Scientists at the University of California, San Diego have produced dramatic images of brain cells forming temporary and permanent connections in response to various stimuli, illustrating for the first time the structural changes between neurons in the brain that, many scientists have long believed, take place when we store short-term and long-term memories.
In a paper published in the November 30 issue of the journal Cell, researchers from UCSD's Divisions of Biology and Physical Sciences describe their achievement, a "Holy Grail" for neuroscientists who have long sought concrete evidence for how nerve connections in the brain are changed temporarily and permanently by our experiences.
"The long-term memories stored in our brain last our entire lives, so everybody had assumed that there must be lasting structural changes between neurons in the brain," says Michael A. Colicos, a postdoctoral fellow at UCSD and the lead author of the paper. "Although there's been a lot of suggestive evidence to indicate that this is the case, it's never before been directly observed."
"While most people assumed that some sort of rearrangement of nerve cell connections took place in the brain, this was extremely difficult to demonstrate experimentally," says Yukiko Goda, a professor of biology at UCSD who headed the research team, which included Michael J. Sailor, a professor of chemistry and biochemistry at UCSD, and Boyce E. Collins, a postdoctoral fellow in Sailor's lab. "Some investigators saw increases in the number of synapses in the brain in response to stimuli, while others saw no changes. There are a billion synapses in a cubic centimeter of brain tissue, so no one could tell for certain whether the statistical comparisons of synapse density between one sample and another showed a real increase."
To resolve this problem, the UCSD researchers focused their attention on individual nerve cells, specifically neurons from the hippocampus -- the portion of the human brain crucial to forming particular types of memory -- and filmed them as their synapses made new connections to other nerve cells in response to electrical impulses.
The ability of the scientists to do this without impairing the normal physiological functions of the cells depended on two new techniques implemented in Goda's lab to study synaptic connections. One was a method of visualizing the rods and filaments of actin -- the girders that make up the cytoskeleton, the internal skeleton of the cell. Using molecular biology techniques, fluorescent versions of actin were constructed and visualized as the neurons grew and changed shape to establish new connections.
The second development, which resulted from a collaboration between Goda and Sailor, was a method of stimulating nerve cells in a manner that mimicked their stimulation in the brain. This involved using the "photoconductive" properties of silicon in a way that allowed the researchers to deliver a short, high frequency burst of electricity to a specific area of a neuron on a silicon chip by simply shining light on that area. Light excitation in that area of the silicon created a narrow pathway through which Colicos and his colleagues could apply a tiny voltage below the chip to target the neuron.
"We stimulate these cells with a short, high-frequency burst," says Colicos, working in Goda's lab. "That type of stimulation is what other researchers believed for many years was the type that formed these connections between neurons."
A key advantage of this method is that it doesn't damage the cell. "Part of the reason people haven't been able to demonstrate this before is that the technology hasn't been available to do this before," says Colicos. "The standard way of stimulating a neuron is to use an electrode. But as soon as you stab the cell with an electrode, it begins to die. So the advantage of this new technique is that we can keep the cells in their physiologically normal state. And when we stimulate the cells of our choice by shining light, we can induce the actual structural changes that occur in the brain -- the formation of these new synapses."
In their experiments, the UCSD researchers discovered that when they stimulated a cell once, the actin inside the cell was activated and temporarily moved toward neurons to which they were connected. The activity in the first cell also stimulated the movement of actin in neighboring neurons, which moved away from the activated cell. Those changes in the cells were temporary, however, lasting for about three to five minutes and disappearing within five to 10 minutes.
"The short-term changes are just part of the normal way the nerve cells talk to each other," says Colicos. "The long-term changes in the neurons occur only after the neurons are stimulated four times over the course of an hour. The synapse will actually split and new synapses will form, producing a permanent change that will presumably last for the rest of your life."
"The analogy to human memory is that when you see or hear something once, it might stick in your mind for a few minutes. If it's not important, it fades away and you forget it 10 minutes later. But if you see or hear it again and this keeps happening over the next hour, you are going to remember it for a much longer time. And things that are repeated many times can be remembered for an entire lifetime."
"It's like a piano lesson," says Goda. "If you play a musical score over and over again, it becomes ingrained in your memory."
In their experiments, which were financed in part by grants from the National Science Foundation and the National Institutes of Health, the researchers observed no changes in these newly formed nerve connections once they were established, indicating that they were permanent.
"Once you take an axon and form two new connections, those connections are very stable and there's no reason to believe that they'll go away," says Colicos. "That's the kind of change one would envision lasting a whole lifetime."
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