Focus October 20-Neurobiology

Neurobiology:

VISUALIZING THE SYNAPTIC CONNECTION



Wade Regehr



The transfer of information from one neuron to the next is a fascinating feat, a message system that relies on an electrical signal releasing a packet of chemical messengers-called neurotransmitters-that travel across the synaptic gap to the receiving neuron. The synapse, which changes constantly, is fundamental to the relay of information throughout the central nervous system.

However, scientists have long been thwarted in directly examining many aspects of the signaling in the mammalian brain due to the tiny size of the synaptic boutons, the club- shaped enlargements on the terminal ends of neurons. But now Wade Regehr, an assistant professor of neurobiology, has developed a novel optical technique for "seeing" the chemical activity that drives this message system, particularly the changing levels of calcium ions within the presynaptic neuron. It is this release of calcium ions and their staying power at the synapse that governs the "strength" of the neurotransmitter message.

Regehr uses fluorescence-microscopy techniques to view and discover changes in presynaptic calcium ions. Using a dye that alters its fluorescence properties when it binds to calcium, he can detect calcium levels and the influx of calcium within the presynaptic neuron. He has used this technique to study synaptic activity in two regions of rat brain, the cerebellum and hippocampus.

"It's presynaptic calcium that is the key player in many of these events," he explains. "Visualizing what goes on at the presynaptic side gives us a new way of looking at synaptic function."

To learn how calcium ions control neurotransmitter release at the presynaptic site, Regehr has focused his research on the synapse between two types of neurons in the rat cerebellum: the granule cell and the Purkinje cell. He and his co-workers, Isabelle Mintz and Bernardo Sabatini, published their most recent results in the September issue of Neuron.

Regehr chose to study the cerebellum as a model synaptic system because of its anatomical design, which he describes as "the most beautiful structure you can imagine. It has very stereotypic architecture, almost like a crystalline array of Purkinje cells." Such a "floor plan" helps Regehr investigate calcium's role in synaptic activity.

"We're now able to look at what's going on pre- and postsynaptically in a synapse in the mammalian brain."
-- Wade Regehr

Granule cells in the rat brain extend their axons outward from the granule layer of the cerebellum into the adjoining molecular layer. There they bifurcate and stretch out towards their target-the dendrites of Purkinje cells located in the molecular layer. The axons extend parallel to the layer's edge, forming a series of fibers. These parallel fibers contain closely spaced presynaptic boutons that contact Purkinje cells with a high level of frequency. Regehr uses fluoresence microscopy to observe calcium influx down this axonal roadway.

To do his research, Regehr manipulates the release of calcium ions in granule cells by stimulating the cells' axons electrically. The fluorescence of the calcium-binding dye enables him to detect the calcium ions as they enter the presynaptic boutons. He then calculates the calcium influx into the terminal by using the change in calcium levels and measures the amplitude of the resulting electrical current in the postsynaptic terminal of the Purkinje cell.

He has found that calcium ions enter the presynaptic terminals through different types of calcium channels. The greater the inflow of calcium ions, the greater the electrical current in the postsynaptic cell. In fact, doubling the calcium influx yields almost six times the electrical signal in the postsynaptic cell, he says.

Regehr next posed the question of whether all the different types of calcium channels in presynaptic terminals participate in synaptic transmission.

To answer this question, Regehr used three agents-cadmium, which is very similar in size to calcium, and two toxins-to block the channels and prevent the calcium entry. In a series of experiments, Regehr bathed the neurons in a cadmium-ion solution separately. He applied one toxin individually, followed by both together, first in one arrangement and then in the reverse. By applying the different baths, Regehr effectively shut down those classes of channels sensitive to these agents.

Again using the dye, he tracked the altered calcium influx. He found that while a sole toxin could shut down certain channels, it never blocked all channels. Calcium ions continued to circulate, indicating that the channels had different sensitivities to the toxins. Regehr concluded that at least three classes of calcium channels exist in the presynaptic terminal-a class sensitive to each toxin and one sensitive to neither-and that they differ from one another pharmacologically. Each type contributes to neurotransmitter release.

This discovery opens the door for possible pharmacologic treatments that might be used to deactivate particular calcium channels in the brain. And if scientists can also learn how each channel type contributes to specific brain functions, then drug treatments might be developed that would be very specific in their effect on brain function.

"If you can modulate each one of the calcium channels, there is a great deal of potential for control," Regehr says.

In addition to his research on synaptic activity in the cerebellum, Regehr is investigating the role of calcium ions in the presynaptic terminal of the rat hippocampus, the section of the brain involved in both long-term memory and in short-term memory lasting from tens of seconds to one or two minutes.

"What we think is going on is that you get a build-up of calcium in these structures, and calcium acts to enhance the strength of transmission from one cell to another," he says. "As long as there's an elevation of calcium, there's a stronger connection. So, in a sense, presynaptic calcium is playing a role in the processes we think are the physiological correlates of short-term memory."

Regehr sees his research as helping to improve our basic knowledge about how the brain works. The visualization method he has developed for observing the activity of cerebellar synapses should have application for studying the activity of synapses in many other parts of the brain as well.

"We're taking a bottom-up approach, looking at how cells communicate with each other and how concentrations of calcium ions change their communication strengths. We hopefully will [eventually] take it up to another level and look at how circuits of neurons can perform certain functions," he says.

--Colleen Sauber