Thursday, June 23, 2011

Blocking Exocytosis Decreases and Delays Structural Plasticity

One of my biggest beefs with "Science"* is the journal publishing culture, where incomplete stories get buried, and cool little findings have no place to be heard or read. However, as Ghandiji says, "Be the change you want to see in the world," so I'm going to present some unpublished data.


* Not the journal


Exocytosis of AMPA receptors is important in LTP


In a previous life, aka grad school, I studed AMPA receptor (AMPAR) trafficking in Ryohei Yasuda's lab. AMPAR trafficking is essential for the most common model for studying learning and memory, LTP (I won't discuss LTP here, but my favourite review was written by Derkach and Soderling).  AMPAR are ligand-gated ionotropic receptors the reside in most excitatory synapses in the brain and mediate a majority of synaptic activity.  They can move around the plasma membrane by diffusion, and are trafficked between endosomes and the plasma membrane by exocytosis and endocytosis.


Given that AMPAR are responsible for LTP, and that they move via diffusion and membrane trafficking, one might wonder which type of movement is necessary during LTP.  A series of papers from the Ehlers lab (defunct) has argued that during LTP, AMPARs are exocytsed, and that this exocytosis is necessary to quickly deliver a large number of receptors to the synapse. Last year they even identified the SNARE responsible for post-synaptic, activity dependent exocytosis: syntaxin-4 (this will be important later).

During my thesis work I used glutamate uncaging to ask where AMPAR are exocytosed during synaptic plasticity, and which signaling pathways led to exocytosis.  In a glutamate uncaging protocol, we use spine volume as a proxy for synaptic strength (there's a long literature linking the two).  If you stimulate a spine with glutamate, it gets bigger quickly, and then relaxes a bit (see Fig. 1 below).  Using this protocol we found that AMPAR are exocytosed in the stimulated spine and surrounding dendrite, and that the Ras-ERK pathway led to exocytosis.

TeTX delays and decreases structural plasticity following glutamate uncaging


You might think that in a paper about AMPAR exocytosis and structural plasticity there'd be a figure about structural plasticity when exocytosis is blocked.  And you'd be partially right: we blocked exocytosis and measured structural plasticity for ~5 min, but not long enough to look at anything like LTP.  It was only after we published that we actually did the full thirty minute experiment.

Under normal conditions, when you uncage gluatamte on a spine, it grows to +300-500% of its initial size ~ 1-2 minutes following uncaging.  Then over the next thirty minutes, the spine size plateaus to a size ~100-200% of its initial size (top left panel, black line).  To block exocytosis, we transfected cells with tetanus toxin (TeTX), which interferes with VAMP-mediated exocytosis.  When I uncaged on cells transfected with TeTX, both the initial size increase, and the late phase structural plasticity were decreased (top left panel, blue line).

Fig. 1: Blocking exocytosis decreases and delays structural plasticity. Methods can be found at Figshare.

When I was doing the experiments, I noticed that the TeTX-expressing cells grew slower than the control cells.  I tried a few ways to quantify that.  First, I normalized both Ctl and TeTX responses to their peak (top right panel), and you can see that on average the TeTX peak structural plasticity is slightly delayed.

To see how fast individual spines grew, I normalized each responses, and plotted the growth during the first five minutes following uncaging (bottom left panels).  You can see that a large majority of the control spines reach their peak size quickly, within 1-2 minutes.  In contrast, only a few TeTX spines reach their peak size within that window, and there are many slow spines that take over two minutes to peak.  The scatterplot of the individual peak times is shown on the bottom right.

How does one interpret this?  The decrease in amplitude is fairly easy: exocytosis is important for structural plasticity, and without it there is less growth.  The delayed growth is a little more complicated.  One theory from the Ehlers lab is that when an endosomes fuses with the membrane during exocytosis, it provides membrane in addition to the more traditional cargoes of receptors and membrane proteins.  Thus when we knock out exocytosis, we have decreased the supply of membrane to the spine, and it takes longer for the spine to grow.  It still can, but it takes more time to suck in membrane from the dendritic shaft.

Syntaxin-4 mediates AMPAR exocytosis during structural plasticity

As mentioned before, the Ehlers lab identified syntaxin-4 as the SNARE involved in activity-dependent exocytosis.  When they were revising their paper, they asked us to do some uncaging experiments, but they published before we could finish.  Anyway, we simply did the same experiment, and uncaged on neurons transfected with syntaxin-4 shRNA. And we found exactly what you'd expect: syntaxin-4 shRNA decreased both peak and late-phase structural plasticity, as well as delayed the peak structural plasticity (right panel).

Fig. 2: Syntaxin decreases spine density as well as structural plasticity. Brief methods at Figshare.

When I was doing the experiments, I noticed that the spine morphology was bizarre.  There was a huge decrease in spine number, but the remaining spines were quite large.  We did not quantify spine size, but we did look at spine number, and found that syntaxin shRNA indeed decreased spine density (left panel).


That's my little bit of unpublished data.  It's a pretty clean set of results, but not quite enough to publish anywhere.  Hopefully someone finds it useful.

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