As always this is meant to be a brief overview of a paper (and the methods used in neuroscience) I happened to have found interesting- for more details please refer to the manuscript itself.



I enjoyed reading a relatively recent paper published in Science from group in Japan (Kazuo Emoto, The University of Tokyo) that suggests a role for Ca2+ signaling in dendritic pruning, a house-cleaning function in neurons that has been shown to be very important in learning and memory, as well as experience and some forms of cognitive deficits (such as autism and neurodegeneration).

Dendrites are highly branched areas of the cell that act as antennae receive information from neighboring cell. Pruning refers to a highly complex, regulated, activity dependent process in which connections that are non-essential to the formation of the developing brain are cleaved.

In pruning, our neurons clean house. Unnecessary connections formed during development are trashed as a way to reduce clutter and improve accuracy and efficiency of signaling. Many of the basic mechanisms behind how pruning works have been identified. Specific cascades of enzymes (caspases) as well as a key self-destruction pathway (proteosome-ubiquitin) work together to rid individual neurons of unnecessary connections. While a lot is known about the processes that regulate the pruning, very little is known about the signaling that tells which dendritic arbors (branches of dendrites) are to be pruned and which of those is to be kept as a part of the neural network.

In their new work, Kanamori et al (2013) use the fruit fly Drosophila Melanogaster, which has four subtypes of dendritic arbors, to ask what local mechanisms in the dendrites are involved in pruning. Specifically they look at local calcium (Ca2+) signaling in a subtype of dendritic arbor called C4da. The “unnecessary” branching on these sensory neurons are generally pruned between 5 – 14 hours after puparium formation (APF).

Methodology

Ca2+ imaging: When neuroscientists want to look at changes in Ca2+ concentrations in neurons/glial cells they often employ a microscopy technique that involves fluorescence imaging. A great primer on fluorescence microscopy can be found here. Using the principle of fluorescence imaging, Kanamori et al (2014) use a genetically encoded Ca2+ indicator (GECI) called GCamp3. Briefly, they can genetically target this Ca2+ sensor to a specific type of neuron and image specifically from their neurons of interest. When they excite the GCamp3 with 488nm light, any GCaMP3 that is bound to Ca2+ will give off fluorescnece around 525nm, which can be recorded by highly sensitive research imaging cameras in real time. Throughout the paper (as is standard in Ca2+ imaging) values are reported as ΔF/F 0 , or change in fluorescence / baseline fluorescence.

Optogenetics: Similar to the way that the GCaMP3 is used, a light sensitive ion channel known as Channelrhodopsin-2 can be expressed in specific cell types. In this case, rather than simply giving off fluorescence that can be recorded, when blue light is absorbed by the chromophore retinal (a component of the ChR2) there is a change in the conformation of the ChR2 cation pore which opens it up and allows non-specific cations to flow in. This results in a depolarization of the cell, which is the primary signal transduction mechanism in neurons. That is to say that light, activating ChR2, can control neuron excitability. In this paper the authors use a specialized method of targeting light (470nm for ChR2) to specific neurons (digital micromirror device, Andor Mosaic 3) in order to precisely control the timing and location of sensory neuron stimulation.

Findings and Discussion

In order to develop an understanding of changes in Ca2+ and how it might correlate to changes in dendrite arborization (pruning) Kanamori et al first established the baseline levels of Ca2+ during these stages of development. Using the Ca2+ imaging described above, they image Ca2+ in C4DA neurons during early metamorphosis from 1 to 8 hours APF and show that there are large changes in Ca2+ in the dendrites of these cells while the Ca2+ in the soma and axons remain relatively stable.

They next show that dendrites that produce the most robust and frequent Ca2+ transients show the highest level of dendritic pruning. That is to say that those most Ca2+ active are the most likely to be pruned, suggesting that Ca2+ is an important mediator in the pruning process (Figure 2).

There are two main sources of Cain neurons: Cacan come in through ion channels on the membrane (such as voltage gated Cachannels, or VGCCs; or Camay be released from internal stores (either ryanodine receptor or IP3 mediated) such the endoplasmic reticulum. They choose to look at the link between CGCCs and the Catransients in the dendritic branches. Using a variety of knock-out and knock down Drosophila, they show that in neurons lacking a key subunit of VGCCs (ca-beta, auxiliary ß-subunit of Cavß) the Catransients are abolished and further in a variety of the VGCC mutants dendritic pruning is compromised.

In a very clever set of experiments they use targeted light to activate ChR2 on specific C4da neurons Kanamori and colleagues asked what contribution intrinsic local excitability had on whether an arbor was pruned or not. They first establish the threshold for triggering Ca2+ transients using ~ 1.1 mW/mm^2 of blue light at the time of 1 hour APF. Then again at 2-3 hour APF they measured the intensity of light to illicit Ca transients as compared to at 1 hour APF. They found that in branches that were bound for pruning, dendritic excitability (as measured as increased sensitivity to ChR2 activation of Ca transients through VGCC’s) was markedly increased (median of 0.44 mW/mm^2 needed to produce Ca2+ transients. In theses experiments they take great care to rule in the VGCC’s, but from what I could tell little attention was given to potential influences of calcium-induced Calcium release. It would be interesting to see if internal stores played a different role as the time APF increased.

Using a measure of the dendrites/cells sensitivity to different intensities of light as a measure of dendritic excitability was a very creative approach, especially to overcome what could be a difficult experiment using traditional electrophysiology.

Conclusions

The authors conclude that they are the first to demonstrate early, localized / compartmentalized Ca2+ transients play a role in dendritic pruning the the drosophila. I liked this paper as it uses very modern techniques to address questions in neuroscience that would be quite difficult to answer without precise Ca2+ imaging and even, in the case of figure 4, optogenetics. The implications may impact our understanding of discrete steps in dendrite and neuronal development, laying a foundation for a better understanding of complex behaviors such as learning. More and more labs are moving to purely optical methods as they tend to allow for macro-scale viewing of complex biology with removing the possible need for invasive perturbation (recording electrodes for instance) of the cell(s).