Deciphering the syntax of Nature article titles

We all want to publish in Nature. Papers in Nature are (supposed to be) the complete package: reliable results that show something novel; cool techniques; a famous corresponding author. And if you want to get one, you need a title that shows you are a refined gentleperson who belongs in the Nature club.

So to help you, dear blog reader, I have scoured the archives of Nature* to decipher the ideal form of Nature titles:

[research-y verb-ing] a neural circuit for [behaviour]

For example in hunger there are: Genetic identification of a neural circuit that suppresses appetite; and Deciphering a neuronal circuit that mediates loss of appetite. Or in anxiety there is Genetic dissection of an amygdala microcircuit that gates conditioned fear. Disambiguate is an underused verb here.

If you're feeling poetic, you can rearrange the elements. For example, you can try putting the neural stuff first, like The neural representation of taste quality at the periphery. Or in olfaction, there's Neuronal filtering of multiplexed odour representations.

If you are particularly concise, you can drop the verb altogether, and just combine a couple nouns with a preposition. The cells and peripheral representation of sodium taste in mice. Perception of sniff phase in mouse olfaction. Distinct extended amygdala circuits for divergent motivational states.

Under NO CIRCUMSTANCES are you to mention the brain region, molecular marker, or techniques you used to [verb] your [behaviour]. If you are studying how photostimulating AgRP neurons induces feeding, don't mention photostimulation or AgRP (Deconstruction of a neural circuit for hunger). Otherwise, you might end up in Nature Neuroscience (AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training).

And, most importantly, keep it short. If you're studying taste receptors, go for something like An amino-acid taste receptor. Stuff like Gustatory expression pattern of the human TAS2R bitter receptor gene family reveals a heterogenous population of bitter responsive taste receptor cells goes in the Journal of Neuroscience.

If you have any other examples of the beautiful Nature titles, write in the comments!

*skimmed the tables of contents

Bonus titles:
The receptors and cells for mammalian taste.
Excitatory cortical neurons form fine-scale functional networksA family of candidate taste receptors in human and mouse
Short-term memory in olfactory network dynamics.The detection of carbonation by the Drosophila gustatory system
The cells and logic for mammalian sour taste detection.
The subcellular organization of neocortical excitatory connections.
The receptors and coding logic for bitter taste
The cells and peripheral representation of sodium taste in mice
The molecular basis for water taste in Drosophila

Channelrhodopsinning: Your light doesn't always do what you want.

Two years ago, I wrote a post about the common mistakes I notice in channelrhodopsin papers. Since then, two labs have developed improved photoactivatable chloride channels for inhibition, binary logic has been introduced to neurons, and you can use one vector to photostimulate and record from a neuron type. Beyond those headline advances, though, were some smaller papers that highlight some of the pitfalls of channelrhopsin use.

As always, given that I've published exactly one paper using ChR2, take these opinions with a Churymov-Gerasimenko comet of salt.

A crumbling pillar

In that post two years ago, I laid out my Two Pillars of Channelrhodopsin: always perform negative controls (I'm still surprised that this actually needed to be said); and always pulse your light.  In particular I was critical of a paper by Kravitz and Kreitzer wherein they used the PINP/phototag technique to record from medium spiny neurons (MSNs) in the direct and indirect pathways of the striatum. To identify, for example, direct pathway MSNs, they expressed ChR2 in those cells, recorded from them, and stimulated with 1 second long light pulses. Units that responded to light with short latency spikes (less than 15ms ) were considered identified. At the time I called this a "crap criterion," because I thought the time window was too long to precisely identify neurons.

A few weeks after my blog post, Kravitz and Kreitzer published a paper in Brain Research expanding on their technique. They wrote,

Medium spiny neurons have two properties that make them unsuited to identification protocols that require high spike fidelity [ed. note: pulsed light stimulation]. First, medium spiny neurons have very low excitability [for an example of MSNs' late firing properties, see the figure below, from this review by Kreitzer], and fire at low spontaneous rates in vivo. It is therefore difficult to drive them to spike reliably and at short latencies without using extremely high-powered illumination... Second, medium spiny neurons have variable membrane potentials which continuously fluctuate between approximately −50mV and −80mV in vivo. As we cannot monitor the membrane potential in our extracellular recordings, we do not know whether the cell is close to spike threshold when we deliver each laser pulse... While it would be simpler if identical techniques could be applied for optogenetic identification across multiple brain structures, it appears that optogenetic identification procedures will need to be optimized for each different brain structure, a situation that occurs with nearly all technical approaches in neuroscience.

