by Michael Mohammadi

Being free from the “chains” of academia I have been able to expand my scientific interests well beyond NMDA receptor signaling and short term memory. I do miss actually producing research from time to time, but I now average 6 papers read in a week which is about double (or more) what I read in grad school so I’m still feel like I’m part of the process. I do have the luxury of spending a lot of time in planes and on trains, both excellent venues for diving into a paper with few distractions (Bose noise canceling headphones are essential!). I have found that it is liberating to be able to read articles from all different fields of science and not be limited to a very specific field or research question. Exploring new research in neuroscience, physiology, physics, optics, imaging and more has really rejuvenated my spirit for science and discovery, a spirit that had faded over the long duration of wrapping up my dissertation. Now that this curiosity and excitement is back and fully charged, I hope to share some of the cool papers I’m reading with you. It is my goal with this “Science in press” series that I review a few papers that I have read in the last few weeks that really stood out. These may be a bit more technical than my other articles, but I hope to keep it accessible to the mainstream reader. As always, questions are encouraged.

For this first installment I tried to cover 5 papers I read recently, but I ended up a bit too excited and went into a lot of detail on paper one. I’ll try to be more concise in the future if it’s more interesting to get into the details let me know, I would enjoy writing either way! So I ended up giving overviews of two recent papers in Nature Neuroscience.

I welcome criticisms and feedback, suggestions on papers to read, as well as corrections to my interpretations or explanations of the experimental design, results or conclusions. I accept I may get things wrong and hope to learn from my readers. Without further ado…

1. Optical control of metabotropic glutamate receptors. Levitz et al, 2013 Nature Neuroscience

I’ll start with a recent paper that employs optogenetics for something other than direct gating of ion channels! Dr. Ehud Isacoff’s group at UC Berkely has been doing some amazing work in the field of molecular engineering with optical probes (among other things). Previous work included a very cool probe called HyLighter (of which some data was acquired with the Mosaic) that is a light-activated glutamate channel that selectively gates K+. In this most recent paper, Levitz et al describe a metabotropic glutamate receptor (mGluR) which is a specific type of G-Protein Coupled Receptor (GPCR; the most abundant receptor type in the body) that they have engineered to respond to specific wavelengths of light which results in a variety of downstream G-protein regulated outputs.

A large part of the current work is based on the G i/o pathway-coupled group II mGluRs, specifically mGluR2 and mGluR3. When activated, these GPCRs can activate adenylyl cyclase, a specific type of K+ channel called GIRK channels, and can also block Ca2+ channels to reduce transmtiter release from a presynaptic neuron. Throughout their work they will show that through activation of these modified GPCRs that respond to optical stimulation (LimGluR2/3), result in precise control of these different pathways. They include some data with engineered group III mGluR (mGluR6), but here I will focus on LimGluR2.



LimGluRs are light-agonized and light-antagonized mGluRs. That is, they are mGluRs modified to respond to very precise wavelenghts of light; specifically they can be activated by UV light (380 nm) and inactivated by green light (500 nm). This is accomplished by attaching a D-maleimide azobenzene glutamate (D-MAG) to the ligand binding domain. The light causes isomerization to the cis state (380 nm light) or isomerization to the trans state (500 nm) light. This shift results in rearrangement/conformation change resulting in subsequent activation of the G proteins (Figures 1 and 2 for more details). They demonstrate clearly that activation/inactivation can happen on very fast time scales (1 ms).

They go on to show a variety of physiological output that can be gated with their LimGluRs. For instance, in cultured hippocampal neurons they can modify the excitability of a cell by giving a normal current step under 500 nm light (deactivated LimGluR, therefore we expect action potential firing at normal membrane potential) and quantifying the number of spikes. When they switch to 380 nm light, they activate the G protein, which hyperpolarizes the membrane, decreasing excitability and therefore reducing the number of action potential (spikes); Figure 5- seen here (violet bars show when they give 380 nm, green bars indicate 500 nm light).

In Figure 6 we see reversible gating of voltage-gated calcium channels and how this can affect excitatory and inhibitory currents, as well as a well described measure of pre-synaptic transmitter release (paired pulse facilitation). We finally see some work in hippocampal slices (live tissue) in Figure 7 where they repeat Figure 5 but under more physiological conditions. The paper finishes by showing that LimGluR2 can modify escape response behavior in live zebrafish larvae (a favorite of the Isacoff lab), importantly showing that without the D-MAG-0 they see no response.

