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Summary: Animals exhibit a repertoire of innate behaviors that result in stereotyped social and sexual responses to the environment. In Drosophila, courtship behaviors governed by pheromonal excitation of peripheral olfactory pathways ultimately activate behavioral circuits in higher brain centers. One pheromone, cVA, elaborated by the male, suppresses male-male courtship but in females, enhances receptivity to courting males. Thus, a single pheromone elicits different behaviors in the two sexes. Richard Axel's lab has developed a neural tracing procedure that employs two-photon laser scanning microscopy to activate the photoconvertible fluorophore, PA-GFP, to demonstrate anatomic dimorphisms in the neural circuit responsive to the pheromone cVA. The observation that cVA activates a sexually dimorphic circuit in higher olfactory centers suggests a mechanism whereby a single pheromone can elicit different behaviors in males and females.

Courtship behavior represents an innate sexually dimorphic behavior that can be observed in naïve animals without prior learning or experience, suggesting that the neural circuits that mediate their behaviors must be developmentally programmed. Despite dramatic behavioral differences between the sexes, surprisingly few anatomic distinctions have been observed that differentiate the male and female brain in any species. In Drosophila, courtship is initiated by the male and involves a complex yet stereotyped behavioral array. Genetic studies strongly implicate Fru^M, a male-specific isoform of the fruitless gene (Fru), in programming male courtship behavior. Fru is expressed in about 2,000 neurons in the fly brain and is observed in subsets of primary sensory neurons as well as in cells in higher brain centers. The expression of this protein in neurons in the olfactory pathway may be particularly revealing, since pheromones thought to activate sensory neurons in the antennae are likely to provide environmental cues initiating the events of courtship. Fru is expressed in three subpopulations of olfactory sensory neurons that converge on three glomeruli within the antennal lobe. Moreover, projection neurons (PNs) that connect these glomeruli in the antennal lobe to higher olfactory centers in the mushroom body and lateral horn also express Fru.

Olfactory neurons expressing the odorant receptor 67d (OR67d) are Fru⁺ and respond to the male-specific pheromone cis-vaccenyl acetate (cVA). Males and females maintain equal numbers of cVA-responsive 67d sensory neurons in the antennae, and these neurons project axons to the DA1 glomerulus. Male flies mutant in OR67d exhibit high levels of male-male courtship but do not suppress male-female courtship. Female flies mutant in the 67d receptor exhibit reduced receptivity to the male's courtship display. These observations immediately pose the question as to how a single pheromone acting through identical sets of sensory neurons can elicit different behaviors in male and female flies. We have asked whether anatomic or functional dimorphism in this circuit might be responsible for the dimorphic behaviors.

	Neurons expressing an odorant receptor gene... Projection neurons extend dendrites... Scents and Sensibility: Towards a Molecular Logic of Perception - A lecture presented at Columbia University RealPlayer required

Tracing Anatomic Dimorphism in a Fru⁺ DA1 Projection Neuron
The approach we have developed to trace the projections of individual PNs in the protocerebrum involves the expression of the photoconvertible fluorophore PA-GFP in projection neurons. PA-GFP normally exhibits low-level background fluorescence, but this fluorescence is enhanced 100-fold after photoconversion with high-energy light. Photoactivation of an individual glomerulus or a single PN provides sufficient spatial resolution and photoconversion energy to visualize the neuronal processes of defined neuronal populations as well as individual neurons in the fly brain.

The development of a combined genetic and optical neural tracing method now permits us to compare the function and topography of projections from Fru⁺ PNs that innervate the DA1 glomerulus in male and female flies. Photoconversion of the DA1 glomerulus and visualization of the pattern of projections of the population of DA1 PNs in the lateral horn in living brains revealed that the projections from the DA1 glomerulus target a compact region in the anterior ventromedial region of the lateral horn. Approximately 80 percent of the projection pattern is similar between males and females. However, Fru⁺ DA1 projections from males exhibit additional axonal branches that extend ventrally and slightly medially. Superposition of the projection patterns of PNs obtained after photoconversion of DA1 taken from 10 male and 10 female flies confirms this observation.

The anatomic dimorphism observed at the level of the population of axons is also exhibited by the axons of single identified neurons. Tracing individual labeled neurons after warping reveals that males have more richly elaborated ventrally directed axonal branches than females and identifies a population of ventral axons that target a unique region of protocerebral space that is male-specific. These data from single-axon tracing along with our observations from populations of DA1 neurons indicate that DA1 PN projections are sexually dimorphic.

Dimorphic Projections are Controlled by Fru^M
We next asked whether the sexually dimorphic pattern of axonal projections in Fru⁺ DA1 PNs is regulated by the fruitless gene. Fru^M mutant flies lack the male-specific Fru isoform and exhibit male-male courtship, a behavior also exhibited by flies deficient in the cVA receptor, OR67d. Males lacking Fru^M retain the expression of OR67d, suggesting that Fru^M mutants detect cVA but that the altered behavioral response results from downstream alterations in the neural circuit. We therefore visualized the axonal projections of single DA1 PNs in Fru^M mutant males and observe that these PNs lack the characteristic male-specific axonal branches and reveal a branching pattern more characteristic of wild-type females.

Females that express the male-specific isoform Fru^M in Fru⁺ neurons exhibit male courtship behavior and diminished receptivity to males. If this male-type sexual behavior results in part from alterations in neural connectivity, we might expect that Fru^M expression in female DA1 PNs would result in a male-specific pattern of axonal branching. Traces of single Fru DA1 PNs in female flies that express Fru^M (Fru-Gal4; UAS-Fru^M) exhibit a striking increase in axonal projections in the ventral male enhanced area. Quantitation of these branches reveals that expression of FruM in females renders their ventral axon branch pattern statistically indistinguishable from that of males. Thus, the analysis of the PN projections of single defined neurons, along with the population analysis, reveals that Fru⁺ DA1 PNs project to different regions of the protocerebrum in male and female flies. Moreover, this anatomic dimorphism in the neural circuit is controlled by the male-specific transcription factor, Fru^M.

Animals exhibit a repertoire of innate sexual and social behaviors that can be observed in naïve animals, suggesting that the neural circuits that mediate these behaviors are developmentally programmed. The existence of programmed neural circuits that govern innate behavioral repertoires implies that these behaviors must be tightly regulated to assure that they occur in an appropriate social context. In Drosophila, courtship behavior is governed by pheromonal excitation of peripheral olfactory pathways that ultimately activate behavioral circuits in higher brain centers. One pheromone elaborated by the male, cVA, suppresses male-male courtship but enhances receptivity of females to courting males. Thus a single pheromone elicits different behaviors in the two sexes. cVA activates the DA1 glomerulus, which is innervated by Fru⁺ PNs that exhibit sexually dimorphic projections in the lateral horn. It is therefore tempting to assume that the anatomic differences we observe in Fru⁺ DA1 projection neurons underlie the distinct behaviors elicited by cVA in the two sexes. This model assumes that the segregation in axonal projections in the two sexes results in different connections with third-order neurons in the lateral horn.

This work is supported in part by grants from the National Institutes of Health and the Mathers Foundation.

Last updated: December 5, 2007