Annals of Underwhelming Papers: The Promising Knockout with No Phenotype

As a wannabe hipster, I love categorizing and listing things.  And what better to classify than the coin of the realm, papers?

For lab meeting this week, a post-doc presented a recent paper about the role of the gene Ano2 in olfaction. Ano2 enocodes a Ca²⁺ activated Cl- channel, which was thought to help amplify the signals from olfactory sensory neurons.  In other words, that it was essential for how your nose communicated with your brain.

So the authors did the sensible thing, and knocked the gene the fuck out. (I should state here, that while I will be flip, I respect the work the authors put in, and mean no disrespect.) The first four figures of the paper are dedicated to Westerns and immunostains identifying where Ano2 is expressed in the brain, where its expressed in the olfactory epithelium, and showing that their knockout technique indeed works.  Which is where the papers gets its first category: the Inception.

Ano2 is in the MOE.  Probably best in the Supplement.
From Billig et al (2011).

Inception was a great movie, but was weighted down by minutes upon decades of exposition.  So you can go multiple layers deep in dreams? And use totems to see if  you're dreaming?  Oh look, two hours have passed, and my ass is getting sore.

An Inception paper is one that spends an unseemly number of figures and text setting up its premise before getting to the meat. Note that the issue here is not performing or showing controls, but rather one of degree: one or two control figures is fine, but we invented supplemental figures for a reason. Figures that are probably best left to the supplement include but are not limited to: North, South, (East,) and Western blots showing that something is knocked in or out; immunostains showing that molecules you thought were coexpressed were indeed coexpressed; fEPSP timecourses showing you can get LTP in your system; immunoblots showing your antibody is specific.

(The Journal of Neuroscience recently stopped accepting supplemental figures, a decision I strongly disagree with (there was some blogosphere  discussion of this). When I read a paper that is not of direct interest, I want to the authors to get to the point concisely, and present only the most essential, interesting figures. I do not want to sift through perfunctory controls like actin stains, or negative results. While these controls are essential, they are best put in a place only reviewers and competitors. Needless to say, the readability of JNeurosci papers has plummeted, and it will be interesting to see if it impacts their citations going forward. (Which is not to say that interesting figures should go in the supplement, as unfortunately sometimes happens, but that is a larger discussion.)

In any case, the paper got a bit more exciting in figure 5, where they performed whole cell patch clamp on the knocked out neurons, and found that Ano2 was essential for Ca²⁺-induced chloride currents.  And in figure 6, they verified this by uncaging Ca²⁺ in cells, and showing that the KO neurons had reduced currents.

Ano2^-/- neurons have smaller currents.
From Billig et al (2011).

Then they tested whether this reduced current was actually functional, and recorded from the olfactory nerve while presenting odors to the mice.  This part got a little weird to my naive eyes, as they presented the odors in fluid phase, and air phase.  As far as I know, mice's nostrils only fill with fluid when they're sick (I think...), so it seemed a unnatural.  Anyway, the olfactory nerve transmitted less voltage in the knockout mice! Ano2 was functional!  With the important exception that it was only for the fluid phase and not the air phase, but what's a chemical phase between friends?

Fluid phase nerve response is reduced in knockout mice.
From Billig et al (2011).

On to the behaviour!  Using an automated olfactometer (somehow the machine that presents odors is called a meter), they trained mice to discriminate between odors.  And they found that the Ano2 ^-/- mice were perfectly able to discriminate between every odor pair and concentration difference they tested.  While Ano2 may be important for some electrophysiological aspect of olfactory sensory  neurons, they aren't essential for function.  Which makes this a Sunshine paper.

Ano2^-/- mice have no trouble discriminating odors. Another KO mice does...
From Billig et al (2011).

Sunshine started as good science fiction suspense movie.  It had a beautiful cast (if only scientists looked like that), unexplained occurrences, and cool special effects. Halfway through the movie I couldn't wait to see how it ended.  Then it ended as a bad horror movie. (An alternative, if clunkier name for the category: Invention of Lying.)

Like Sunshine, this paper started well (if Inceptionally slowly), and going through figure 7 I couldn't wait to see how it ended.  Until I saw there was no phenotype, and this ion channel isn't important in this system.  Kinda disappointing (but not horrifying).

In the discussion, the authors mention that humans with this gene deleted have no olfactory impairment, which should have been a tipoff.  And they hypothesize that, "The expression of Ca²⁺-activated Cl− channels in mammalian OSNs may be an evolutionary vestige from freshwater animals." Yeah.

As I said, it's a solid piece of work.  Shame about the phenotype.  The authors have my sympathy.