Neurons communicate across synapses. Scientists at the Picower Institute for Learning and Memory at MIT sought to determine how neurons sustain their capacity to set up and sustain this communication. Their new study in fruit flies provide revelations about how neurons set up and sustain this vital infrastructure.
Their findings are published in the journal eLife in a paper titled, “Regulation of presynaptic Ca2+ channel abundance at active zones through a balance of delivery and turnover.”
“Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs),” wrote the researchers. “The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery, and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here we describe how the AZ levels of Cacophony (Cac), the sole VGCC mediating synaptic transmission in Drosophila, are determined.”
“Calcium channels are the major determinant of calcium influx, which then triggers vesicle fusion, so it is a critical component of the engine on the presynaptic side that converts electrical signals to chemical synaptic transmission,” said Troy Littleton, senior author of the new study and Menicon professor of neuroscience in MIT’s departments of biology and brain and cognitive sciences. “How they accumulate at active zones was really unclear. Our study reveals clues into how active zones accumulate and regulate the abundance of calcium channels.”
The more scientists knocked out a protein called alpha2delta with different manipulations (right two columns), the less Cac calcium channel accrued in synaptic active zones of a fly neuron (brightness and number of green dots) compared to unaltered controls (left column).
“Modulation of the function of presynaptic calcium channels is known to have very important clinical effects,” Littleton said. “Understanding the baseline of how these channels are regulated is really important.”
Using the model system of fruit fly motor neurons, the researchers employed a wide variety of techniques and experiments to show for the first time the step by step process that accounts for the distribution and upkeep of calcium channels at active zones.
Using another technique that artificially prolongs the larval stage of the fly the researchers were also able to see that given extra time the active zone will continue to build up its structure with a protein called BRP, but that Cac accumulation ceases after the normal six days.
“It was revealing that the neuron had very different rules for the structural proteins at the active zone like BRP that continued to accumulate over time, versus the calcium channel that was tightly regulated and had its abundance capped,” Karen Cunningham, a graduate student in Littleton’s lab, said.
The findings showed there must be factors other than Cac supply or changes in BRP that regulate Cac levels so tightly. Cunningham turned to alpha2delta. When she genetically manipulated how much of that was expressed, she found that alpha2delta levels directly determined how much Cac accumulated at active zones.
In further experiments, Cunningham was also able to show that alpha2delta’s ability to maintain Cac levels depended on the neuron’s overall Cac supply. That finding suggested that rather than controlling Cac amount at active zones by stabilizing it, alpha 2delta likely functioned upstream, during Cac trafficking, to supply and resupply Cac to active zones.
Littleton said his lab is eager to build on these results. Now that the rules of calcium channel abundance and replenishment are clear, he wants to know how they differ when neurons undergo plasticity.