was already chosen when the scaffold was removed, so the experiment tests maintenance, not choice: identity has to be actively held.

The cut-axon result in the opening points the same way. In a 2008 study from Frank Bradke’s laboratory, with Susana Gomis-Rüth and Corette Wierenga, cutting the axon so that a stump shorter than 35 micrometres remained let a dendrite become the axon, while longer stumps regrew. The authors linked the difference to stable microtubules in the distal axon and did not test the initial segment. Myelin and nodes of Ranvier, which repeat much of the AIS machinery along the axon, come in that lesson.

Design problem

You grow rat hippocampal neurons until they are fully polarized. In one neuron you cut the axon less than 35 micrometres from the cell body. In another you cut it farther than 35 micrometres from the cell body. Predict what grows out in each case, and say what the pair of results tells you about where axon identity is stored.