Beware, surgery screws with your mouse's mind!

In 2002 two groups published apparently contradictory results in the same issue of Nature. One group, lead by Wen-Biao Gan, said spines heads were essentially stable in the cortex when followed for long periods using in vivo 2P imaging. Svoboda's group had an article that said "the opposite". Each group used H-line mice, the difference was that Gan imaged through a thinned skull, whereas Svoboda through an implanted window.

Gan and colleagues have now attempted to solve the puzzling difference between these 2 landmark papers. They have found that even a carefully installed craniotomy can change profoundly the structure of glia close to the volume of surgery (see image above, Fig 3S from their paper- doi:10.1038/nn1883). (a) is an image of GFP-micro glia taken through the skull, and (b) is from a mouse with a 'hole in its head'. The structure of the micro glia is quite different. In experiments with GFAP-GFP astrocytes they see a significant increase in GFAP, implying "reactive astrocytes" are produced by surgery. One can easily imagine that glia restructuring could enhance spine-head loss in the cortex. They conclude: "Our findings suggest that the choice of cranial window type for in vivo imaging is the major factor contributing to previous discrepancies observed in spine dynamics under both normal and sensory deprivation conditions."

"Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex." Nature Neuroscience (2007) doi:10.1038/nn1883.

Grutzendler, J., Kasthuri, N. & Gan, W.B. Nature 420, 812–816 (2002).

Trachtenberg, J.T. et al. Nature 420, 788–794 (2002).

Electroporation of Ca dyes in vivo.

A collection of scientists at Yale and Huazhong Universities have developed a method for loading of neurons in vivo. No great shakes you might say? Well it turns out they have managed to load dextran-conjugated fluorescent Ca dyes into neurons so that one can see Ca dynamics in spines heads! The figure is 6C from their paper showing spines and line-scan imaging of electrically evoked fluorescence changes. I have to point out they need to average 8 traces to this poor S/N, even with a high affinity Ca indicator (Kd 200 nM, 0.02 mM). I would think this dye would be saturated within the spine head, but clearly it is not (labs that patch clamp cells ex vivo use lower affinity dyes such as fluo-4FF (0.15-0.5 mM), or OGB-5N (0.5 mM) but see much stronger signals during synaptic inputs).

These quibles aside, This is important technical accomplishment, as it is the first report of imaging spines heads so clearly with a Ca dye. They also claim to image boutons, which is even more amazing. Frankly, I am not used to looking at boutons in vivo (or ex vivo) so I find those data hard to judge. The spines look lovely though.

In Vivo Simultaneous Tracing and Ca Imaging of Local Neuronal Circuits.
Nagayama, et al., Neuron (2007) 53:789-803. DOI 10.1016/j.neuron.2007.02.018

The Munich group published a very nice paper on their technique last year:

Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo.
O Garaschuk, R-I Milos, A Konnerth Nature Protocols (2006)
1:380-386.