Summary: Researchers reveal how neurons create and sustain the critical infrastructure that allows seamless neurotransmission.
source: Bakeware Institute for Learning and Memory
The nervous system works because nerve cells communicate via connections called synapses. They “talk” when calcium ions flow through the channels into “active regions” laden with vesicles carrying molecular messages.
Electrically charged calcium causes vesicles to “fuse” into the outer membrane of presynaptic neurons, releasing their communicative chemical payload to the postsynaptic cell.
In a new study, scientists at the Picower Institute for Learning and Memory at MIT provide several discoveries about how neurons create and maintain this critical infrastructure.
“Calcium channels are the main determinant of calcium influx, which then leads to vesicle fusion, so it is an important component of the motor on the presynaptic side that converts electrical signals into chemical synaptic transmission,” said Troy Littleton, senior author of the new study. in eLife and Menicon Professor of Neuroscience in the Departments of Biology and Brain and Cognitive Sciences at MIT.
How it stacks up in active areas wasn’t really clear. Our study reveals clues about how active regions accumulate and regulate the abundance of calcium channels.”
Neuroscientists wanted these clues. One reason is that understanding this process can help reveal how neurons change the way they communicate, a capacity called “plasticity” that underlies learning, memory and other important brain functions.
Another reason is that drugs such as gabapentin, which treat conditions as diverse as epilepsy, anxiety and neuropathic pain, bind a protein called alpha2delta that binds closely to calcium channels. By revealing more about the exact function of alpha2delta, the study better explains the effect of these treatments.
The more scientists knocked out a protein called alpha2delta with different treatments (two columns on the right), the less calcium channel Cac accumulated in the synaptic active areas of the fly neuron (brightness and number of green dots) compared to the unmodified controls (left column).
“It is known that modulation of presynaptic calcium channel function has very important clinical implications,” Littleton said. “It’s really important to understand the basis for how these channels are organized.”
Karen Cunningham, a postdoctoral researcher at the Massachusetts Institute of Technology, led the study, which was her doctoral thesis in Littleton’s lab. Using the Drosophila motor neuron model system, I used a variety of techniques and experiments to show for the first time the step-by-step process that accounts for the distribution and maintenance of calcium channels in active regions.
cover 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 ‘cacus’ or Cac) is so important, flies literally can’t live without it. So instead of eliminating Cac via the fly, Cunningham used a technique to knock it out in just one group of neurons. By doing this, I was able to show that even without Cac, the active areas grow and mature naturally.
Using another technique that artificially lengthens the larval stage of the fly, I was also able to see that with additional time the active region would continue to build its structure with a protein called BRP, but that Cac buildup stops after the normal six days.
Cunningham also found that moderate increases in decreases in the supply of available Cac in neurons did not affect how much Cac ended up in each active region. Most intriguingly, she found that while the amount of Cac is proportional to the size of each active area, it hardly budges if she takes in too much BRP in the active area. Indeed, for each active region, neurons appear to impose a fixed ceiling on the amount of Cac present.
“He was revealing that neurons had very different bases for structural proteins in the active region such as BRP that continued to accumulate over time, versus a calcium channel that was tightly regulated and its abundance restricted,” Cunningham said.
The team’s model shows the factors that regulate the abundance of CAC in the active regions. Active Zone scaffold development and Cac delivery via alpha2delta increases it while its rotation maintains a cap on it. Cac biosynthesis hardly increases its abundance.
The results showed that there must be factors other than Cac supply or changes in BRP that very tightly regulate Cac levels. Cunningham switched to alpha2delta. When I genetically manipulated how much was expressed, I found that alpha2delta levels directly determined how much Cac accumulated in the active regions.
In other experiments, Cunningham was also able to show that alpha2delta’s ability to maintain Cac levels depends on Cac supply to neurons in general. This result suggests that rather than controlling the amount of Cac in the active regions through its stabilization, it is likely that alpha 2delta acts upstream, during Cac trafficking, to supply and re-supply Cac to the active regions.
