The fear chemical?

We often talk, often rather vaguely, about instincts and how they shape our behavior (my instinctive reaction was…, etc.).  Predator-prey interactions are one place where instincts are real, and really matter. A cat that doesn’t realize that a little scuttling squeaky thing is also a good meal probably won’t be welcome in the barn of a corn farmer.  Similarly, if a mouse doesn’t know to avoid the lair of a fox without having to be trained in avoidance, that mouse is at severe risk of not getting a chance to pass on its genes to its progeny. Hard-wired responses to the smell of predators are well documented, but not well understood.  A new paper from a multi-disciplinary collaboration led by our close neighbor Stephen Liberles (and including our even closer neighbor Bob Datta) has identified one of the chemical components that lead to this response (Ferrero et al. 2011. Detection and avoidance of a carnivore odor by prey.  PNAS doi/10.1073/pnas.1103317108).

Ever since the pioneering work of Linda Buck and Richard Axel, we’ve known, roughly, where our experience of smell comes from: volatile odorants are detected by a large class of receptors expressed in the neurons of the olfactory epithelium.  Each neuron expresses just one receptor. A scent is typically a mixture of many odorants, each of which may bind to and trigger the activity of several receptors; the combination of neurons activated by a given scent is unique to that scent, and so each scent sends a distinct set of signals to our brains.  As a result of this combinatorial detection, we can discriminate an extremely wide range of scents with a relatively limited set of receptor molecules.  Stephen Liberles and Linda Buck later identified a second set of receptors that detect amine odorants, the “trace amine-associated receptors” or TAARs.  These receptors may detect some of the important signals mammals use to communicate with each other about, for example, their state of sexual receptiveness.  But in most cases the question of which odorant a specific receptor responds to has yet to be answered.  There’s no easy way to guess or deduce this: what you have to do is try various possible odorants and see which ones activate the receptors.  Luckily, since all odorant receptors respond to activation by causing an increase in the level of the second messenger cyclic AMP (cAMP), this is now possible to do in vitro: you express your receptor of interest in an ordinary cell line, making sure that the cell line has the appropriate machinery to connect the receptor to the adenyl cyclase that makes cAMP.  Then you add a cAMP-responsive reporter gene.  And then you try every possible odorant you can think of, and look for blips in reporter gene expression.  Then you move on to the next odorant receptor, and do it all again.

Because the TAAR receptors are thought to detect odorants that may be in mammalian bodily secretions such as urine, urine was one of the first places Liberles and Buck originally looked for odorants that would activate TAAR receptors.  The new paper identifies a mouse odorant receptor, TAAR4, that specifically responds to urine from predators such as bobcats and mountain lions.  (One cannot help feeling sorry for the person who has the job of collecting bobcat urine, though there may be worse jobs).  Other receptors in the same family respond equally to the urines of multiple species.  Ferrero et al. therefore reasoned that there might be a specific chemical present in the urine of predators that mice detect (at least in part) using this receptor.

Chemical fractionation of bobcat urine and mass spectrometry came up with a candidate for the chemical that’s responsible for the urine’s activity: 2-phenylethylamine (hereinafter PEA).  The receptor responds strongly to pure PEA, but doesn’t respond at all to benzylamine, which is only one CH2 group different.  Now the question becomes, is TAAR4 a specific bobcat avoidance receptor, or does it detect the presence of predators more generally?  Ferrero et al. collected a wide range of urine samples from all kinds of beasts, big and small, and found that carnivores almost always have more PEA in their pee than other creatures.  The only exception, oddly, is the ferret, which has about the same amount of PEA in its urine as does the llama. Apart from the ferret, there looks to be a pretty clear division between predators and non-predators based on PEA concentration.  We don’t yet know why — perhaps production of high levels of PEA is a result of a carnivorous diet, or perhaps it results from the way a metabolism is tuned to cope with a carnivorous diet (a subtle but real difference).

In any case, a mouse that has sensitive receptors for PEA would seem to have the potential for a significant evolutionary advantage.  Smelling PEA is not enough, though — you would also want the PEA receptors to be hooked up to a strong “run away” response, one that is so hard-wired that the mouse doesn’t stop to think about it.  Is that the case for this odorant?  The dorsal area of the olfactory epithelium has been shown in other studies to be associated with aversive, a.k.a. “run away”, behavior. Using imaging of the activity of sensory neurons in tissue slices, detected by the presence of transient calcium spikes, the authors show that PEA activates a significant number of neurons in exactly this region.  Consistent with this, when a rat is put in a box with PEA spotted in one corner it spends significantly less time in that corner than in any of the others — as it does when the corner is instead decorated with lion urine.  PEA has a similar kind of effect on mice.  The response appears to be one of fear, not just dislike: rats exposed to PEA show high levels of corticosterone, indicating that they’re stressed by the smell.  The authors, cautiously, don’t claim that PEA definitely induces fear, though; there are more conclusive tests for fear responses that they haven’t yet done.

At this point in the paper the authors have shown us that PEA is sufficient for the TAAR4 response.  But is it necessary?  This is not such an easy question to answer.  Luckily there’s an enzyme, monoamine oxidase, that semi-selectively oxidizes PEA and some other aromatic amines.  Adding monoamine oxidase to lion urine brought the PEA levels down to a point where they were undetectable; and the “rat in the corner” test also indicated that this oxidized urine no longer induced avoidance responses.  When PEA is spiked back into the oxidized urine, all the unpleasantness comes flooding back: rats avoid PEA-spiked oxidized lion urine just as assiduously as they avoid lion urine that hasn’t been manipulated at all.

Ferrero et al. point out that PEA is almost certainly only part of the story.  In the neuronal imaging studies they performed in tissue slices, not all of the neurons that responded specifically to lion urine (as opposed to giraffe urine) also responded to PEA.  Also, other odorants are known to induce species-specific aversion responses; for example the chemical 2,5-dihydro-2,4,5-trimethylthiazole, which is produced by foxes, induces an avoidance response in rats and mice.  But PEA is clearly a major part of the aversion signal in predator urine, at least for mice and rats.  This is a big step forward for researchers who are interested in studying the neural pathways that are responsible for instinctive behavior: now they have a chemical tool for selectively triggering the important aversive response.  Perhaps it is also a big step forward for those of us who live in old houses that are frequently invaded by mice as winter sets in.  If I had the option of spraying my basement with PEA (which to humans, apparently, smells only mildly unpleasant at full strength) as a precautionary measure instead of possibly having to call the exterminators in, I almost certainly would. Look for spray bottles of “mouse-be-gone” in a hardware store near you next winter.

Ferrero DM, Lemon JK, Fluegge D, Pashkovski SL, Korzan WJ, Datta SR, Spehr M, Fendt M, & Liberles SD (2011). Detection and avoidance of a carnivore odor by prey. Proceedings of the National Academy of Sciences of the United States of America PMID: 21690383