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Nature Methods - 2, 599 - 605 (2005)Published online: 21 July 2005; | doi:10.1038/nmeth777
Many memorable journal articles are those that present a new method to the science community. For this week’s TBT I’m posting about an article that literally made the experiments in my own thesis possible. Anne Taylor and colleagues published a novel way to compartmentalize cultured neurons in Nature Methods, 10 years ago (yikes!). One of the fundamental properties of neurons is that they consist of molecularly and functionally distinct compartments. Axons, cell bodies, and dendrites all have their unique roles to ensure proper growth and maintenance of the neuron, as well as for completing the neuron’s mission to transmit information from one neuron to another. The complexity of this compartmentalization is really quite beautiful; and can really be quite difficult to understand. This article presents a microfluidic chamber as a way to isolate (and thus study) separate parts of the cell, in vitro.
The paper describes their fabrication of the microfluidic device; essentially, the device is made up of two narrow chambers that are separated by tiny microgrooves. It is a simple design that takes advantage of the properties of diffusion—when there is a volume difference between the chambers the high fluidic resistance of the microgrooves (given by their “micro” size) produces a small but sustained flow from one chamber to the other. Importantly, this unidirectional flow will counteract any diffusion of small molecules (or drugs or growth factors, etc.) from the lower volume chamber. The establishment of flow allows for manipulation of one compartment and one compartment only. For example, one can ask if the action of a neuronal survival factor is necessary in axons or in cell bodies. The especially novel advantage of this method is that the chamber is made of a clear plastic polymer (PDMS) and is affixed to a glass coverslip, which allows for immunocytochemistry and live cell imaging in a compartmentalized culture system. In this paper specifically, they used the chamber as a model for studying axonal injury and regeneration. The authors showed that cutting the axons (axotomy) resulted in a host of proteins being upregulated in the cell body (despite the cell body being protected from injury), thus illustrating the axonal environment of a cell does have important effects on the biochemistry of the cell body.
The fun of a TBT is that we can look back and see which publications ended up being impactful in our field. This paper is an example of a methods paper that has really helped the field advance. This microfluidic system has been shown to be quite adaptable; many different versions of these chambers now exist for asking unique questions not only about injury and regeneration but also about synapses. And one day soon my own paper will be adding to the scientific conversation of how neuronal compartments compare, thanks to this novel method!
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"Progress in science depends on new techniques, new discoveries and new ideas, probably in that order." — Sydney Brenner
Bread, beer, and the flu are only a few of the many ways microbes welcome us to their worlds. By looking into the microbial realms, modern biology has found the genome-engineering tools to put the right transgenes in the right cells. These tools have changed the way researchers study neural circuits. Nowadays, to study the neurons responsible for specific behaviors, researchers typically screen through a library of pre-engineered animals, each expressing transgenes in a distinct group of neurons, with the hope that one of these neuronal populations controls the behavior of interest. Together with other tools, this library approach accelerated the discoveries of specific neurons controlling feeding, drinking, fighting, and parental behaviors. However, despite great effort, this strategy is still limited by the transgenic lines available and helpless when the relevant neuronal populations do not share a common genetic locus. For example, what if one wishes to study how higher-order, olfactory-processing neurons respond to the smell of apple pie? How can we control these pie-smelling neurons if we don’t know who they are?
Manipulating specific neurons without genetic access is like dancing with ghosts that cannot be seen. However, if we know enough about those ghosts, we may be able to monitor them indirectly by, for example, examining their surrounding air flow with smoke. In the case of neurons, the air flow is calcium and the smoke is c-fos. c-fos belongs to a group of genes called immediate early genes1, which are turned on quickly and transiently by elevated calcium in the cell, a proxy for neural activity. In the pie case, the pie-smelling neurons will become active when presented with a pie, and the resulting build-up of calcium in these neurons will turn on c-fos only in these cells. In this way, researchers can take advantage of the calcium-dependent c-fos promoter to drive expression of cell-manipulating tools, such as the light-activated channel Channelrhodopsin-22,3, in neurons that respond to the pie smells. In this way, researchers can gain control of the pie-smelling neurons, and study their circuitry by re-activating them after the initial labeling, only this time with light instead of the pie.
What else can be done with these c-fos-derived tools? Virtually anything that can be done with standard genetic tools. Researchers have already used optogenetics to both activate and inhibit the labeled neurons2–4. In the future, one might be able to use c-fos-driven reporters of cell activity to have a slow-motion view of the dynamics of only the labeled neurons during a behavior. One could use genetically encoded gene-manipulation tools to knock down or knock out genes in labeled neurons. One could use c-fos tools to study anatomy in an activity-dependent manner. One could even use c-fos derived tools to discover new drug targets by performing molecular profiling in neurons active in the process of interest.
Sometime in the future, calcium-dependent genetic tools may also be used to treat patients with hyperactive neurons (e.g. seizure). One could use c-fos to deliver hyperpolarizing channels only in hyperactive excitatory neurons. In this way, by allowing cell-specific tailoring of hyperpolarization drive, one could selectively suppress the activity of seizing neurons while leaving the rest to function normally.
- Sheng, M. & Greenberg, M. E. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4, 477–485 (1990).
- Gore, F. et al. Neural Representations of Unconditioned Stimuli in Basolateral Amygdala Mediate Innate and Learned Responses. Cell 162, 134–145 (2015).
- Liu, X. et al. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature 484, 381–5 (2012).
- Tanaka, K. Z. et al. Cortical representations are reinstated by the hippocampus during memory retrieval. Neuron 84, 347–54 (2014).