A Science Driven Life

An un-edited blog about science, discovery, technology, travel and the occasional whiskey

Targeting Light: Two recent papers that use active illumination!

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Screen Shot 2014-03-20 at 5.02.10 PMIn my day job I get to travel quite a bit and visit labs around the world who use methods in microscopy in imaging.  These labs range from neuroscience to developmental biology and everything in between, and many of these labs have very specific needs when it comes to light delivery.  The applications I most often work with can involve from high power lasers for ablation and thrombosis, or FRAP or photoconversion.  I’m very focused on optogenetics and uncaging, as well as any type of imaging, especially the use of genetically encoded indicators (for voltage, ions, temperature, pH, etc).  

While many people tend to use a simple configuration for single spot/point illumination, or full field illumination (and we provide these tools as well) where we are unique is in systems that allow for “targeted” or “active illumination.”  There are many methods for targeting light (possibly a good topic for an upcoming review) but the two of the main methods used are either using a galvo-mirror system (very fast mirrors that move a single spot of light in X-Y to create regions with the ability to target to a diffraction limited spot) or a digital micromirror device, or DMD.  

The DMD can be thought of as the chip that is in a typical overhead or presentation projector- a chip that has 100’s of thousands (or millions) of micromirrors that can switch in any shape/pattern (in <20 usec) allowing for diffraction limited light patterning with simultaneous regions in real time (see image below).  Using a DMD on a microscope, a researcher can select any pattern or shape of patterns on their sample, and deliver light to multiple regions simultaneously with fast switching in between (5000 Hz).  In the field of optogenetics, DMDs are becoming a very powerful and often necessary tool, especially in preparations where cells expressing the optogenetic probe or sensor are densely distributed and detailed spatial information is desired.

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Here I’ll briefly outline two recent papers that highlight the power of using targeted illumination (one with a commercial galvo-based device, one with a commercial DMD system) to tease apart spatial and temporal aspects of complex behavior and networks.  Those looking for all the details, I’ve linked the papers below.

Jain et al 2014 – Journal of Neuroscience – Mirror Movement-Like Defects in Startle Behavior of Zebrafish dcc Mutants Are Caused by Aberrant Midline Guidance of Identified Descending Hindbrain Neurons

The first comes from the lab of Michael Granato at the University of Pennsylvania who uses zebrafish (which have a very well characterized neuroanatomy) as their model organism for a variety of work in neuroscience and developmental biology.

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In this recent paper (Jain et al. 2014) Granato and colleagues looked at what are called “mirror movements”- where voluntary movements in one hand or finger (unilateral) is mimicked (unintentionally) in the opposite side of the body.  This is common in early development, but if it persists into adulthood it is referred to as Mirror Movement Disorder (MMD).  Mutations in the dcc gene, which encodes a receptor (netrin-1; a transmembrane protein of the immunoglobulin superfamily) that is important for guiding neuronal processes across the midline, (see reference Srour et al., 2010) has been linked to MMD.  Jain et al use the Zebrafish hindbrain as a model system to identify the role of a variety of neurons that could play a role in the deficits seen in MMD.

They first show that a mutant called “spaced out” (spo) that carries a mutation in dcc which causes disruption in naetrin-1 binding.  In these same mutants, they observe a behavior that is like the mirror movement – when one side of the body is touched an involuntary turn happens on the other side.  They go on to ask what part of the circuit may be involved in this miscommunication in the dcc mutants.  (again, this is a brief review and the curious reader is urged to check out the whole paper!)

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Using targeted ablation (essentially killing with a high power laser from the MicroPoint system) of specific ipsilateral projection neurons (a variety of MiD neurons- see those above pre-ablation and post-ablation from Jain et al, Figure 5), allows Jain et al to dissect apart the contributions of specific cells and cell types that lead to these involuntary turns.  By selectively ablating individual cells then testing the behavior, they can show which cells are involved in the mirroring pathway.  Granto and colleagues find that removing the inappropriately connected projection(s) in the dcc mutants results in restoration of unilateral behavior (no more mirroring) following a stimulus.   That is to say, that the targeting of light to individual neurons in the circuit is essential to tease apart what one single cell, or multiple cells may be doing in the complex motor circuit.

Cho & Sternberg, 2014 – Cell – Multilevel Modulation of a Sensory Motor Circuit during C. elegans Sleep and Arousal

The second paper for discussion is from the lab of Paul Sternberg (C. elegans!) at CalTech.  Julie Cho from the Sternberg Lab focused some of her PhD work on the question of how sensory and interneurons interact in the C. elegans during sleep and arousal.  C. elegans is a great model system as the sensory motor circuit is quite well understood and has a simple architecture.

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Interestingly, C. elegans will “sleep” (lethargus; 2-3 hour period of reduced activity and sensory responding) after activities such as eating, as well as after each stage of larval development (4 stages total).  They will respond less to sensory stimuli (avoidance behavior) as well during this period.  Cho & Sternberg knew from previous work that a sensory neuron called ASH was responsible for this avoidance behavior and designed a set of experiments to determine what part of the signaling pathway (ASH and its interneurons) mediate sleep and arousal.

Cho & Sternberg first use traditional chemical stimuli (Cu+) and look at responses (Ca2+ responses, they primarily use GCamp3) in ASH, as well as AVA and AVD (interneurons) and show that activating ASH results in increased Ca2+ in ASH neurons during awake and lethargus, but decreased Ca2+ during lethargus.  They go on to show that the activity between the two interneuron types (AVA and AVD) are synchronous during the awake state, but the AVA and AVD are asynchronous during lethargus (Figure 2, Figure 3).

To determine if the sensory modulation during lethargus is at the level of the ASH neuron or down stream, Cho & Sternberg switch to worms that have the optogenetic activator Channelrhodopsin-2 (ChR2) in the ASH neurons (remember, ChR2 is activated by blue light, and when active, opens a non-specific cation channel which leads to depolarization of the cell).  Using ChR2 allows for very direct activation of the ASH cells and downstream neurons.   By using targeting illumination of just the ASH (Mosaic DMD system) they show that it is in fact down stream (at the level of AVD and AVA) of the sensory neuron that is responsible for modulating the sensory state (Figure 4) and again this seems to be a function of synchronous vs asynchronous firing.  In a final, very clever experiment, they vary concentrations of all-trans-retinal (the cofactor that is required for ChR2) as well as light intensity, in young and adult worms, and show that the sensory modulation is highly reversible and it requires both the AVD and AVA interneurons.

Light Targeting!

These are just two examples of how precision targeting of light, in both space and time, is changing the way that researchers can study both complex networks and pathways.  It is an exciting time in science: as our research tools continue to improve, so will our ability to perturb and interrogate complex systems will, allowing us to better address and answer some of the most fundamental and important questions in biology.

Image credits:  Zebrafish, C. elegans both are from Wikipedia Commons.

Written by Michael Mohammadi

March 20, 2014 at 16:04

One Response

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  1. […] live organisms (c. elegans) and in vivo in rodents.  As researchers demand greater and greater spatio-temporal control of signaling pathways it’s safe to assume those identifying and developing these new tools are up to the […]


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