Glutamate dynamics in a retinal microcircuit

This research project was motivated by fundamental questions about how the nervous system works. The nervous system is comprised of individual cells - called neurons - that communicate with each other at specialized junctions called synapses. Information flows from one neuron to another neuron via the release of neurotransmitters. Glutamate is the primary excitatory neurotransmitter in the central nervous system and is responsible for most of the fast transfer of information that occurs between neurons at synapses. But glutamate can also diffuse out of the synapse - a phenomenon referred to as "glutamate spillover". Once outside the synapse, glutamate can activate a different population of neurotransmitter receptors called "extrasynaptic" receptors. This spillover mode of signaling appears to be an important yet relatively unexplored mechanism of cell-to-cell communication in the central nervous system. The main focus of this research project was on a group of neurons in the retina of the eye that are organized into a "microcircuit". There are many different microcircuits in the retina, each thought to perform a series of computations that are responsible to process a particular type of visual information. The microcircuit that we work on is responsible for processing visual signals in the dark and allows for the excellent night vision that most mammals, including humans, posess. Dysfunction in this microcircuit can lead to diseases that cause night blindness. In this project, using a combination of sophisticated optical imaging and electrophysiological techniques, we have explored the hypothesis that spillover of glutamate and the subsequent activation of receptors that lie outside the synapse, play a physiological role in mediating vision in the retina. Earlier in the project, we discovered that specific glutamate receptors this microcircuit are compromised in diabetes (Castilho et al, 2015a; Castilho et al 2015b). We have recently published a characterization of a second type of glutamate receptor found on these neurons (Zhou et al., 2016). Our ongoing goal is to have a precise understanding of the mechanisms that underlie signaling in the healthy nervous system that will lead to clearer and potentially therapeutic insights into the diseased and damaged brain.

A basic goal of neuroscience research is to understand the neural basis of behavior. This requires a detailed study of the synaptic and functional organization of the brain and represents one of the greatest challenges for modern neuroscience. This projec t will exploit recent advances in optical imaging and electrophysiological recording to visualize and access constituent elements of microcircuits deeply embedded in morphologically intricate tissue, allowing for precise correlations of morphology and fun ction at the cellular and subcellular level. This project investigates the hypothesis that spillover of synaptically released glutamate plays a physiological role in signal transmission in the central nervous system. For this, I will use a specific, well -characterized microcircuit in the rod pathway of the mammalian retina. The rod pathway is responsible for processing visual signals in the dark and provides excellent night vision for most mammals, including humans. Dysfunction in this pathway leads to d iseases that cause night blindness. Retinal microcircuits are more numerous and complex than previously thought and provide a series of necessary computations for later visual processing. As such they provide useful model circuits to investigate basic que stions of synaptic transmission and signal processing. My colleagues and I have previously found that glutamate released from rod bipolar cells can "spill out" from the synapse and activate a presynaptic transporter that exerts a negative feedback action. Here, I plan to determine the spatio-temporal concentration profile of glutamate spillover during synaptic activity. In addition, I will investigate the functional role of this spillover by determining its effect on postsynaptic NMDA and AMPA receptors. This will be accomplished by combining patch-clamp electrophysiological recording and multi-photon excitation microscopy with recently developed optical sensors for extracellular glutamate.