Videos of AMPA-R Insertion

One of the mechanisms -- perhaps even the primary mechanism -- by which synapses in the brain are potentiated -- made more sensitive to activation -- is the insertion of more AMPA receptors (AMPA-R) into the synapse. AMPA-R are glutamate-activated, cation (Na and Ca) channels that are really the business end of creating the electrical activity in the post-synaptic cell. Their insertion is activated by the Ca admitted by another cation channel, the NMDA receptor. Basically long-term potentiation (LTP) -- which we believe is the primary storage form of memories on a cellular level -- of synapses results when activated NMDA receptors cause the insertion of AMPA-R into the synapse.

Whew. Who says you can't boil down synaptic physiology into a paragraph? That is some good summarizing, Son.

Anyway, the insertion of AMPA-R in the synapse is not something that we have hitherto been able to watch in real time. That is until Yudowski et al. at UCSF found a way.

The way that they image AMPA-R insertion is really quite clever. We have known for several years how to tether fluorescent proteins to the proteins we want to image. You force a cell to express your protein construct -- through a process called transfection -- that you have modified to have an additional fluorescent portion attached. This fluorescent molecule can be visualized using a special microscope.

The trouble with AMPA-R is that you want to see only those receptors that have been inserted into the membrane. This is the clever part. The researchers attached a fluorescent molecule that is sensitive to pH. It only fluoresces when it is exposed to 7.4 pH. They bathe the cells in this solution in the extracellular fluid allowing them to visualize only inserted AMPA-R.

It works like this. The uninserted AMPA-R do not fluoresce because they are inside the cell and not exposed to the right pH. However, once the cell is activated and the AMPA-R are inserted into the membrane they begin to fluoresce because the fluorescent tag is now exposed to the extracellular solution which has the right pH.

As they say: badass.

The researchers cultured rat hippocampal neurons, and transfected them with their construct. They would then image the cells and form videos of AMPA-R being inserted under different conditions.

Here are some of their videos. (All of these videos are in the supplementary data section of the paper.) What you are looking for are short, bright lights. That is a bundle of AMPA-R being inserted. This one almost looks like twinkling Christmas lights.

Incidentally, to get these videos to work you need a really fast camera. They took 10 frames/sec. Here is another.

That is all fine and good, but what is the significance, you ask? Well the authors found some interesting things. For example, we had assumed that insertion of AMPA-R was directly in the post-synapse. It turns out that this is not the case. Rather, the AMPA-R are inserted in the membrane beside the synapse, and then they float laterally in the membrane into their appropriate locations. This was not a result that you would anticipate.

That result is shown in this video. In this video, the researchers counter-stained for a protein called PSD-95 -- a marker for the post-synaptic density -- shown in red. What you can see is that the bright dots are not over the red densities. The red densities are on what are called dendritic spines which look like little mushrooms. The bright spot appears on the shaft connecting them which is the dendrite. Thus, insertion occurs on the dendritic shaft or on the cell body, not directly into the synapse.

The authors summarize the significance of their results thusly:

To our knowledge, the present results are the first to directly visualize discrete membrane trafficking events mediating surface delivery of AMPARs. Our results unambiguously resolve both constitutive and regulated (NMDA receptor-dependent) components of exocytic insertion of GluR1-containing AMPARs in both hippocampal slice and dissociated culture preparations. Discrete insertion events were observed under basal conditions, and were substantially increased in frequency by two chemical stimuli that produce a net increase in surface AMPAR expression at steady state. Furthermore, our results reveal the existence of kinetically distinct exocytic modes mediating both constitutive and activity-dependent surface delivery of AMPARs.

Previous studies have concluded variously that surface insertion occurs in the dendritic shaft, exclusively in the cell body, or directly into spines. They also differ on whether surface AMPAR insertion was proposed to occur in a regulated manner or constitutively. Such divergent conclusions are understandable given the previous inability to observe discrete AMPAR insertion events as they occur, and considering that the distribution of surface AMPARs can be rapidly modified by lateral movement or endocytosis. The present results, by directly resolving individual AMPAR insertion events in real time, show clearly that exocytic insertion of AMPARs occurs both in the cell body and throughout the full length of the dendritic shaft. Furthermore, in both compartments, our data identify constitutive and regulated components of exocytic insertion. The proportion of persistent exocytic events observed in dendrites was higher than that observed on the cell body. Nevertheless, estimates of absolute event frequency suggest that exocytic events occurring in both membrane domains make a significant contribution to net surface insertion of AMPARs. So far, we have not observed "hot spots" of repeated SEP-AMPAR insertion in either the cell body or in dendrites. Given that our rapid image series are limited for practical reasons to 1 min in length, however, we cannot rule out the existence of such regions of increased exocytic probability if repeated events occur >1 min apart.

We were surprised to note that, in multiple experiments in which hundreds of discrete exocytic events were visualized, exocytic insertion of AMPARs directly into spines was never observed. We cannot presently exclude the existence of such a direct exocytic route for synaptic AMPAR delivery, as suggested in an elegant recent study in which mutational inhibition of exocytic fusion produced an accumulation of intracellular AMPARs in spines at steady state. Direct exocytic insertion to the spine could be missed by our methods if these events have relatively low surface fluorescence intensity. Nevertheless, considering the remarkably high peak surface fluorescence of the many exocytic events that our methods did resolve (and that all of these events occurred clearly outside of synaptic spines and postsynaptic sites marked by PSD-95), the present data argue strongly that a major pathway mediating both constitutive and regulated surface insertion of AMPARs is via exocytosis of AMPAR-containing vesicles occurring extrasynaptically in the dendritic shaft.

Given that many discrete exocytic events mediating surface AMPAR insertion were observed outside of synapses, how are receptors subsequently delivered to synaptic spines? One possibility is that surface delivery to spines occurs by lateral partitioning from a diffuse extrasynaptic receptor pool supported by long-range lateral diffusion. (Emphasis mine. Citations removed.)

Congratulations to the authors on this incredible technical advance and beautiful work.

The complete citation for this paper:

Guillermo A. Yudowski, Manojkumar A. Puthenveedu, Dmitri Leonoudakis, Sandip Panicker, Kurt S. Thorn, Eric C. Beattie, and Mark von Zastrow. "Real-Time Imaging of Discrete Exocytic Events Mediating Surface Delivery of AMPA Receptors"
J. Neurosci. 27: 11112-11121; doi:10.1523/JNEUROSCI.2465-07.2007

Hat-tip: Faculty of 1000