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A male mouse listens and sniffs as an animal enters his territory. His sensory systems reveal that it is another mouse, but this is not enough to dictate an appropriate behavioral response. Is the intruder male? Should he fight to defend his territory? Fighting would be a mistake if the intruder were a receptive female. There is no hesitation in his response. Pheromone cues released from the other mouse ensure that social interactions such as territorial aggression, sexual behaviors, onset of puberty, and maternal responses are executed without error (1).

How does the brain recognize important features of the environment that require a behavioral response? The central nervous system (CNS) is responsible for encoding accurate biological representations of the external environment that dictate and modify an animal’s behavior. Tremendous progress has been made in identifying the molecular and neurological mechanisms that encode environmental stimuli, but little is known about how the CNS uses this information to direct specific output behavior. In general, primary sensory information is first gathered and organized, then sent to the cortex to generate perception, and finally appropriate behavior is initiated. The intermediate step of cortical perception, or awareness of the environment, allows the animal to modify behavior on the basis of past experience. From an experimental standpoint, however, this learning and memory step confounds investigations aimed at decoding neuronal pathways responsible for behavior. In contrast, the circuitry of the vomeronasal organ (VNO), an unconventional olfactory system that is implicated in pheromone mediation in terrestrial vertebrates (1, 2), is subcortical without direct relays to centers of perception. VNO circuitry terminates at neuroendocrine factor–releasing neurons in the hypothalamus, influencing levels of steroid hormones to regulate many social behaviors. As expected from this circuitry, a defined environment evokes a predictable response, whether the animal is a novice or has experience.

The precise role of the VNO in initiating pheromone response has been difficult to interpret. Conventional ablation experiments collaterally damage other important olfactory centers, and VNO neurons are likely to be rapidly replaced from a pool of precursor cells present in the adult (3). To bypass these limitations, we recently engineered mice with a disruption in the putative ion channel TRPC2 (4) that is expressed exclusively in VNO sensory neurons (5). In wild-type mice, urine pheromones stimulate a specific increase in the firing rate of VNO neurons (6). In contrast, VNO neurons of the mutant TrpC2^-/- animals fail to respond to the presence of mouse pheromone stimuli. This suggests that TRPC2 activity is essential for VNO neurons to respond to pheromone cues. Mutant mice that are unresponsive to pheromones provide a tool to clearly address the function of the VNO in initiating social behaviors.

Surprisingly, the onset and display of sexual behavior of the mutant animals is intact in both males and females. Additionally, there are no defects in maternal response and pup nursing from TrpC2^-/- animals, suggesting that the VNO does not mediate stimuli that initiate these behaviors. The mutant animals, however, show a dramatic change in their aggressive behavior.

In the wild, mice live in small social groups consisting of one male, several females, and juveniles (7). The presence of an adult male intruder initiates aggression from the resident male (8). This territorial aggressive behavior can be recapitulated in the lab by singly housing adult males and introducing an adult male intruder. The pheromone cue responsible for the evoked aggression has been shown to be present in adult male urine because the presence of females, juveniles, or even castrated adult males does not initiate resident aggression (9). A castrated adult male intruder that is swabbed with a small amount of adult male urine is sufficient to stimulate resident aggression in control animals (10). TrpC2^-/- males display motor programs necessary for tactile-evoked aggression, yet they are completely devoid of pheromone-evoked aggressive behavior. What is the mechanism behind this phenotype? Low testosterone levels generally correlate with decreased aggression (11); however, the TrpC2^-/- animals produce normal levels of testosterone, suggesting that the phenotype is due to a defect in detection of cues by the sensory neurons that initiate aggressive behavior.

Other aspects of VNO function have been discovered from analysis of the TrpC2^-/- animals. Interestingly, the TrpC2^-/- males substitute sexual behavior toward male intruders for aggressive behavior. This behavior, from courtship vocalizations to mounting, is similar to that normally observed from wild-type males toward females. When given a choice, mutant males mount both estrus females and males with equal frequency. This response suggests that the inability to respond to pheromones does not alter sexual preference but rather creates behavior that is indifferent to cues that indicate mating partner gender. Importantly, this failure to recognize gender accounts for all aspects of the TrpC2^-/- phenotype: both lack of aggression toward males and indiscriminate sexual behavior.

Behavioral analysis of TrpC2^-/- animals that are unable to respond to natural pheromone stimuli suggests a model whereby the VNO does not simply generate preprogrammed motor patterns to specific cues but rather regulates behavioral output initiated through other sensory systems. Audio, visual, tactile, and main olfactory cues generate the perception of an intruder mouse in the environment, whereas information from the VNO regulates a gender-appropriate response: aggression if the intruder is a male or sexual courtship as default behavior. How is this information integrated? At the circuit level, neurons project from the VNO to the amygdala, a common relay for sensory processing that may provide a physical location for the regulation of gender-evoked behavior mediated by the VNO upon information gathered from the other sensory systems.

The VNO circuitry provides a system to understand the neurobiology that encodes environmental stimuli and evokes complex behavior. About 500 different pheromone receptors are expressed in the VNO, suggesting a tremendous amount of coding potential beyond gender recognition. Additionally, natural environments are composed of a blend of pheromones from various individuals. Candidate VNO functions include behaviors that influence sexually dimorphic responses, allow recognition of an individual, or are species specific. A molecular investigation of VNO circuitry has the power to illuminate how multiple sources of instructive information are organized to appropriately regulate complex behavior.

References

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2. M. Halpern, Annu. Rev. Neurosci. 10, 325 (1987).
   
3. C. J. Wysocki, J. J. Lepri, J. Steroid Biochem. Mol. Biol. 39, 661 (1991).
4. L. Stowers, T. E. Holy, M. Meister, C. Dulac, G. Koentges, Science 295, 1493 (2002).
5. E. R. Liman, D. P. Corey, C. Dulac, Proc. Natl. Acad. Sci. U.S.A. 96, 5791 (1999).
6. T. E. Holy, C. Dulac, M. Meister, Science 289, 1569 (2000).
7. G. A. Van Oortmerssen, Behaviour 38, 1 (1971).
8. J. A. Maruniak, C. J. Wysocki, J. A. Taylor, Physiol. Behav. 37, 655 (1986).
9. R. A. Mugford, N. W. Nowell, Nature 226, 967 (1970).
10. P. M. Driver, D. A. Humphries, Nature 226, 968 (1970).
11. F. Sluyter, G. A. van Oortmerssen, A. J. de Ruiter, J. M. Koolhaas, Behav. Genet. 26, 489 (1996).