Day and night, breakfast and dinner, winter and summer, wake and sleep…our lives are dominated by interacting rhythms in our environment and our behavior. Why is it that we sleep at night and not during the day? Why are heart attacks and strokes more common in the morning than the evening? How do animals adapt to winter and summer? Why do we get jet-lag, and what is it, exactly? All these questions revolve around a central subject in neuroscience: circadian clocks. During day 1 of the Society for Neuroscience annual meeting in San Diego, CA, I was treated to a nanosymposium (timely insights in circadian regulation) highlighting new and exciting research in this area. Chaired by Steven Brown and Alessandra Porcu, this session covered all aspects of circadian biology, from behavior to neuronal circuits, and from synapses to molecules.
The suprachiasmatic nuclei (pictured above) serve as the master clocks controlling mammalian circadian rhythms (Credit: Jeremy C. Borniger, PhD; Stanford)
Here, I highlight a few of the talks that I found the most interesting. Unfortunately, I am not able to cover everything, and some really cool stuff slipped through! That’s the downside of this immense conference…there’s never enough time to see everything!
Two talks on the same protein (one in flies and the other in mammals) grabbed my attention. These talks were given by Masashi Tabuchi and Benjamin Bell, two researchers from Johns Hopkins University. During Masashi’s talk, he described a potential mechanism by which the protein Wide Awake (WAKE) regulates sleep/wake cycles in flies.
WAKE regulates sleep quality through appropriate timing of neural firing codes (Credit: Tabuchi et al., 2018; Cell)
He showed that irregular neural firing rates during the day (regulated by WAKE) promote arousal while regular firing patterns during the night promote sleep.
Ben Bell followed up his talk by taking their findings in flies to mammals, describing a mammalian ortholog to the fly WAKE protein (called mWAKE). mWAKE is highly enriched in the master clock, the suprachiasmatic nucleus (SCN) suggesting it plays a role in circadian time keeping or regulation.
Unlike in flies, knockout of mWAKE in mice only caused mild problems in sleep/wake states. However, through measuring locomotor activity, the researchers found that these mice were extremely hyperactive (>5 times more active than wild-type mice). Curiously, this trait (phenotype) only came about during the dark phase (mice are nocturnal, so active during the dark phase). To investigate this further, the researchers examined the firing rates of SCN neurons during the day and night. Normal mice have a large difference between the night and day in SCN firing rates, with peak neural activity occurring during the day. However, mWAKE knockout mice showed no difference between day and night, with firing rates remaining high all the time!
Additionally, cells lacking mWAKE showed blunted responses to the inhibitory neurotransmitter GABA, and this lack of inhibition may explain the hyperactive profile mice lacking mWAKE had. Finally, they examined (using an mWAKE reporter mouse) where mWAKE expressing cells project to throughout the brain. They found that cell bodies were distributed throughout the brain, in all major arousal centers. Importantly, they seemed to be discrete from other neuromodulator systems present in these areas, like hypocretin/orexin neurons in the lateral hypothalamus, or histamine neurons in the tuberomammillary nucleus.
Significant more research is required to fully understand the role this protein plays in sleep/wake states. Is it a ‘master regulator’ of arousal? Does it interact with every ‘arousal center’ differently or does it have a distributed ‘homogenous’ effect across the brain. When does mWAKE start to express during development? Does this coincide with changes to sleep-wake behavior during early age? I’m excited to follow this story going forward!
Expansion Microscopy - ‘Just add Water’
Microscopes are getting beefier and beefier, more complex and expensive, with the sole purpose of being able to see tiny, tiny things just a little bit better. Enter ‘expansion microscopy’, an idea that literally works in the opposite direction to that goal. Instead of ‘zooming in closer’, expansion microscopy aims to ‘blow things up’ in order to see the (once) tiny details (like synapses, or nuclear pores…) on a conventional microscope. Remember those dinosaurs that would expand when you added water as a kid? I sure do…and expansion microscopy works pretty much the same.
Although this technology has been around for a few years, it is just getting started in terms of its ease of use, applicability to different samples (proteins, RNA, DNA, lipids…), and support community. All info on this fascinating technique is available at ExpansionMicroscopy.org.
First described by Edward Boyden and colleagues at the MIT Media Lab in 2015, expansion microscopy is rapidly being applied across fields, species, and disciplines to examine extremely fine structures at the nanoscale (10-20 nm).
Expansion microscopy allows for uniform expansion of a biological sample. Here, we see a brain slice (in panel B) which has been weaved into a polymer mesh with biomolecular anchors. When the polymer is expanded (‘Just add water’), it pulls the biomolecules along with it, maintaining the relative spacing between structures. In (C ) we can see that same brain slice ‘expanded’, revealing tiny pieces of biology previously too small to see(Credit: Chen et al., 2015; Science).
Ed Boyden provided a ‘state of the art’ summary of expansion microscopy to date at a minisymposium today titled “new observations in neuroscience using superresolution microscopy” chaired by Michihiro Igarashi. He gave a quick overview on how they developed the idea that was to become expansion microscopy, through adapting old techniques from the early 1980’s. Next, he discussed the problems of ‘expansion’, the primary one being ’ how can we evenly expand a sample without losing valuable spacial relationships between proteins, DNA, RNA etc…? To overcome this problem they needed to develop biomolecular anchors, which link the molecular target to the polymer mesh. In this way, isometric expansion of the mesh results in the same for the anchored sample.
Using this technique, many researchers have expanded tissues to look at things like synaptic proteins and microtubules at a much finer detail than what was previously possible with conventional confocal microscopes. Others have adapted the technique to work with in situ hybridization, allowing for expansion and quantification of RNA. Dr. Boyden’s lab is also working on expanding non-soft tissues, like bone, and using expansion microscopy in the clinic to diagnose and investigate cancer in unprecedented detail (so called ‘expansion pathology’).
By combining expansion microscopy with RNA visualization (ExFISH) and sequencing (MERFISH), hundreds of transcripts can be examined simultaneously in situ!
Towards the end of the talk, Dr. Boyden highlighted some open questions in the field. These questions focused on a few primary themes:
Can we validate expanded samples below 10-20 nm?
Is expansion ‘pulling’ synapses apart, leading us to false conclusions?
Can we use this technique to probe protein-protein interactions?
Whats the smallest thing we can expand? Can we expand a virus? A DNA origami??
How much can we expand a sample while maintaining all relevant spatial relationships?
To take the last question, Dr. Boyden’s team reasoned, if we can expand something once, why not twice, or thrice?? They put samples through an iterative process allowing for expansion up to 20x the original size!! (shown below)
Iterative Expansion Microscopy allows for sample expansion up to 20x! Panel A shows dendritic spines without expansion, panel B shows the same at 4.5x expansion, and panel C shows dendritic spines at 20x expansion after the iterative process is complete (Credit: Chang et al., 2017; Nature Methods)
A cool side effect of expansion is that it involves filling the sample with water, making it essentially transparent, and useful for long-range circuit mapping at high detail or speeding up techniques like light-sheet microscopy. We are only at the surface of what is possible with this and other super-resolution techniques. I look forward to all the exciting things to come!
That’s my two cents for day 1. Keep an eye out for more coverage of some of the coolest stuff at SFN 2018!