How Neurons Build and Maintain Their Capacity to Communicate - Neuroscience News

Summary: Researchers reveal how neurons organize and maintain the vital infrastructure that enables seamless nerve transmission.

Source: Picower Institute of Learning and Memory

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

The electrically charged calcium causes the vesicles to “fuse” to the outer membrane of the presynaptic neuron, releasing its communicative chemical charge to the postsynaptic cell.

In a new study, scientists at The Picower Institute for Learning and Memory at MIT provide some revelations about how neurons organize and maintain this vital infrastructure.

“Calcium channels are a major determinant of calcium influx, which then triggers vesicle fusion, so they are important components of the machinery on the presynaptic side that converts electrical signals into chemical synaptic transmissions,” said Troy Littleton, senior author of the new study. in eLife and Menicon Professor of Neuroscience in MIT’s Departments of Biology and Brain and Cognitive Sciences.

“How they stack up in the active zone is really unclear. Our study reveals clues about how active zones accumulate and regulate calcium channel abundance.

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

Another is that drugs such as gabapentin, which treat conditions as diverse as epilepsy, anxiety and nerve pain, bind to a protein called alpha2delta that is closely linked to calcium channels. By revealing more about the exact function of alpha2delta, this study better explains what influences the treatment.

The more scientists removed a protein called alpha2delta by different manipulations (two right columns), the fewer Cac calcium channels were obtained in the synaptic active zone of fly neurons (brightness and number of green dots) compared to the unchanged control (left column).

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

MIT postdoc Karen Cunningham led the research, which was her doctoral thesis work in Littleton’s lab. Using a fruit fly motor neuron model system, he used a wide variety of techniques and experiments to demonstrate for the first time a step-by-step process explaining the distribution and maintenance of calcium channels in the active zone.

Hats in Caca

Cunningham’s first question was whether calcium channels were necessary for the active zone to develop in the larva. The fly’s calcium channel gene (called “cacophony,” or Cac) is so important, flies simply can’t live without it. So, instead of rapidly immobilizing Cac, Cunningham used a technique to immobilize it in just one population of neurons. By doing so, he was able to show that even without Cac, the active zone grows and matures normally.

Using another technique that artificially lengthens the fly larval stage, he was also able to see that with the additional time given, the active zone would continue to build its structure with a protein called BRP, but Cac accumulation stopped after six normal days.

Cunningham also found that a moderate increase from a decreased supply of available Cac in neurons did not affect how much Cac ended up in each active zone. Even more strangely, he found that while the number of Cacs scaled with the size of each active zone, it barely moved if he took up a lot of BRP in the active zone. Indeed, for each active zone, neurons seem to impose consistent limits on the amount of Cac present.

“It was revealed that neurons have very different rules for structural proteins in the active zone such as BRP that continue to accumulate over time, versus calcium channels that are tightly regulated and whose abundance is restricted,” Cunningham said.

Regular refresh

The team’s model shows factors that regulate Cac abundance in the active zone. Development of the Active Zone scaffold and delivery of Cac via alpha2delta increased it while turnover continued to limit it. Cac biosynthesis hardly increases abundance.

The findings suggest that there must be factors other than Cac supply or changes in BRP that regulate Cac levels so tightly. Cunningham switched to alpha2delta. When he genetically manipulated how much was expressed, he found that the alpha2delta level directly determines how much Cac accumulates in the active zone.

In further experiments, Cunningham was also able to show that the ability of alpha2delta to maintain Cac levels was dependent on the overall Cac supply of the neuron. The findings suggest that instead of controlling the amount of Cac in the active zone by stabilizing it, alpha 2delta likely functions upstream, during Cac trading, to supply and supply Cac to the active zone.

Cunningham used two different techniques to see that supply was occurring, resulting in a measurement of its level and timing. He chose shortly after a few days of development to photograph the active zone and measure Cac abundance to ascertain the landscape. He bleached the Cac’s fluorescence to remove it.

