Neurons are a fundamental computational unit in the nervous system, and their ability to process and store information relies on their ability to generate and sustain functional differences between dendritic compartments. The complex morphology of the neuron, with its elaborately branched dendrites onto which impinge thousands of individual synapses, requires that highly specialized mechanisms exist for localizing, maintaining, and removing proteins at the synapse. Such mechanisms are crucial for the initial establishment of postsynaptic specializations during synaptogenesis and for activity-dependent changes in synaptic strength that underlie experience-dependent plasticity. Moreover, alterations in the trafficking or stabilization of neurotransmitter receptors at the synapse may contribute to pathologic changes in neurologic and psychiatric disease.
Trafficking of Ionotropic Glutamate Receptors to and from the Synapse
One of our major lines of ongoing research in the lab is the study of the molecular and cellular mechanisms that regulate the trafficking of ionotropic glutamate receptors to and from the synapse. We are currently studying two types of glutamate receptor: the AMPA- and NMDA-type glutamate receptors. These glutamate-gated ion channels mediate the vast majority of excitatory synaptic transmission in the mammalian brain, play a central role in synaptic plasticity, and are implicated in numerous neurologic and psychiatric diseases, ranging from stroke, epilepsy, and central nervous system (CNS) injury, to schizophrenia and alcoholism. Our interest stems from the fact that the insertion and removal of glutamate receptors at the synapse plays a central role in synaptic plasticity, learning, memory, and brain development. Recently, we have found that AMPA-type glutamate receptors undergo activity-dependent endocytosis and recycling at the postsynaptic membrane, and we have identified the major endocytic compartments and kinase/phosphatase signaling pathways that regulate AMPA receptor sorting. Our studies have revealed that recycling endosomes are the major internal source of AMPA receptors during long-term potentiation (LTP) at hippocampal synapses, opening new avenues of research into the protein machinery on endosomes that responds to plasticity-inducing stimuli. We have recently developed and employed super-resolution optical imaging approaches to study the movement of single glutamate receptors within synapses, and have shown that activity of single synapses slows AMPA receptor diffusion.
Membrane Trafficking in Dendrites and Dendritic Spines
We are also studying the dynamics and regulation of membrane-trafficking machinery in dendrites and dendritic spines. The two major membrane-trafficking pathways in cells are the secretory pathway and the endocytic pathway.
Secretory trafficking. The secretory pathway—consisting of the endoplasmic reticulum (ER), the Golgi apparatus, and the trans-Golgi network—is the route by which all newly synthesized integral membranes and secreted proteins reach the plasma membrane and is the location of most lipid biosynthesis in cells. Despite extensive study in model cell systems, the organization and regulation of secretory organelles in neurons (which have cell surface areas 10,000 times or more greater than typical animal cells) has received limited study. We have initiated a series of studies identifying the cellular secretory machinery that participates in ER-to-Golgi and post-Golgi trafficking in dendrites. Using live-cell imaging, we have identified a novel distributed organization of ER exit sites and Golgi "outposts" in neuronal dendrites capable of transporting newly synthesized membrane proteins. This distributed mode of ER-to-Golgi traffic is unique among mammalian cells and highlights the special requirements of neurons in controlling membrane composition and surface levels of ion channels and neurotransmitter receptors. We are investigating the molecular mechanisms that generate and localize Golgi outposts in dendrites and the functional consequences for localized secretory trafficking on neuronal architecture, dendrite composition, and synaptic function.
Endocytic trafficking. Endocytosis of cell surface molecules, including glutamate receptors, is essential for nearly all aspects of neuronal function, yet remarkably little is known about where and when such endocytosis occurs over the complex dendritic arbor. We have recently discovered a specialized membrane domain in dendritic spines dedicated for clathrin-mediated endocytosis that we have termed the "endocytic zone." This novel microdomain lies adjacent to the postsynaptic density (PSD) and represents a new class of membrane specialization that likely serves to control the local complement of receptors and other membrane proteins at the synapse. More recently, we have found that exocytosis from recycling endosomes mediates plasticity-induced growth of dendritic spines, indicating a novel requirement for spatially localized membrane recycling in microstructural brain plasticity. We are investigating the cellular mechanisms by which localized spine endocytic cycling is established and regulated during synapse formation and plasticity, and how establishment of the postsynaptic endocytic zone contributes to synapse formation and maintenance.
Manipulating the Activity of Genetically Defined Populations of Neurons
Our third area of ongoing research is the development of mouse models to manipulate the activity of genetically defined populations of neurons in vivo. Mapping functional circuits has been a major goal for both cellular and systems neuroscience for decades. To date, much of the work addressing these questions consists of anterograde or retrograde labeling techniques, in conjunction with independent electrophysiological recordings. It has been challenging to combine these two approaches into a single experiment by genetically specifying a population of cells to be simultaneously monitored by a fluorescent reporter and manipulated to elicit precise electrophysiological activity. A holy grail of neurobiology has been to incorporate spatial and temporal control over neural activity through genetic manipulation of neuronal subsets.
We have recently described transgenic mice in which CNS neurons express the algal light-gated ion channel channelrhodopsin-2 (ChR2) and demonstrated precise regulation of neuronal activity in living animals by focal illumination of cells transgenically expressing ChR2. We have demonstrated a high degree of temporal resolution and fidelity in response properties that can be obtained both in vitro and in vivo. We have subsequently applied this mouse model to the problem of olfactory cortical integration by presenting focal light stimulation in the olfactory bulb while recording electrophysiological responses in both the bulb and piriform cortex. By varying the size and area of bulb photostimulation, we have found that olfactory information processing relies on a high degree of mitral cell convergence and integration onto the piriform cortex. Thus, engineered ChR2 mice can be utilized for the precise and rapid activation of genetically defined populations of mammalian neurons in vivo. This model system thus enables controlled neuronal stimulation for exploring complex brain circuitry. We are designing next-generation mouse models for complementary optical and chemical genetic activation of neuronal subsets in the intact mammalian brain.
To study receptor trafficking and dendritic vesicular transport, we use a variety of molecular and biochemical techniques. State-of-the-art live-cell optical imaging is used to examine the dynamic features of dendritic membrane trafficking. In particular, high-resolution confocal, two-photon, and single-molecule microscopy, together with time-lapse video imaging, photostimulation, photobleaching, and photolytic uncaging are used to directly visualize and measure membrane-trafficking events in neuronal dendrites. For functional analysis, electrophysiological recordings, in both cultured neurons and brain slices, are used to determine the effect of cellular events and protein interactions on synaptic transmission and neuronal activity. We are also engineering mouse lines to visualize and manipulate neural activity and synaptic composition, whose functional consequences are assessed using in vivo electrophysiological recordings and behavioral analysis. These varied techniques allow us to examine the molecular regulation of protein-trafficking events and the functional consequences for neural circuit plasticity.
