Molecular Biology of Pheromone
Detection in Mammals: From Genes to Behavior
Pheromones have evolved in all animal phyla, including
mammals, to signal the sex and the dominance status of animals
and to promote mating and social rituals among conspecifics.
In mammals, pheromones are primarily detected in a distinct
olfactory structure opposed to the ventral nasal septum,
the vomeronasal organ (VNO). VNO-derived signals are directly
targeted to discrete loci of the amygdale and hypothalamus
that elicit innate behavioral and neuroendocrine programs.
The ability to associate VNO chemosensory stimulation with
specific behavioral arrays and defined hormone changes provides
a unique opportunity to uncover the neural basis of mammalian
behavior.
The identification of the pheromone receptors is essential
for further investigation of VNO function. Work performed
in my lab has been instrumental in identifying novel families
of candidate pheromone receptors as well as molecules unique
to the VNO that are likely to be associated with pheromone
detection.
The isolation of the olfactory receptor genes in mammals
and in Caenorhabditis elegans has led to breakthroughs
in our understanding of olfactory sensory coding. Similarly,
the molecular and functional characterization of VNO-specific
receptors and signaling molecules is likely to provide insight
into the logic of the pheromone-evoked responses in the
mammalian brain (Dulac 2000).
Several years ago, as a postdoctoral fellow in Richard
Axel's lab (HHMI, Columbia University), I developed a procedure
to generate CDNA libraries from individual neurons (Dulac
and Axel 1995). The construction of single-cell libraries
is invaluable in the nervous system because neurons, even
within the same neural structure, display heterogeneous
molecular properties and neural connectivity. The ability
to investigate specific gene expression in individual cells
provides a powerful too[ to analyze the molecular basis
of neuronal identity. We have used this approach to discover
different classes of VNO sensory neurons, to characterize
their receptor properties, and to proceed with analysis
of olfactory development and function.
Our cloning efforts have led to the identification of large
and divergent families of candidate pheromone receptors
in the VNO (Dulac and Axel 1995; Herrada and Dulac 1997;
Pantages and Dulac 2000). We estimate that the receptor
gene families contain as many as 400-500 putative pheromone
receptors subdivided into distinct subgroups. This exceeds
previous estimates and suggests that a remarkable molecular
and cellular complexity is required for pheromone detection.
What is the molecular and functional significance of this
organization? Our recording of the VNO neurons shows that
large fractions of the VNO neuronal population are activated
by natural sources of pheromonal stimuli (Holy et al. 2000).
The absence of any clustering of the neuronal response types,
together with recent evidence of the narrow tuning of the
VNO neuronal activation by defined compounds points to the
activation of multiple receptor populations by large but
distinct sets of pheromonal cues. Furthermore, we have uncovered
a wiring diagram of the VNO fibers within the anterior accessory
olfactory bulb (AOB) that appears perfectly suited to accomplish
the integration of multiple receptor inputs (Belluscio et
al. 1999).
We propose a model of pheromone information processing
in which the VNO acts as a sensor for a variety of chemical
cues and the AOS mitral cells function as coincidence detectors
to ensure the pheromone response is specific to the species,
the sex, and the individual.
Sensory transduction in the VNO appears unrelated to that
in the vertebrate olfactory and visual systems: the putative
pheromone receptors of the VNO are evolutionarily independent
from the odorant receptors and, in contrast to vertebrate
visual and olfactory transduction, vomeronasal transduction
is unlikely to be mediated by cyclic nucleotide-gated channels.
We hypothesized that sensory transduction in the VNO might
involve an ion channel of the TRP (transient receptor potential)
family, members of which mediate cyclic nucleotide- independent
sensory responses in Drosophila and C. elegans
(Liman et a]. 1999). We isolated a CDNA (rTRP2) from rat
VNO encoding a protein of 885 amino acids that is equally
distant from vertebrate and invertebrate TR P channels (10-30
percent amino acid identity). The rTRP2 MRNA is exclusively
expressed in VNO neurons, and the protein is highly localized
to VNO sensory microvilli, the proposed site of pheromone
sensory transduction. The specific expression of TRP2 in
the VNO, together with the absence of a cyclic nucleotide-mediated
response, suggests parallels between vomeronasal sensory
transduction and light-induced signaling in the Drosophila
eye.
Genetic ablation of the TRP2 channel, a candidate-signaling
molecule in the mouse VNO, allowed us to assess VNO-mediated
sensory responses and behaviors directly. We found that
TRP2 deficiency eliminates the sensory activation of VNO
neurons by urine pheromones. Moreover, the absence of VNO
function has striking behavioral effects. TRP2-/- male mice
appear unable to recognize the sexual identity of their
conspecifics: they fail to display the pheromone-evoked
aggression toward male intruders that is normally seen in
wild-type males and, remarkably, they display courtship
and mounting behavior indiscriminately toward males and
females (Stowers et al. 2002). Our data contradict the established
notion that VNO activity is required for the initiation
of male-female mating behavior in the mouse and suggest
instead a critical role in ensuring sex discrimination.
The identification of a large number of putative pheromone
receptor genes grouped into several divergent gene families,
together with the collection of multiple receptor projections
within individual glomeruli, suggests that the pheromone-evoked
response is likely to involve patterns of activity across
the receptor population. In collaboration with the lab of
Markus Meister (Harvard University), we reasoned that such
a distributed population code should be observed by simultaneously
recording the activity of a large number of VNO neurons
in response to natural stimuli. Using a flat array of 61
extracellular electrodes, we have obtained the simultaneous
recording of action potentials from large subsets of VNO
neurons (Holy et al. 2000). Our study revealed several features
of VNO neuronal activation. First, VNO neurons respond to
components of urine by increasing their firing rate. In
accord with our proposed model of VNO signaling that parallels
that of Drosophila phototransduction and involves
a channel of the TRP family, we showed that the chemosensory
activation requires phospholipase C function. Moreover,
unlike most other sensory neurons, VNO neurons do not appear
to adapt under prolonged stimuli. This surprising feature
of the VNO response may be physiologically related to the
necessity of detecting minute amounts of pheromonal cues
and to the long-lasting impact of pheromone detection on
the organism.
Remarkably, the full-time course of VNO spiking in response
to the stimulus concentration can be captured by a first-order
kinetic. By directly quantifying the neuronal response to
a given chemical stimulus, we generated a simple quantitative
model of the neuronal response. This enabled us to demonstrate
that subsets of VNO neurons are strongly selective for either
male or female urine, while other neurons appear to recognize
pheromones that vary between individuals of the same sex.
The population recording of VNO neurons provides a powerful
tool to investigate the complex sensory recognition involved
in the pheromone-evoked response: the discrimination of
the species, the sex, the familial status, or the individual
differences among animals.
