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How neurons create and maintain their ability to communicate

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Summary: Researchers are uncovering how neurons create and maintain the vital infrastructure that ensures uninterrupted neurotransmission.

Source: Picower Institute for Learning and Memory

The nervous system works because neurons communicate through connections called synapses. They “talk” as calcium ions flow through channels into “active zones” filled with vesicles carrying molecular messages.

The electrically charged calcium causes the vesicles to “fuse” with the outer membrane of presynaptic neurons, releasing their communicative chemical cargo into the postsynaptic cell.

In a new study, scientists at MIT’s Picower Institute for Learning and Memory have made several discoveries about how neurons create and maintain this vital infrastructure.

“Calcium channels are the main determinant of calcium influx, which then triggers vesicle fusion, so they are a critical component of the mechanism on the presynaptic side that converts electrical signals into chemical synaptic transmission,” said Troy Littleton, senior author of the new study. in electronic life and Menicon, Professor of Neurology in the Departments of Biology, Brain, and Cognitive Sciences at MIT.

“How they accumulate in cores was really unclear. Our study provides insight into how active zones accumulate and regulate the number of calcium channels.”

Neuroscientists wanted these clues. One reason is that understanding this process can help understand how neurons change the way they communicate, a capability called “plasticity” that underlies learning, memory and other important brain functions.

Another thing is that drugs like gabapentin, which treats conditions as diverse as epilepsy, anxiety and neuralgia, bind to a protein called alpha-2-delta, which is closely associated with calcium channels. By revealing more about the exact function of alpha2delta, the study better explains what these treatments are impacting.

The more scientists knocked out a protein called alpha-2-delta using various manipulations (two columns on the right), the less calcium channels Cac accumulated in the synaptic active zones of the fly neuron (brightness and number of green dots) compared to unmodified controls (left column) .

“Modulating the function of presynaptic calcium channels is known to have very important clinical effects,” Littleton said. “Understanding how these channels are regulated is really important.”

MIT postdoctoral fellow Karen Cunningham led the research, which was her doctoral thesis at Littleton’s lab. Using a model system of fruit fly motor neurons, she used a wide range of methods and experiments to show for the first time the step-by-step process that is responsible for the distribution and maintenance of calcium channels in active zones.

Cap on Cac

Cunningham’s first question was whether calcium channels are necessary for the development of active zones in larvae. The fly calcium channel gene (called “cacophony” or Cac) is so important that flies literally cannot live without it. So instead of knocking out Cac on the fly, Cunningham used a technique to knock it out in just one population of neurons. Thus, she was able to show that even without Cac, cores grow and mature normally.

Using another method that artificially prolongs the fly’s larval stage, she was also able to see that for additional time, the core would continue to build up its structure with a protein called BRP, but Cac accumulation would stop after the usual six days.

Cunningham also found that a modest increase in the amount of available Cac in a neuron did not affect how much Cac ended up in each active zone. Even more curiously, she found that while the amount of Cac did increase with the size of each core, it barely changed if she removed a lot of BRP in the core. Indeed, for each active zone, the neuron seemed to set a constant limit on the number of Cacs present.

“It turned out that the neuron had very different rules for structural proteins in the active zone, such as BRP, which continued to accumulate over time, compared to the calcium channel, which was tightly regulated and its abundance was limited,” Cunningham said.

Regular update

The team model shows the factors that regulate Cac abundance in active zones. The development of the Active Zone framework and the delivery of Cac via alpha2delta increases it, while staff turnover holds it back. Biosynthesis of Cac hardly increases the abundance.

The results indicated that there must be factors other than Cac intake or changes in BRP that regulate Cac levels so tightly. Cunningham turned to alpha2delta. When she genetically manipulated its expression, she found that alpha2delta levels directly determined how much Cac accumulated in active zones.

In further experiments, Cunningham was also able to show that the ability of alpha2delta to maintain Cac levels depends on the neuron’s overall supply of Cac. This discovery showed that instead of controlling the amount of Cac in the cores by stabilizing it, the alpha-2 delta likely functioned upstream during the transport of Cac to supply and replenish Cac in the cores.