After 24 h, he visualized the Cac fluorescence again to highlight only the new Cac delivered to the active zone during that 24 h. He saw that during the day there were Cac deliveries in almost all active zones, but the one day’s work was indeed only a fraction compared to what had been built over the previous few days.

Moreover, he could see that the larger active zone produced more Cac than the smaller one. And in flies with mutated alpha2delta, delivery of new Cac very little.

If the Cac channel is indeed being continuously supplied, then Cunningham wants to know at what rate the Cac channel is removed from the active zone.

The more scientists removed a protein called alpha2delta by different manipulations (two right columns), the fewer Cac calcium channels were obtained in the synaptic active zone of fly neurons (brightness and number of green dots) compared to the unchanged control (left column). Credit: Littleton Lab/MIT Picower Institute

To determine it, he used a staining technology with a photo-transformable protein called Maple that is tagged with a Cac protein that allows it to change color with a flash of light at a time of his choosing. That way he can first see how much Cac has accumulated at any given time (shown in green) and then turn on the light to turn that Cac red.

When he checked back five days later, about 30 percent of the red Cacs had been replaced with new green Cacs, indicating a 30 percent turnover. When he reduces the rate of Cac delivery by altering delta alpha2 or reducing Cac biosynthesis, the Cac turnover stops. That means a large amount of Cac is delivered daily in the active zone and that turnover is driven by new Cac deliveries.

Littleton said his lab was eager to develop 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 incoming new information requires neurons to adjust their communication to increase or decrease synaptic communication.

See also

He said he also wanted to track individual calcium channels as they are made in the cell body and then travel down the nerve axon to the active zone, and he wanted to determine what other genes could influence Cac abundance.

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

Funding: The National Institutes of Health and the JPB Foundation provided support for this research.

About this neuroscience research news

Author: David Orenstein
Source: Picower Institute of Learning and Memory
Contact: David Orenstein – Picower Institute of Learning and Memory
Picture: Image credited to Littleton Lab/MIT Picower Institute

Original Research: Open access.
“Regulation of presynaptic Ca2+ channel abundance in the active zone through balance of delivery and turnover” by Troy Littleton et al. eLife

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Abstract

Regulation of the abundance of presynaptic Ca2+ channels in the active zone through a balance of delivery and turnover

Ca. tension door²⁺ channels (VGCC) mediate Ca²⁺ entry to trigger the release of neurotransmitters at special presynaptic sites called the active zone (AZ). VGCC abundance in AZ regulates possible neurotransmitter release (P_r), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery, and recycling work together to establish VGCC AZ abundance, experimentally isolating these distinct regulatory processes is difficult.

Here we describe how the AZ levels of Cacophony (Cac), the only synaptic transmission mediating VGCC in Drosophiladetermined.

We also analyzed the association between Cac, a conserved VGCC 2δ regulatory subunit, and Bruchpilot core AZ scaffold protein (BRP) in constructing functional AZ. We found Cac and BRP were regulated independently on AZ growth, as Cac was negligible for AZ formation and structural maturation, and BRP abundance did not limit Cac accumulation. Moreover, AZ stopped accumulating Cac after the initial growth phase, whereas BRP levels continued to increase with extended development time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP does not have this buffer.

To investigate the mechanisms that determine the abundance of AZ Cacs, photoconversion of FRAP and intravital Cacs was used to separately measure delivery and turnover in individual AZs over a period of several days. Cac delivery occurs widely across the AZ population, correlates with AZ size, and is limited by 2δ.

Although Cacs did not undergo significant lateral transfer between neighboring AZs during development, removal of Cacs from AZs did occur and was driven by the delivery of new Cacs, resulting in a cap on Cac accumulation in mature AZs.

Together, these findings reveal how Cac biosynthesis, synaptic delivery, and recycling regulate VGCC abundance in individual AZs during synapse development and maintenance.

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