Isacoff and his team are world leaders in this field and I would highly recommend a read-through of this paper (especially the details of how they engineered the LimGluRs) as well as their previous work. There are three things I really like about this most recent paper: 1) this is a non-opsin based method for optogenetics and focuses on GPCRs, 2) unlike the opsin-based-GPCRs, LimGluR is highly reversible on a very fast time scale (1 ms). 3) the use of optogenetics to control GPCR responses is one of the most exciting directions the field is heading. GPCRs are the most abundant receptors in the body and can modulate a variety of ion channels and signaling pathways.

2. Saccadic eye movements evoked by optogenetic activation of primate V1. Jazayeri et al, 2012 (Nature Neuroscience, 15:10, p. 1368)

Ok, I didn’t mean to do it but I did. Another paper on optogenetics. It’s just so hard to get away from, the majority of the papers I’m reading are within this field, and frankly, some of it is so “sci-fi” and cool I can’t resist…so here we are.

I first read this paper at the end of February going into a talk at the University of Washington in Seattle. I thought it would be interesting to point out some of the work using optogenetics that was ongoing at UW. This paper comes from the lab of Dr. Gregory Horwitz and is (to my knowledge) the first paper using optogenetics to modify behavior in a primate.

Jazayeri et al. (2012) uses two monkeys in which they have used Adeno-associated virus (AAV) to target the ChR2 gene in the primary visual cortex (V1). Using fluorescence staining (ChR2-mCherry; figure 3 seen here) they show that the ChR2 was expressed where they expected (near the injection site) and in sufficiently high expression. The ChR2 was most densely expressed in Layer 4B, with some ChR2 in Layers 5 and 6 as well (much less than 4B). They do suggest that the high expression in specific layers could be a result of their viral vector (AAV1) and I would expect that they, and other groups will investigate this more thoroughly in the future.



Now to the exciting part; the basis of their experiments is a specific type of conditioning they use to train the monkeys to pay attention at the end of a visual stimulus. The monkeys heads are immobilized and the researchers used a custom recording system to track the positions (saccades) of their eyes on-screen. They are then given a 0.5-1.0 second pulse of light in the center of their visual field (this is called the Fix trial). At the end of the trial they receive a liquid reward which coincides with the point of light disappearing. In half of the trials, they are given the same central fixation point of light and the same reward, but also optical stimulation in V1 for 0.10-0.25 s (130 ms after disappearance of central fixation point via optical fiber near the site of injection on the dura mater). The idea here is that over time, they will pair the central fixation point (CFP) with a reward and the optical stimulation will train their V1 to the CFP. In a second group of experiments, called Tar, the monkeys are given a reward only after they made a saccade to a visual target that appeared after the disappearance of the Fix interval (100 ms later). Again, they add a Fix + Op group, in which they apply optical stimulation is paired with the reward for a correct saccade.



They propose the following hypothesis: Using optical stimulation they could “bias saccades toward the receptive field of the optically stimulated neurons.” To test this hypothesis they analyze the saccade endpoints in trials both with and without optical stimulation (Figure 1C). Jazayeri et al. show that the are able to train the monkeys to fix on a central point, then using optical stimulation in V1, the monkeys will saccade to a specific endpoint (with very prcise grouping) based on where they were trained to respond. That is, they use behavioral conditioning to teach the monkeys’ brain to respond very specifically to an optical (ChR2) stimulus! (A nice figure is supplementary figure 4 in which they show that even if they change the position of the visual saccade endpoint, optical stimulation of ChR2 will bias their saccade back to the receptive field of the stimulated neurons).

In the only other figure in this Brief Communication, Jazaryeri et al. show some multiunit recordings (electrophysiology data) that show a variety of responses to stimulation of ChR2 with both excitation and suppression of firing depending on recording sites (Figure 2A-D). One explanation they offer for the fact that the normally excitatory ChR2 acted sometimes as inhibitory is that the SYN1 promoter they use may target ChR2 to both excitatory and inhibitory neurons. This possibility could be explored with more advanced staining/fluorescence methods.

Overall I like the paper a lot. It is simple in its hypothesis, design and conclusions, and opens the door for a wide variety of follow-up studies in the primary visual cortex as well as other brain regions. As the first paper using optogenetics to modify primate behavior, I think it is quite effective.

In closing…

I hope you found this as exciting as I have and urge you to click on links throughout the page (no commercial interest other than the Andor link) for more information and details. As I continue these reviews of current science in my Science in Press series, I hope to expand to other areas of neuroscience and biology, as well as optics and some physics. As always, please feel free to suggest papers and topics for review!

Cheers!