DURHAM, N.C. -- Infinitesimal particles of gold have enabled
neurobiologists to track down key molecules in the machinery of
"entry points" in neurons -- offering clues to the organization
of a region that has thus far remained largely unknown neuronal
territory.

The researchers -- from Duke University Medical Center and
the University of North Carolina -- used electron microscopy to
locate molecules tagged with targeted antibodies attached to
gold particles -- rendering the molecules' precise location
visible.

The findings by the researchers, led by Michael Ehlers,
M.D., of Duke and Richard Weinberg, Ph.D., of the University of
North Carolina at Chapel Hill, were published online Aug. 22,
2004, in the journal Nature Neuroscience. Other co-authors are
Bence Rácz, Ph.D., of UNC and Thomas Blanpied, Ph.D., of Duke.
The research was sponsored by the National Institutes of
Health, the National Alliance for Research on Schizophrenia and
Depression, Christopher Reeve Paralysis Foundation and the
Broad Foundation.

Their studies aimed to understand how receptors on the
surface membranes of nerve cells undergo a recycling process
called endocytosis, in which the receptors are drawn into the
interior of the neurons to be recycled.

These receptors are proteins that are activated by bursts of
signaling chemicals, called neurotransmitters, launched from
another, transmitting neuron. Such activation triggers a nerve
impulse in the receiving neuron. Changes in the strength of a
neuron's response to such chemical signals depend on the number
of receptors on the dendritic spine surface. And the strength
of such connections is key to establishing the neural pathways
through the brain that are the basis of learning and
memory.

The neurotransmitter "receiving stations" on the neuron are
mushroom-shaped dendritic spines that festoon its surface. The
signaling regions between neurons are known as synapses, and
the receiving membrane on the dendritic spine is known as the
postsynaptic membrane.

A key mystery about dendritic spines, said Ehlers, has been
where on their surface such recycling of receptors takes
place

"It has been known for some time that signal reception takes
place in a small region of the spine membrane known as the
postsynaptic density," he said. "But the postsynaptic density
comprises only 15 percent of the membrane area. What happens in
the remaining 85 percent of the spine's membrane has been
almost completely unknown," he said.

"One way that connections in our brains are weakened is by
removing receptors from synapses, but where this removal occurs
has been unclear. Defining this 'microanatomy' of dendritic
spines is thus quite fundamental to understanding how neural
connections are formed and restructured as our brains develop,
change, and age," he said.

According to Ehlers, it has been believed that receptors to
be recycled "uncouple" from the postsynaptic density and move
across the fatty membrane to an "endocytic zone." In this
unidentified zone, molecular machinery attaches to the
receptor, draws it into a bubble-like vesicle and transports it
to machinery where it is either recycled or destroyed.

To attempt to map such zones, the researchers decided to
trace the precise location of three key molecules known to play
central roles in endocytosis:

-- clathrin, the protein that stitches together like a
soccer ball to create the vesicle that buds from the
membrane,

-- AP-2, the adaptor molecule that grabs onto receptor cargo
and attaches it to clathrin, and

-- dynamin, the protein that drives the machinery that
pinches the vesicle off from the cell membrane, freeing it to
travel to the recycling machinery.

The researchers attached gold particles to antibodies that
specifically targeted each of these proteins, and used electron
microscopy to search for these molecules in rat brain tissue.
Their tracking revealed that each of the molecules concentrates
in specific lateral zones of the spines.

"If you think of the spine as a roughly spherical structure
with the synapse at 12 o'clock, we found that these endocytic
molecules concentrate at zones at 3 o'clock and 6 o'clock,"
said Ehlers. He said that these concentrations mark the spots
at which the membrane is internalized by endocytosis and the
receptors drawn in. And even when the spines are larger or
smaller, the distances expand or shrink so the zones stay at
the same relative positions.

"While we still don't fully understand how this zone is
established or how molecules move through this zone into the
cell interior, with these findings, we are beginning to see a
level of organization that we didn't know existed," said
Ehlers.

"These findings imply a hidden level of organization on the
dendrite that's yet to be revealed," said Ehlers. "This
specialized endocytic zone is only the second known membrane
specialization in dendritic spines."

What's more, he said, the existence of zones in the
postsynaptic membrane mirrors a similar organization known to
exist on the "presynaptic" terminals on the transmitting
neurons that launch bursts of neurotransmitter.

Ehlers also said that the findings of organization on
dendritic spines could have broader implications in
understanding signaling between nerve cells.

"It's well known that many kinds of receptors -- not just
neurotransmitter receptors -- undergo downregulation by
endocytosis," said Ehlers. "These include receptors involved in
learning and memory, tolerance to medications, or reactions to
drugs of abuse. So, I think our findings regarding the spatial
organization of endocytosis will be relevant in understanding a
wide range of such processes."