MSNs are really hard to stimulate. Recordings from an MSN in response to current injection. MSNs only fire an action potential after prolonged stimulation.

Since then, I have recorded from MSNs in GPR-88 mice using an optrode, and I can confirm that neurons respond robustly to long square pulses (>10ms), but not to trains of short pulses. I was wrong, and I apologize to the Kreitzer lab.

Another interesting aspect of their review was the low yield. They recorded ten mice, and 143 well-isolated single units, and from that recorded only 19 PINP units. Some of the low yield was due to the difficulty of unit identification: of the multiunits recorded, around half were light responsive. (In my test mouse, my yield was slightly higher, ~7 cells, but perhaps my criterion are less strict.)

While a yield of ~2 units per mouse seems low, if you run the numbers it makes sense. In many brain regions, half of the units you record will be from non-target neurons, and of the target population only a fraction of the neurons will express ChR2 at a high enough concentration to be usable. So in the end, only ~10-30% of the units you record, if you are doing everything right, will express ChR2. If you record 20-30 units / mouse, which seems reasonable to me, you would end up with a high of 2-9 neurons per mouse. If your brain region is smaller, these yields would drop.

Channelrhodopsin can inhibit too

While the Kreitzer lab showed how long light pulses can work, former Duke post-doc, now Baylor PI, Ben Arenkiel put out a paper that highlighted why, in general, I prefer pulsed light. They expressed channelrhodopsin in a wide variety of neuron types throughout the brain (mitral cells, cortical pyramidal cells, interneurons, and more), and patched onto them. While they were recording, they stimulated with trains of light, varying the frequency and the duration of each pulse (from 1 ms to 49 ms (or near constantly)).  They found that, as you increase the pulse duration, stimulation fidelity decreases, and you in fact can begin to inhibit neurons.

Recording from CRH neuron in the olfactory bulb. They stimulate the neuron at 20Hz with light pulses (top) or current injection (bottom); and vary the width of the pulses from 10ms (left panels) to 49ms (right panels, ~100% on time). At the short pulse width, the neurons are able to follow the 20Hz stim. With the long stimulus, the neurons do not fire action potentials, and are actually inhibited. From Herman, et. al. (2014)

The idea that prolonged stimulation could cause inhibition is not new - we've known about shunting inhibition for a while - but now we have evidence that it can happen with channelrhodopsin. More intriguing, the response depended on neuron type. Most neurons, presumably with low basal firing rates, were inhibited by prolonged light, but a subset of fast-spiking neurons were actually even more excited by constant photostimulation.

Plot of firing rate versus pulse duration for two types of neurons photostimulated at 20Hz. Most neurons decrease their firing rate at long pulse duration, like the cortical neurons shown in D. Some, however, increase their firing rate (E). From Herman, et. al. (2014)

My conclusion from this paper is the same as that from Kreitzer's review, "optogenetic identification procedures will need to be optimized for each different brain structure." You need to record from your neurons of interest in slice, or if you can't, ask those weirdo slice physiologists down the hall for a solid. You need to know how fast you can stimulate reliably, and how long your light pulses should be. Otherwise, you might be inhibiting the neurons you are trying to stimulate, or making them fire faster than you thought you were.

Spikes != synaptic release

So you've recorded from the cell type you want to stimulate, and ran the cells through their paces to choose a stimulation paradigm. You're good to go, right?

A couple weeks ago in joint lab meeting, a post-doc presented data where she recorded from a pair of neurons: one neuron expressing ChR2, and the other downstream. She stimulated at 10-20Hz for a short while, and saw something like this:

In the middle trace, the cell expressing channelrhodopsin easily follows the 10Hz train. However, in the bottom trace, the downstream cell receives a strong EPSC for the first light induced action potential, but the EPSCs get smaller as the vesicle pool is depleted, until they all fail. Then, after the stimulus is ended, there is a refractory period where synaptic activity is absent. 

This is, of course, an obvious result if you think about it. Yet I hardly ever see it mentioned. Just because your neuron can follow your channelrhodopsin stimulation doesn't mean that it's actually releasing vesicles.

