June 05, 2003
"Kiss-and-Run" Rules the Inner Lives of Neurons
Neurons transmit chemical signals in a fleeting
“kiss-and-run” process, which in large part determines how
quickly neurons can fire, according to new studies by Howard Hughes
Medical Institute researchers.
The transfer of information between nerve cells occurs when
chemicals called neurotransmitters are released into the synapse, the
junction between neurons. Electrical impulses in the neuron cause tiny
vesicles loaded with neurotransmitters to move to the tip of the nerve
terminal where they are released.
“The advance that we have made is to figure out a way of imaging individual vesicles so that we can measure the time course of single-vesicle events.”
Charles F. Stevens
In an article published in the June 5, 2003, issue of the journal
Nature, HHMI investigator Charles
F. Stevens and Sunil Gandhi, both at The Salk Institute, reported
that they have devised a technique that permits them to visualize
individual vesicles after they have released their cargo. The new
findings are significant, said the researchers, because they answer
questions about the rate at which synaptic vesicles can be recycled.
This rate determines how much information nerve cells can transmit.
Stevens and Gandhi have identified three distinct ways in which a
used vesicle can be retrieved from the surface of the nerve cell once
it has released its cargo. The fastest of these, called the
“kiss-and-run” mode, takes less than a second; the slower
“compensatory” mode takes up to 21 seconds; and the
“stranded” mode leaves the vesicle stuck at the surface
until the next nerve impulse triggers its retrieval.
According to Stevens, the latest findings settle lingering questions
about how vesicle retrieval occurs. Early electron microscopy images of
vesicles in synapses were interpreted as either a kiss-and-run model or
one in which the vesicle is completely incorporated into the cell
membrane, to be drawn back into the cell.
“The advance that we have made is to figure out a way of
imaging individual vesicles so that we can measure the time course of
single-vesicle events and immediately answer these questions,”
said Stevens.
The optical recording technique devised by Stevens and Gandhi
involves genetically modifying a gene for one type of vesicle protein
to incorporate a special form of green fluorescent protein. This
modified fluorescent protein, developed by other researchers, does not
fluoresce under acidic conditions normally present in vesicles fully
loaded with neurotransmitter. However, when the vesicle releases its
payload, the interior becomes less acidic and the vesicle glows a
bright green.
Thus, said Stevens, by imaging individual vesicles in cell cultures
of neurons, it is now possible to detect how and when vesicles release
their cargo at the synaptic membrane.
“Among the minor observations we made was that vesicles can
re-acidify themselves in less than half a second,” said Stevens.
“We also observed that the proteins in the vesicle are maintained
together, so that when a vesicle is taken back in from the membrane,
the same proteins are still there, even if the vesicle had been fused
with the membrane for quite a while.
“And the third thing that was surprising is that all vesicles
across different preparations have basically the same number of these
tagged protein molecules,” said Stevens. “This means that
they are either saturated or there is some mechanism for counting the
proteins.”
The major observations from their studies, said Stevens, are that
are three modes of vesicle release and retrieval from the membrane.
“One is what you could call classical, when the vesicle opens to
the outside world, stays open for about eight seconds, and then is
taken back in at random times extending out to twelve or fourteen
seconds,” he said. This finding confirms previous theories about
modes of vesicle recycling, he said.
“However, sometimes if the vesicle failed to be
re-internalized to be reused again by about fourteen or fifteen
seconds, sometimes it got stuck there,” said Stevens. In this
“stranded” mode, the vesicle remained stuck until another
nerve impulse caused it to be zipped into the interior of the neuron to
be recycled. Presumably, stranding occurs because vesicle recycling
depends somehow on the level of calcium in the nerve cell, which rises
precipitously during a nerve impulse, and drops afterward, said
Stevens.
“The third recycling mode we observed was a kiss-and run-mode
that happened very rapidly, in less than half a second,” said
Stevens. “Also, we showed experimentally that in this mode there
was a `fusion pore' formed where the vesicle contacted the
membrane,” he said.
Stevens and Gandhi also found that vesicles appear to adjust their
mode of recycling based on the probability that a given synapse will
trigger the release of a vesicle's cargo. Vesicles in synapses with a
low-release probability are more likely to use the rapid kiss-and-run
mode, he said, while those vesicles in a higher-probability synapse use
the slower compensatory mode.
Future studies will seek to determine the molecules responsible for
recycling and how structures such as the fusion pore form. The
researchers will also explore the role of calcium in recycling, as well
as the advantages to the nerve cell of using the kiss-and-run recycling
mode.
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