Visualizing synaptic tagging and capture

A set of two articles recently came out in Science that directly visualize two different (and likely complementary) approaches to synapse specific delivery of gene products. Plasticity at specific synapses (input specificity — we’re restricting the discussion to the dendrites of the post-synaptic neuron) requires proteins (eg. new AMPA receptors) to get to those post-synaptic compartments and membranes. But the intructions for these new proteins are contained in the nucleus with the rest of the genome. Clearly, new proteins synthesized in the soma can’t just be sent everywhere, since only specific inputs (eg. particular dendritic spines) need these new proteins. How does this happen? Hence, the postulated synaptic tag.

Two approaches

Broadly, there are two approaches to synaptic tagging: 1) mRNA is distributed widely and translated locally at tagged locations; 2) protein products are distributed widely in the bodies of dendrites but only contact/off-load at tagged synaptic specializations. This News & Views gives a nice overview of the two papers, which find approach 1) in Aplysia cultures with sensorin mRNA and approach 2) in rat hippocampal neurons with Vesl-1S/Homer-1a protein. It amazes me that both were found pretty much simultaneously, but that might have more to do with the use of the photoconvertible Dendra2 protein than anything else.

With both approaches, we still don’t know why mRNA/protein is directed to a certain location. That is, we can visualize synaptic tagging but we don’t know what is the tag, its ontogeny, or the mechanism of tagging. But that might not be so important to understanding more about neural function. These new tools might allow us to image plasticity at many synapses at once, perhaps even in vivo. But before that, more work is needed… does the optical signal (from the Dendra fusion protein) correlate with degree of potentiation? Can we detect plasticity in the opposite direction, ie. synaptic depression, through another tag?  (As a sidenote to approach 1), the use of 5′ and 3′ UTRs as a sort of molecular zipcode is also intriguing.)

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Neurotubes music videos

Heesoo Kim sent me a note that The NeuroTubes have released a set of neuroanatomy music videos. All of them are wacky and neat… here’s a clip of Proud to Be a Neural Tube (which achieves the impressive feat of rhyming notochord with neuropores):

Sunday afternoon reading: Genetic tools "primer"

I came across this fantastic review of tools for the Genetic Dissection of Neural Circuits in Neuron a few days ago. It’s by Liqun Luo, Ed Callaway, and Karel Svoboda. I highly recommend it, as it spans the gamut from genetic targeting (recombination, binary logic, viral delivery) to circuit reconstruction (super resolution LM, EM, brainbow) to activity modulation and functional mapping (uncaging, artificial GPCRs, light-gated channels, MIST). I don’t think I’ve ever seen quite a review of so many cutting edge neurotechnologies in one place. I can’t recommend this piece enough really. For me, with my lack of molecular expertise, the first sections on combinatorial gene targeting/expression techniques were great, pulling together Gal4, Cre/Flp, and Tet systems into a unified framework, along with more general concepts like site-directed integration, enhancer-trap, and repressor trap (eg. Thy1 mice).

sCRACM: ChR2 circuit mapping

As has become a hallmark of the Svoboda lab, this new paper in Nature (advance online publication) combines several cutting edge technologies (rAAV-delivered ChR2, most prominently, and 2-photon 1-photon laser stimulation) to do some interesting synaptic physiology.

The subcellular organization of neocortical excitatory connections : Article : Nature.

They used ChR2 (with TTX and 4-AP to block action potentials) to find where on the dendritic tree particular inputs synapsed onto L3 and L5 cells and to measure the strength of those inputs. ChR2 depolarizes the input axon locally (60um spot diameter) at points of (potential) axodendritic contact. If you’ve heard the term “potential synapse” before, then think of this technique as a way of checking potential synapses and seeing if there really is an actual synapse there.

The technique allowed them to map on a L3 barrel cortex pyramidal cell where different thalamic inputs (VPm, POm) and cortical inputs (M1, barrel L2/3, barrel L4):

screenshot001

sCRACM stands for subcellular ChR2-assisted circuit mapping.

Plant neuroscience

Plants Found to Show Preferences for Their Relatives – NYTimes.com

Two amazing things here:

  1. Plants missing photosynthetic enzymes of their own that migrate directionally toward “victim” plants. This behavior has an uncanny resemblance to axon guidance. Make sure to view the time-lapse video in the NYT article. Here’s an image from the PSU website:

  2. Plants capable of identifying kin and “being nice” to kin while going into a competitive mode of root growth with non-kin. Amazing.

It refreshing to see this kind of interesting behavior without any neurons involved. It makes me think (realize) that the idea of a neuron or a neural system has many components and there might not be any good reason to assume that a single cell must have all of those properties or none of them. Something like a neuron-like cell that’s not a neuron in the classical sense. Anyone know of other examples?

Split GFP reconstituted: A dynamic synapse label

This new technique from Cori Bargmann’s lab is one of the neatest that I’ve seen in a while. The authors split GFP into two pieces, expressing one piece presynaptically and the other postsynaptically. This creates functional (ie. fluorescing) GFP only at sites of synaptic contact where the protein can reconstitute. They call the technique GFP Reconstitution Across Synaptic Partners (GRASP). Check out an example labeling here:
GRASP labeling figure
The neurons are expressing mCherry in the cytoplasm but GFP is expressed only at the site of synaptic contacts where the split GFP peptides can be reconstituted into a complete GFP fluorophore.

Real-time STED to visualize vesicle dynamics

Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement

Just the optical engineering alone here deserves mention: 28 frames per second at 62nm resolution (well below the diffraction limit of 260nm for light of the wavelength used)! STED (or stimulated emission depletion, developed in Stefan Hell’s group) is ideal for visualizing synaptic vesicles, whose small size (~50nm) has typically confined them to the domain of electron microscopists. The ability to get high-speed STED allowed the researchers to track individual vesicles and their path dynamics. They conclude that vesicle movement has both motor-driven and diffusive components (ie. a biased random walk). I’m sure with more time and more analysis there will be a lot of interesting applications for this kind of real-time vesicle tracking. Perhaps in the near future we will have single vesicle “minis” monitored at multiple sites through microscopy instead of just one or two sites electrophysiologically…

Here’s the resolution difference between STED and confocal for a single vesicle:
Sted vs. confocal vesicle picture

And, for those of you with ~$1.25M lying around, you can now purchase a STED setup directly from Leica!