So be careful out there channelrhodopsinners. Choose the right stimulus paradigm, make sure it's not inhibitory, and hope that the axon terminals can keep up with the action potentials. Your light doesn't always do what you want.

References:

Herman AM, Huang L, Murphey DK, Garcia I, & Arenkiel BR (2014). Cell type-specific and time-dependent light exposure contribute to silencing in neurons expressing Channelrhodopsin-2. eLife, 3 PMID: 24473077

Kravitz, A., Tye, L., & Kreitzer, A. (2012). Distinct roles for direct and indirect pathway striatal neurons in reinforcement Nature Neuroscience, 15 (6), 816-818 DOI: 10.1038/nn.3100

Kravitz AV, Owen SF, & Kreitzer AC (2013). Optogenetic identification of striatal projection neuron subtypes during in vivo recordings. Brain research, 1511, 21-32 PMID: 23178332

Questions of Taste

Some time ago, Neuroecology asked people in the twitterverse what the biggest questions in their field are. While I'm no longer working on taste, these are the three big questions to my mind. Take these with a rock of salt, as I'm not 100% up to date on the literature, and these are just my opinion.

0. To what extent, and where, is taste a labeled line system versus a combinatoric one?

This is the grand debate in the field. On one hand you have Charles Zuker pushing the idea that labeled lines - that each of the basic taste qualities is represented separately with its own "line" - extend from taste cells in the periphery to deep into cortex in the form of segregated hotspots. On the other hand, everyone else in the field believes that taste is a combintarial sense, which means that the lines for individual tastes begin to overlap early in the system, perhaps even in taste cells tongue, and certainly in the brainstem and cortex. (There is an interesting inside/outside dichotomy in people's opinions. Most people outside the taste field assume Zuker is correct, while everyone inside assumes he's wrong.)

I personally think the evidence right now is that there are labeled lines in the periphery which combine in the central nervous system, probably near the NTS. But the three questions below are aimed at answering this overarching question in the periphery and cortex.

1. What in God's name is going on in the taste bud?

Due to the work of Zuker, Margolskee, and others, we have identified the basic taste receptors and the cells that express those receptors. To wit, there are GPCR expressing cells ("type II") that are responsible for sweet, umami, and bitter; sour seems to be detected by PKD channels in presynaptic "type III" cells; and salty seems to be detected by ENaCs on glial-like "type I" cells. Simple enough, yes?

None of these cells form conventional synapses.

The three main types of taste receptor cells in the taste bud. Note that none of the cells forms a canonical synapse with afferent fibres.
From Chaudhari and Roper, 2011

I believe the figure above is accurate and reflects the current understanding of connections in the taste bud. Glial-like cells do not appear to have direct connections with other cells in the taste bud, and may signal via glia-like mechanisms. Receptor cells are wrapped by afferent nerves, but do not form synapses with the nerves. Rather, they appear to release ATP which activates the fibres. And pre-synaptic cells do indeed form synapses, but it's not clear whether they actually synapse directly onto the afferent fibres. There is also some cross-talk between the receptor and pre-synaptic cells in the form of serotonin and P2Y.

How the cells in the taste bud interact with each other and send information to the central nervous system will have fundamental implications on question zero. If the cells are talking to each other in the taste bud, it means the labeled lines are not independent at the first step. And, well, knowing how your sensation is transduced is fundamentally important.

2. Is bitter a single labeled line?

In 2000, the Zuker lab published two papers reporting that the T2R family of GPCRs is the bitter taste family. In Adler et. al. (2000), they asked the question of how many of these T2Rs are expressed in a given taste cell. To measure this they performed in-situ hybridization with 2, 5, or 10 probes for bitter receptors. They hypothesized that if bitter taste cells only expressed a subset of T2Rs, the 10 probe stain would reveal more cells than the 2 or 5 probe stain. They wrote,

"the demonstration that different mixtures of 2 or 5 probes detected as many positive cells as the mix of 10 suggests that each positive cell expresses nearly the full complement of T2Rs. If we assume that each receptor signals via the same pathway, and that the patterns of receptor expression delineate the logic of taste coding, these results indicate that there be limited functional discrimination between T2R-positive cells."

If this were true, one would hypothesize that our discrimination between foods comes from olfaction.

