In vivo imaging of adult neurogenesis
Supplementary Methods:
Imaging (controls)
Although in clear optical chambers I could image down to the granule cell bodies
imaging was limited to more superficial layers. Specifically, all images that were used for
analysis were taken from either the external plexiform layer (usually no more than 250 microns
deep) or the glomerular layer. To verify that laser power had no detrimental effects on the
results, a control experiment was conducted where spines were imaged at twice the normal
scanning resolution (0.11 m/pixel) for 11 consecutive imaging sessions for a total of 90
minutes. No detrimental effects were evident (see supplemental movie 3). In addition, the
different dynamics observed for different neuronal types rules out specific effects of
photodamage. To test whether sufficient resolu1tion was achieved, I compared between in vivo
(two-photon) and fixed (confocal) preparations. No major changes were evident (see
Supplemental Fig. 7). Quantitative analysis of spine distributions in mature GFP labeled cells
(both 45 and 90 DPI) was not significantly different between in vivo and fixed tissue (in vivo:
0.36±0.08 spines/m, n=8 dendrites; fixed: 0.3±0.09 spines/m, n=7 dendrites). Finally, cranial
window preparation by thinned bone or partial bone removal might influence the results of longterm imaging data. These different methods have been thoroughly discussed before (see
discussion in 1 and also supplemental information in 2). Here, I normally imaged via thinned bone
which had sufficient image quality. In several cases I removed a small piece of the bone, in
which case the bone grew back within several days. Because the dynamics of dendrites were not
different for given neuron along consecutive days it seems that the surgical procedure had no
direct effect. Taken together, it is unlikely that photodynamic damage, problems of resolution or
tissue health influenced the conclusions of the results.
Intracellular dye fillings
In order to compare the morphology of newborn cells to pre-existing neurons we used
intracellular filling of randomly selected cells around glomeruli (n=10 neurons). For this
purpose, mice were anaesthetized as described in the methods (see main text) and decapitated.
Olfactory bulbs were dissected out into an ice-cold oxygenated solution containing (in mM): 85
NaCl, 25 NaHCO3, 22 glucose, 70 sucrose, 3.5 MgSO4, 2.5 KCl, 1 NaH2PO4, and 0.5 CaCl2.
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In vivo imaging of adult neurogenesis
Bulbs were embedded in agarose and sliced coronally at 400 m using a vibrotome (VT1000s,
Leica). Slices were equilibrated for at least 1 hr in an oxygenated solution containing (in mM):
125 NaCl, 25 NaHCO3, 15 glucose, 3 KCl, 2 CaCl2, 1.3 NaH2PO4, and 1 MgCl2, and
transferred to the recording chamber. Patch-clamping of PGNs was done under infrared video
microscopy using glass electrodes filled with 2 mg/ml Alexa Fluor 488, or Alexa Fluor 568
(sodium salt, Molecular Probes) dissolved in 150 mM KCl and 10 mM HEPES (pH 7.4). After
achieving whole-cell patch-clamp configuration, 10–100 pA negative current steps (50 ms, 4 Hz)
were applied for 10-30 min. Slices were fixed in 4% PFA (30 min), washed in PBS and then
imaged with the two-photon microscope on glass slides. One cell per slice was injected. Neurons
were then reconstructed as described above. Although this method ensures high quality filling,
slicing has the risk of cutting dendrites which was clear in some cases. Also the 3D whole-cell
morphology is probably affected by the procedure. Therefore, comparison was limited to
qualitative 2D comparison of the dendritic trees.
Immunocytochemistry
Mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde and the
brains prepared for histology by soaking them in 30% sucrose. OB’s were sectioned coronally
and the SVZ was sectioned either coronally or sagitally on a sliding microtome (40 m slices).
For immunolabeling, slices were processed with the following antibodies: Rabbit ant-GFAP
(1:1000) and Mouse anti-NeuN (1:500) from Chemicon (Temecula, CA), rat anti-BrdU (1:200)
from Serotec (Oxford, UK). Secondary antibodies were: goat anti-rabbit Cy5 (1:200) and goat
anti mouse-cy3 (1:200), both from Chemicon, and biotinylated rabbit anti-rat (1:100) from
Vector labs (Burlingame, CA). Amplification was carried out using Texas Red streptavidin
(5g/ml, Vector labs). BromodeoxyUridine (BrdU) was injected i.p. (50 g/g body weight
dissolved in dH2O). BrdU staining was performed on free floating sections pretreated for DNA
denaturation (by incubation in 2N HCL at 37°C for 30 minutes). For in vivo imaging of
astrocytes I used SR101 3.
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In vivo imaging of adult neurogenesis
Supplemental references
1.
Zuo, Y., Lin, A., Chang, P. and Gan, W.B., Development of long-term dendritic spine
stability in diverse regions of cerebral cortex. Neuron, 2005. 46(2):181-9.
2.
Holtmaat, A.J., et al., Transient and persistent dendritic spines in the neocortex in vivo.
Neuron, 2005. 45(2):279-91.
3.
Nimmerjahn, A., Kirchhoff, F., Kerr, J.N. and Helmchen, F., Sulforhodamine 101 as a
specific marker of astroglia in the neocortex in vivo. Nat. Methods, 2004. 1(1):31-7.
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