Spine structural change - Nothing to do with physiology ?

Today's journal club was about Shrinkage of dendritic spines associated with LTD, Neuron. 2004 Dec 2;44(5):749-57. from Poo's lab.

Blocking of cofilin phosphorylation blocks spine shrinkage without affecting LTD, and blocking PP1 blocks LTD without blocking spine shrinkage. Both are blocked by calcineurin blocker. These suggest the LTD inducing stimulation activates the calcineurin pathway, and the pathway branchs out to produce two different phenomena, LTD and spine shrinkage.

Clearly spine structural change is NOT required by the change in synaptic strength. This is probably the same for LTP and spine enlargement: I heard from several guys that the washout by patch-clamping often blocks selectively the spine enlargement but not LTP.

Then why the spine structure changes ? One possibility is the structural change is just a side effect: because the actin polymerization is regulated by a balance of kinase/phosphatase activity, LTP/LTD inducing stimulation, which changes this balance, can change the sturcture of spines. Or, maybe this is important for late-phase LTP: larger spines can contain more resource to maintain LTP.

Background subtraction in calcium imaging

"For analysis of calcium transients, the fluorescence background was subtracted from the fluorescence intensity averaged over the line" -- XXX et al., XXXX, 2005

2-photon calcium imaging became popular as a tool to quantify calcium signaling in neurons. In a lot of papers published, fluorescence signal is subtracted by surrouding background fluorescence. However, generally this is a WRONG idea.

----Quote from Yasuda et al., 2004 Science STKE ------
Relating fluorescence and [Ca²⁺] requires subtraction of background from the fluorescence signal. Because neuronal compartments such as spines and boutons are typically smaller than the excitation volume of 2PLSM, the background calculation is complicated. Background fluorescence is commonly estimated by measuring fluorescence far from the spine, F_B. ΔF/F_o is defined by Eq. 12, where F and F_{o, raw} are the raw fluorescence signals during the response and baseline periods.

ΔF/F_o = (F-F_o,raw)/(F_o,raw - F_B)) (Eq.12)

Because the spine volume is smaller than the excitation volume, F, F_o,raw, F_raw, and F_B can be expressed by Eq. 13, where V_sp and V_ex are the spine volume and the excitation volume outside the compartment, b_sp and b_ex are background fluorescence intensities inside and outside the compartment per unit volume, respectively, and f_o and f are the fluorescence intensities from fluorophore in the compartment per unit volume before and after the stimulation.

F = (f+b_sp)V_sp + b_exV_ex
F_{o, raw} = (f_o + b_sp)V_sp+b_exV_ex
F_B=b_ex (V_sp + V_ex)

Because the excitation volume is V_sp+ V_ex, F/F_o is defined by Eq. 14.

ΔF/F_o = (f-f_o)/(f_o-(b_ex-b_sp)) (Eq.14)

Therefore, this type of background subtraction gives the correct value only if b_sp = b_ex, that is, if background is exactly the same inside and outside spine. Unfortunately, this is rarely the case. Under some conditions, depending on the excitation wavelength, spatially heterogeneous background fluorescence is excited. More importantly, it is often the case that small quantities of indicator spilled in the extracellular space during patching produce substantial background fluorescence in the brain slice. In this case, background subtraction always results in an underestimate of F_o and a resulting overestimate of F/F_o. Moreover, this error depends on the size of the compartment; it is larger for smaller compartments. In our view, quantitative measurements demand conditions in which the background is not significantly different from the dark noise of the PMT. If background fluorescence is higher than the dark noise of the PMT, the data are severely compromised for analysis of the amplitudes and time-courses of transient changes in [Ca²⁺].

Effects of coverslip on PSF

We use water-dip type objective for our two-photon microscopy. However, if there is a coverslip between the objective and the sample, the PSF(Point spread function) is degraded significantly.
This is because the these objectives are not designed for this situation.

The refractive index of water is 1.33, and coverslip is 1.52. Thus if there is a coverslip between a sample and an objective, an incident light is refracted by the coverslip and make a focus at a different point than the focal point without the coverslip. The shift (D) can be calculated as:


D(θ) = a(tanθ - tanθ')/tanθ'

Where N/N' = sinθ / sinθ' (N: refractive index of water, N': of coverslip), and a is coverslip thickness (typically 170 μm).