The next year the Roper lab (Caicedo and Roper, 2001) performed calcium imaging on taste buds from intact tongues, blind to cell type. They applied five different bitter compounds, at varying concentrations, and found that individual cells responded to only a subset of the different bitters. In other words, single cells were able to discriminate between different bitter compounds, which would imply each cell has a different complement of bitter receptors. This sort of result has been found in recordings from the NTS and PBN.

Receptive field of different taste cells (y-axis) to five different bitters (x-axis). Many cells respond primarily to one bitter compound (black blocks), while some cells respond to two or more bitters (blue, green, and red blocks).
From Caicedo and Roper, 2001.

Over the last decade, the Meyerhof lab has been investigating bitter receptors, and has performed two different tests that indicate bitter taste cells only express a subset of T2Rs. In Behrens et. al., 2007, they performed in-situ experiments similar to Adler, and found that going from one bitter receptor probe to two probes roughly doubled the number of labeled bitter cells. In Voigt et. al., 2012, they generated T2R131-GFP, and found that GFP only labeled ~50% of the gustducin-labelled bitter cells (gustducin is a common downstream messenger of the bitter GPCRs).

At this point, I would normally write off the bitter-is-a-single-line theory as Zuker being Zuker. Except bitter discrimination behaviour supports the idea. To test bitter discrimination, experimenters first normalize the bitterness by choosing concentrations that elicit similar aversion for each bitter. Then they ask animals to discriminate between these two bitter compounds. In both flies (Masek and Scott, 2010) and rats (Spector and Kopka, 2002), the subjects fail to discriminate between equally aversive bitters, supporting the idea that there is a single bitter labeled line.

Rats discriminating between two tastes. Rats are able to discriminate bitters (Qui: quinine; Den: denatonium) from salts (NaCl and KCl). However, when asked to discriminate between two bitters (final testing sessions), the rats are not able to discriminate. From Spector and Kopka, 2002.

These experiments are not completely clean to my mind. One might imagine that there are two ranges of bitter tastes: at high concentrations, bitters taste bad and are aversive; and at lower concentrations, bitter tastes may be less aversive, and more informative. By testing at aversive concentrations, they may be saturating receptors, and not be in the discriminatory range. These experiments, of course, are hard to design.

Personally, I lean towards the idea that bitter is a multi-lined label. The physiology is straightforward. Humans who are anosmic are still able to discriminate between tastes. Yet no one has demonstrated convincing behaviour that bitters can be discriminated without olfaction.

3. How do cortical neurons respond to mixtures of tastes? And mixtures of taste and smell for that matter?

When you eat a hamburger, you don't just taste sweet ketchup, umami meat, and sour pickles; the different taste lines merge together, and combine with the smell of the toasted bun, and non-taste oral cues like the temperature and texture of the ground meat, to form a whole. Which is a long way of saying that while "taste" is a single sense, flavour involves multisensory integration.

Unlike the previous two questions, there really isn't that much to talk about in terms of past research. A couple papers have looked at how binary mixtures are represented in the brainstem and cortex, but they've all been (rightfully) published in "specialty" journals. It is a difficult question to study: done well, one would want to record many neurons' responses to large numbers of mixtures. Hopefully someone can manage it sometime soon.

References

Adler, E., Hoon, M. a, Mueller, K. L., Chandrashekar, J., Ryba, N. J. P., & Zuker, C. S. (2000). A novel family of mammalian taste receptors. Cell, 100(6), 693–702.

Caicedo, A., & Roper, S. D. (2001). Taste Receptor Cells That Discriminate Between Bitter Stimuli. Science, 291(5508), 1557–1560. doi:10.1126/science.1056670

Chaudhari, N., & Roper, S. D. (2010). The cell biology of taste. The Journal of Cell Biology, 190(3), 285–96. doi:10.1083/jcb.201003144

Masek, P., & Scott, K. (2010). Limited taste discrimination in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 107(33), 14833–8. doi:10.1073/pnas.1009318107

Spector, A. C., & Kopka, S. L. (2002). Rats fail to discriminate quinine from denatonium: implications for the neural coding of bitter-tasting compounds. The Journal of Neuroscience, 22(5), 1937–41.

Voigt, A., Hübner, S., Lossow, K., Hermans-Borgmeyer, I., Boehm, U., & Meyerhof, W. (2012). Genetic labeling of Tas1r1 and Tas2r131 taste receptor cells in mice. Chemical Senses, 37(9), 897–911. doi:10.1093/chemse/bjs082