Cold Spring Harbor, NY, June 15, 1999 -- A team of researchers at Cold Spring Harbor Laboratory (CSHL) and the National Institute of Health (NIH), in Bethesda, Md., report in the June 11 issue of Science that receptors for the excitatory neurotransmitter glutamate are rapidly transported when nearby nerve cells are stimulated. The unleashed receptors move from inside nerve cells to the surfaces of dendritic spines, tiny protrusions from brain neurons, where synapses are most common. And because synapses are the junctions for communication among nerve cells, glutamate receptor delivery to synaptic sites may be a key molecular event for the increased synaptic transmission that occurs during learning and memory.
"People have been trying to understand how synapses strengthen for a long time," says Roberto Malinow of CSHL. "This paper presents a new model for the postsynaptic changes that are important for long-term potentiation (LTP)." This long-lasting increase in synaptic transmission is thought to underlie the formation of new memories, particularly in the hippocampus, an evolutionarily primitive part of the cerebral cortex. Many researchers also find that dendritic spines-stubby, mushroom-shaped projections from nerve cell branches called dendrites-are frequently the sites of synaptic contact between incoming nerve axons from other brain regions and nerve cells that reside in the hippocampus.
The new studies are important because they represent the first direct observation of the movement of key neurotransmitter receptors to probable sites of synaptic contact during the kind of neural stimulation that simulates learning in a live animal.
In their recent experiments, Malinow, Karel Svoboda, also of CSHL, and their colleagues observed the delivery to dendritic spines of a component of one kind of glutamate receptor, called AMPA receptors (for a-amino-3-hydroxy-5-methyl-4-isoxazole propionate). Specifically, the researchers found that the redistribution of the AMPA receptor subunit GluR1 (glutamate receptor subunit 1) requires the activation of a second kind of glutamate receptor called NMDA receptors (N-amino-D-aspartate). Among neuroscientists who study learning and memory, NMDA receptors have become notorious; many argue that their activation is required for inducing LTP and forming certain kinds of memories.
The new findings provide a possible mechanism by which AMPA receptors in postsynaptic neurons also contribute to synaptic strengthening. Stimulation of presynaptic neurons, which release glutamate as a neurotransmitter, may trigger the delivery of AMPA receptors to synaptic sites on postsynaptic neurons, thereby providing more places for glutamate to bind to and excite the cells.
"Seeing is believing," says Svoboda. "You have glutamate receptors tagged with a label and can actually see them move, which is what people imagined must happen during LTP. But you don't believe it until you see it."
To study the rapid deployment of glutamate receptors to dendritic spines following synaptic stimulation, Malinow, Svoboda, and their colleagues maintained slices of hippocampus from neonatal rats in long-term culture. They visualized the movement of AMPA-type glutamate receptors by tagging GluR1 receptors with green fluorescent protein (GFP). Yasunori Hayashi, a postdoc in Malinow's lab, helped to fuse the genes encoding GFP and GluR1 in a way that allowed them to be incorporated into the non-pathogenic Sindbis virus and injected into cultured hippocampal slices. Neurons that became infected with the genetically altered virus then synthesized the glutamate receptor subunit tagged with the brightly fluorescent protein.
Two to three days later, Song-Hai Shi, a graduate student in Malinow's lab from the State University of New York at Stony Brook (SUNY Stony Brook), worked with Svoboda to use time-lapse, two-photon laser scanning microscopy to see the labeled glutamate receptors in the living nerve cells. (Svoboda developed the high-tech imaging system and used it, in another collaboration with Malinow and Mirjana Maletic-Savatic of SUNY Stony Brook, to study the growth of dendritic filopodia from hippocampal neurons. They published those results in the March 19, 1999, issue of Science.)
Initially, in unstimulated neurons of the CA1 region of the hippocampus, most (88 percent) of the tagged glutamate receptors were distributed intracellularly throughout the shafts of dendrites. The researchers used two methods to observe the initial, even distribution of receptors: two-photon laser imaging of the fluorescence-tagged GluR1, and electron microscopy of immuno-gold labeled receptors, a series of experiments done by collaborators Ronald Petralia and Robert J. Wenthold of the National Institutes of Health.
But within 15 minutes after the researchers delivered a brief, high-frequency stimulus to nearby nerve cell axons, the fluorescently labeled glutamate receptors redistributed in two ways. Some moved to the surface membranes of dendritic spines and others formed clusters within the shafts of dendrites near the bases of spines. The clusters, says Malinow, may represent "a local reserve of glutamate receptors" that will be delivered to the spines over time to maintain a greater number of receptors at synaptic sites.
Another paper in the June 11, 1999, issue of Science by Peter Seeburg and Bert Sakmann of the Max-Planck Institute for Medical Research in Heidelberg, Germany, and their collaborators, and a third paper in the May issue of Nature Neuroscience, also describe molecular changes that are important for LTP. A news article by Marcia Barinaga in Science details the collective impact of the three papers on this field of research.
"We have found this phenomenon," says SUNY Stony Brook grad student Song-Hai Shi about the rapid delivery of glutamate receptors to dendritic spines after high-frequency stimulation. "Now the next step is to dissect the mechanism."
"We really want to be able to say that glutamate receptor movement contributes to an increased synaptic signal during LTP," says Malinow. He and his colleagues want to show that if AMPA-type glutamate receptors are delivered to active synapses, their delivery makes the synapses stronger. Or, if no synapses preexist at those sites, the researchers want to demonstrate that the arrival of the glutamate receptors creates an active synapse.
Cold Spring Harbor Laboratory is a private, non-profit basic research and educational institution with programs focusing on cancer, neurobiology, and plant biology. Its other areas of research expertise include molecular and cellular biology, genetics, structural biology, and bioinformatics. Starting in the fall of 1999, the Laboratory's new Watson School of Biological Sciences will offer an innovative Ph.D. program for a small group of exceptional students. Located on the north shore of Long Island, 35 miles from Manhattan, the Laboratory was founded in 1890 as a field station for the study of evolution. Today, the Laboratory is headed by Director Bruce Stillman and President James D. Watson.
For more information, visit the Laboratory's Web site at http://www.cshl.edu, or call the Office of Public Affairs at 516-367-8455.