With glutamate receptors, it’s all about location and timing

An electron micrograph showing fusion of synaptic vesicles. Mouse hippocampal neurons were stimulated once and then frozen milliseconds later using the “flashzap-and-freeze” approach.

Shigeki Watanabe, associate professor in the Department of Cell Biology at Johns Hopkins School of Medicine, recently received a 2020 Johns Hopkins Catalyst Award, which provides $75,000 to support research over the next year. Watanabe’s lab studies the rapid morphological and molecular changes of neurons to better understand the mechanisms of synaptic transmission and plasticity, as well as the pathogenesis of neurodegenerative disorders.

“[Neurotransmitter] release occurs on a millisecond time scale and can’t be easily captured on video,” Watanabe explained. “My colleagues and I have developed a ‘zap-and-freeze’ approach that can capture images of membrane dynamics over these millisecond time scales”.

From this method, the Watanabe lab creates a “flipbook” of the time series during neuronal communication. The sequence helps to visualize the moments when synaptic vesicles fuse with the membrane to release neurotransmitter and when these vesicle membranes are recycled at synapses. Watanabe is trying to find out how these processes are regulated by proteins and signaling lipids.

In the proposed research, Watanabe and his colleagues will follow up on their recent discovery that neurotransmitter released at different intervals activates specific types of receptors.

“In the mammalian central nervous system, glutamate is the major excitatory neurotransmitter,” Watanabe said. “There are several types of receptors that receive this signal, including AMPA-type and NMDA-type glutamate receptors. These two types of receptors are important for synaptic plasticity: AMPA receptors determine the strength of synaptic signaling, while NMDA receptors serve as a switch for the plasticity. The properties of these receptors are quite different, but in both cases, the affinity to glutamate is rather low so as to keep the signaling transient. Thus, where glutamate is released relative to the receptors is thought to be important for how signals are transmitted at synapses.”

To better observe the timing and location of synaptic vesicle exocytosis relative to glutamate receptors, the Watanabe lab developed a method that uses a high affinity of polyhistidine for nickel to label receptors in hippocampal mouse neurons with gold particles. For each experimental scenario, neuronal cultures were stimulated once and high-pressure frozen at different time points after stimulation. About 100 images were collected with electron microscopy and analyzed.

“The results from these experiments indicated that AMPA receptors are localized toward the edge of postsynaptic receptive field, while the NMDA receptors are distributed toward the center,” he said. “Intriguingly, glutamate is first released near AMPA receptors and then near NMDA receptors following an action potential. Computer modeling suggests that this temporal and spatial organization of release sites and receptors allows a better activation of NMDA receptors. Thus, this organization may have implications for the induction mechanism of synaptic plasticity.”

In its current research, the Watanabe lab is aiming to reveal the molecular players that organize the release sites to the receptors using novel cryo-electron microscopy approaches.

Watanabe added that preliminary data for this research was gathered from a Neurobiology course that he teaches each summer at the University of Chicago’s Marine Biological Laboratory at Woods Hole, MA.

“The course is organized around extensive hands-on experimental sections with students,” Watanabe said. “The results described above were started as course projects and carried on in my lab. The students in the course collected preliminary data and made it to the author list in a paper currently in press and also another paper in revision.”