If optics are at the near-axis, or sinθ ~ tanθ, D does not depend on the incident angle θ. However, for a high NA objective, this is not the case. For example, for NA=0.9 water-dip objective, maximum θ is Asin 0.9/1.33~42^o. In this case the shift D is 42 μm compared to 24 μm at the near axis, the focus position differs by δD~18 μm in z-axis.For NA=0.5, δD~3 μm. Thus, a coverslip degrades PSF significantly.

Therefore, in my opinion, for an in vivo application, one should underfill the backfocal plane with the laser to save laser power.

Pokemon

These guys can't be serious. They named a proto-oncogene as Pokemon = POK erythroid myeloid ontogenic factor....

Recording from axons

-Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons J. Neurosci., 25(2):454-463;

-Determinants of Action Potential Propagation in Cerebellar Purkinje Cell Axons
J. Neurosci., 25(2):464-472 (Mike Häusser's lab)

Amazing technique! They performed simultaneous measurements of action potentials at soma of Purkinje Cells and their axon to quantify the reliability of axnal spike propagation. If a cell spikes regularly, the axonal propagation is reliable even when the frequency exceeds 200 Hz. Interestingly, complex spike pattern such as bursts associated with CF activtion causes unreliable propagation. The reliability is determined by inter-spik-interval (for <2ms ISI, the second one is often skipped) and membrane voltage.

2-photon papers (Karel's recommendations)

"Anyone doing 2-photon should have read and understood these key papers." (Karel)

Key Reviews
Denk, W., and Svoboda, K. (1997). Photon upmanship: why multiphoton imaging is more than a gimmick. Neuron 18, 351-357.

Microscope building
Mainen, Z. F., Maletic-Savatic, M., Shi, S. H., Hayashi, Y., Malinow, R., and Svoboda, K. (1999). Two-photon imaging in living brain slices. Methods 18, 231-239.

Tsai, P. S., Nishimura, N., Yoder, E. J., White, A., Dolnick, E., and Kleinfeld, D. (2002). Principles, design and construction of a two photon scanning microscope for in vitro and in vivo studies. In Methods for In Vivo Optical Imaging, R. Frostig, ed. (CRC Press), pp. 113-171.

Imaging in scattering tissues
Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J., and Charpak, S. (2001). Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J Neurosci Methods 111, 29-37.

Lichtman's talk

Jeff Lichtman gave a talk about competition between axons connecting to a single neuromuscular junction on 1/10 here at Cold Spring Harbor Lab. His group has been imaging axons expressing YFP or CFP, and directly measure the battle between two axons: both axons try to take more synapse area. Interestingly the fate of the competition is determined globally: if a yellow axon loose the battle at one synapse to a blue axon, most likely all of other synapses of the yellow axon also loose to the blue axon. Furthremore, the one loosing battles always innovating more synapses. Thus, it seems like a single axon has only a limited amount of resource, and if one keep winning to have more synapses, the one run out of resources, and start to loose other battles. Interesting questions would be 1) what kind of resource limits the number of synapses and 2) how experience of synapses affect battles. It would be important to measure activity of synapses of winning and loosing axons to solve these questions.

Papers (Neuron 1/6/5)

-NMDA Receptor-Dependent Activation of the Small GTPase Rab5 Drives the Removal of Synaptic AMPA Receptors during Hippocampal LTD (José Esteban's et al.) Neuron, Vol 45, 81-94

Congratulations, José ! It might be fun to see interactions between Rab5 and the C terminus of rabaptin-5 (last 74 amino acids) using our FLIM system.

-Dendritic Spine Heterogeneity Determines Afferent-Specific Hebbian Plasticity in the Amygdala (Andreas Lüthi et al.) Neuron, Vol 45, 119-131

It is always great to see a paper citing my paper! They identify spines connected by thalamic and cortical affarents by calcium imaging. These spines are functionally and morphologically distinct. it surprises me a bit that one can do this kind of experiment. Looking for a stimulated spine is difficult especially if one stimulates far from a patched neuron. It is a kind of needle-in-haystack problem: only a small fraction of spine is activated to show a rapid and stochastic (some times release probability is less than 0.1) calcium responses.

Cloning note

I had a chat with my colleague about why Clontech's vectors produce much weaker protein expression than other CMV-based vectors (such as pCI, pcDNA3, pRK5) do, even though they use the same CMV promoter ? It seems like following features of the clontech vectors make them less efficient:
-Clontech vectors use early SV40 polyA signal instead of late SV40 signal. The late SV40 is ~5 times stronger than the early-SV40 (According to Promega note).
-Clontech vectors do not have introns between the promoter and the SV40 polyA signal. It is widely believed the presence of introns is required for optimized protein expression.