Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes

Forgetting is a common experience in our everyday life. Yet much remains unknown about how we remember, and about why our memories sometimes fail us. The brain contains 80 to 100 billion nerve cells or neurons, which communicate with one another at junctions called synapses. At a synapse, one neuron releases a chemical message, which must diffuse across a small gap, and then activate proteins called receptors on another neuron. If the first neuron activates the second repeatedly, the second cell responds by inserting additional receptors into its membrane at the synapse. This strengthens the connection between the two neurons. Strengthening of synapses is thought to be one of the key mechanisms underlying learning. To confirm this, it would be helpful to be able to monitor the movement and position of individual receptors at synapses. However, the space between the two nerve cells at at synapse, called the synaptic cleft, is no more than 40 nanometers wide. This is about 25 times thinner than a human hair, and too small to be seen with light microscopy. Electron microscopy can visualize synapses, but does not work in living tissue. The only other option is to attach a fluorescent label – either a dye or a man-made crystal called a quantum dot – to a protein found in synapses and monitor the resulting fluorescence. Though the probe must be small enough to pass through the synaptic cleft to do this. Using fluorescence microscopy, researchers have examined the distribution in synapses of proteins called AMPA receptors, which have a key role in memory. Multiple studies have shown groups of AMPA receptors gathered outside synapses. This has led to the suggestion that during learning, AMPA receptors wait outside the synapse until a space becomes available within the synapse’s membrane. However, this has yet to be confirmed directly, in part because conventional fluorescent dyes and quantum dots are too bulky to enter synaptic clefts when bound to a receptor. Lee et al. have now developed a quantum dot that is only 10 nanometers wide and therefore small enough to enter the synaptic cleft with an AMPA receptor attached. These small quantum dots were then used to label AMPA receptors in neurons collected from rats and then grown in a petri dish, which provided a completely new view of synapses. The images show that the majority of AMPA receptors in neurons circulate within confined domains – a little like holding pens – inside the synapse, rather than waiting outside as previously assumed. Labeling the receptors with smaller 4-nanometer-wide fluorescent tags produces a similar picture. Further work is still need to determine how AMPA receptors get into the synapse and contribute to new memories.