Cunningham used two different methods to monitor the occurrence of resupply, producing measurements of its extent and timing. She chose Moment after a few days of development to photograph active areas and measure cak abundance to ascertain the landscape. She bleached that Cac fluorescence to erase it.
After 24 hours, I re-imagined Cac fluorescence to highlight only new Cac delivered to the active areas during those 24 hours. She saw that during that day there was Cac delivery across almost all active areas, but one day’s work was actually a fraction of what was generated over several days before.
Furthermore, she could see that the larger active areas accumulated more Cac than the smaller ones. And in flies harboring the alpha2delta mutation, there was very little new Cac delivery at all.
If the Cac ducts were indeed constantly being replenished, Cunningham wanted to know how quickly the Cac ducts were being removed from the active areas.
To determine this, she used a staining technique with a photoswitchable protein called Maple that was tagged with a Cac protein that allowed her to change color with a flash of light at a time of her choosing. This way she can first see how much Cac has accumulated in a given time (shown in green) and then flash a light to turn Cac red.
When I checked back five days later, about 30 percent of the red kak had been replaced with new green kak, indicating a 30 percent turnover. When Cac delivery levels were reduced by alpha2 delta mutation or reduced Cac biosynthesis, Cac turnover ceased. This means that a large amount of Cac is inverted each day in the active areas and that the rate of circulation is being driven by the delivery of new Cac.
Littleton said his lab is eager to build on these findings. 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 adjust their connections to extend or decrease synaptic connectivity.
He is also keen to track individual calcium channels as they are made in the cell body and then move down the axon to active regions, and he wants to identify other genes that may influence Cac abundance, he said.
In addition to Cunningham and Littleton, the other authors of the paper are Chad Sauvola and Sarah Tavana.
Financing: The National Institutes of Health and the JPB Foundation provided support for the research.
About this research in Neuroscience News
author: David Orenstein
source: Bakeware Institute for Learning and Memory
Contact: David Orenstein – Becker Institute for Learning and Memory
picture: Image credited to Littleton Lab / MIT Picower Institute
original search: open access.
“Regulation of presynaptic Ca2+ channel abundance in active regions through homeostasis of delivery and turnover” by Troy Littleton et al. eLife
Regulation of presynaptic Ca2+ channel abundance in active regions through homeostasis of delivery and turnover
voltage gated Ca2+ Ca .-mediated channels (VGCCs)2+ Flux to stimulate neurotransmitter release at specialized presynaptic sites called active areas (AZs). The abundance of VGCCs in AZs regulates the potential for neurotransmitter release (ss), one of the main presynaptic determinants of synaptic strength. Although biosynthesis, delivery, and recycling cooperate to create an abundance of AZVGCC, isolating these distinct regulatory processes experimentally has been difficult.
Here we describe how AZ levels of Cacophony (Cac), the only VGCC transporter that mediates synaptic transmission in fruit flybent.
We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit α2δ and the Bruchpilot core AZ scaffold protein (BRP) in establishing a functional AZ. We find that Cac and BRP are independently regulated upon growth of AZs, as Cac is dispensable for AZ formation and structural maturation, and abundance of BRP does not limit Cac accumulation. In addition, AZs stop accumulating Cac after an initial growth stage, while BRP levels continue to increase due to the extended growth time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, while BRP lacks this buffering.
To investigate the mechanisms that determine AZ Cac abundance, the intravitreal photoconversion of FRAP and Cac was used to measure delivery and rate separately in individual AZs over a multi-day period. Cac delivery occurs widely across the AZ population, correlates with the size of AZ, and is rate restricted by α2δ.
Although Cac does not undergo significant lateral transfer between adjacent AZs over the course of development, Cac removal from AZs occurs and is promoted through delivery of new Cac, creating a ceiling for Cac accumulation in mature AZs.
Together, these results reveal how Cac biosynthesis, synaptic delivery, and recycling determines the abundance of VGCCs in individual AZs during synapse development and maintenance.
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