Cunningham used two different methods to observe replenishment, measuring its scale and timing. She chose a moment after several days of development to photograph the active zones and measure the abundance of Cac to define the landscape. She bleached the Cac fluorescence to erase it.

After 24 hours, she re-imaged the Cac fluorescence to highlight only the new Cacs that had been delivered to the cores in those 24 hours. She saw that during that day CAC was delivered to almost all active zones, but the work in one day was really only a fraction of what had accumulated a few days before.

Moreover, she could see that more Cac accumulated in large cores than in smaller ones. And flies with mutated alpha2delta generally had very few new Cacs.

If the Cac channels were indeed constantly replenished, then Cunningham wanted to know how fast the Cac channels were being removed from the active zones.

The more scientists knocked out a protein called alpha-2-delta using various manipulations (two columns on the right), the less calcium channels Cac accumulated in the synaptic active zones of the fly neuron (brightness and number of green dots) compared to unmodified controls (left column) . Credit: Littleton Laboratory/Picower Institute, Massachusetts Institute of Technology

To determine this, she used Maple photoconvertible protein staining technology tagged with Cac protein, which allowed her to change color with a flash of light at a time of her choice. So she could first see how much Cac had accumulated in a certain amount of time (shown in green) and then flash a light to make Cac turn red.

When she returned five days later, about 30 percent of the red Cacs had been replaced with new green Cacs, suggesting a 30 percent turnover. When she reduced Cac delivery by alpha2-delta mutation or reduced Cac biosynthesis, Cac turnover ceased. This means that a significant amount of Cac is flipped in cores every day, and this turnover is driven by the delivery of new Cac.

Littleton said his lab is keen to use these results. Now that the rules for calcium channel abundance and replenishment are clear, he wants to know how they differ when neurons undergo plasticity—for example, when new incoming information requires neurons to regulate their communications by increasing or decreasing synaptic connections.

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He said he was also eager to track individual calcium channels as they form in the cell body and then travel down the nerve axon to active zones, and he wants to determine what other genes might influence Cac abundance.

In addition to Cunningham and Littleton, Chad Sauwola and Sara Tavana are the other authors of the article.

Financing: The National Institutes of Health and the JPB Foundation provided support for the study.

About these neuroscience research news

Author: David Orenstein
Source: Picower Institute for Learning and Memory
Contact: David Orenstein – Picower Institute for Learning and Memory
Image: Image courtesy of Littleton Lab/MIT Picower Institute.

Original research: Open access.
“Regulation of the number of presynaptic Ca2+ channels in active zones through a balance of delivery and turnover” Troy Littleton et al. electronic life


Abstract

Regulation of the number of presynaptic Ca2+ channels in active zones through a balance of delivery and turnover

Potential dependent Ca2+ channels (VGCC) mediate Ca2+ influx to trigger the release of the neurotransmitter at specialized presynaptic sites called active zones (AZs). The abundance of VGCC in the AZ regulates the likelihood of neurotransmitter release (PR), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery, and recycling interact to establish VGCC AZ abundance, experimental isolation of these distinct regulatory processes has been difficult.

Here we describe how levels of AZ cacophony (Cac), the only VGCC mediating synaptic transmission in Drosophilaare determined.

We also analyzed the relationship between Cac, the α2δ conserved regulatory subunit of VGCC, and the Bruchpilot AZ basic scaffold protein (BRP) in establishing functional AZ. We found that Cac and BRP are independently regulated in growing AZs, since Cac is essential for AZ formation and structural maturation, and BRP does not limit Cac accumulation. In addition, AZs stop accumulating Cac after the initial growth phase, while BRP levels continue to increase with increasing development time. AZ Cac is also protected from a modest increase or decrease in biosynthesis, while BRP does not have this buffering.

To explore the mechanisms that determine AZ Cac abundance, in vivo FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery is widespread in the AZ population, correlates with AZ size, and is rate-limited by α2δ.

Although Cac does not undergo significant lateral transfer between neighboring AZs during development, removal of Cac from AZs does occur and is stimulated by the delivery of new Cacs, generating a limitation of Cac accumulation in mature AZs.

Together, these findings show how Cac biosynthesis, synaptic delivery, and recycling determine the abundance of VGCCs in individual AZs throughout synaptic development and maintenance.

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