We carried out the first global, voxelwise analysis of structural differences in an insect brain. This allowed us to identify extensive sex-specific volume differences missed in earlier studies of Drosophila
. These are so reliable that brain shape can identify the sex of individual animals, which can be of experimental benefit. The strong overlap of these volumetric differences with dimorphic arbors of fru+
neurons suggests a role in male behavior. Early studies of sex mosaics showed that the superior protocerebrum is a key regulator of courtship behavior [25
]. Our results strongly support that localization and identify specific regions in the superior protocerebrum likely to regulate both male and female behavior.
At the circuit level, we have built a 3D atlas containing 62 fru+
neuroblast lineages in the central brain and 51 in the VNC. These numbers reveal an impressive heterogeneity in the developmental origin of fru+
neurons. The central brain consists of the cerebrum (made by 100 neuroblasts, 57 of which are fru+
) and the smaller subesophageal ganglion (80 neuroblasts, 5 of which are fru+
]. Few of these lineages, or their component neurons, have previously been described in adult flies, making this study a substantial contribution to understanding the development and neuroanatomy of the fly brain.
In contrast to the preliminary conclusion that the fruitless
circuit is anatomically largely isomorphic [9, 17
], we show that one-third of fru+
neuronal clones are dimorphic. Previous work on sexual dimorphisms in insects has focused on changes that increase the sensitivity of sensory systems to visual [39
] or olfactory [40
] cues from conspecifics. However, we find that most dimorphisms are in the arbors of fru+
higher interneurons of the protocerebrum. In those cases where arbors are denser in males than females but still similarly located, the effect could be to increase the strength of connections between the same groups of neurons; this could be analogous to the increases in sensory sensitivity mentioned above, increasing the gain for some signals without fundamentally altering circuit logic. However, all 21 dimorphic clones described in have arbors in locations where there are no processes in the opposite sex; in these cases, it seems very likely that different groups of neurons are connected in the two sexes. The sex-specific projection differences generated during development may therefore be somewhat analogous to the jumper switches in an electronic circuit—a few specific locations where connections can be made or altered in order to change the logic of a largely conserved circuit. Pursuing this analogy, engineers place jumper switches at key locations in order to efficiently alter circuit logic. Can we propose any logic to rationalize the location of fru+
The olfactory system provides an excellent example to explore how these circuit level dimorphisms may regulate dimorphic behavior. In the periphery, fru+
olfactory sensory neurons are more numerous in males [9
]. This could enhance sensory sensitivity but cannot explain, for example, why the male pheromone cVA should be repulsive for males but stimulatory for females [21
]. We propose that changes in connectivity between second- and third-order olfactory neurons, largely because of shifts in the dendritic arbors of fru+
lateral horn neurons, result in the rewiring of sensory signals detected by both sexes. We have identified several populations of lateral horn neurons that show precise overlap with the axons of cVA-responsive PNs. This includes two populations with overlap only in males and another with female-specific overlap. We therefore hypothesize that these lateral horn neurons are the first elements in the olfactory pathway that show different responses to the same stimulus in males and females.
In flies, odors are represented in the first two stages of the olfactory system by the activity of about 50 channels (corresponding to the glomeruli of the antennal lobe). Odors usually activate many channels, and different odors can activate the same channel. PNs show little integration of information across olfactory channels, in clear contrast to third-order neurons [41
]. Although cVA is a rather specific signal known to activate only two channels [21, 36
], it occurs in a variety of behavioral contexts [42
]. Third-order neurons that integrate cVA signals with olfactory signals from other PNs may therefore respond with increased selectivity to behaviorally relevant odor objects such as male and female flies. It would make sense to restrict substantial anatomical dimorphism to such neurons capable of selective integration. More generally, we hypothesize that it would be efficient to alter connections involving higher-order neurons that robustly represent a relevant feature of an external stimulus (e.g., the presence of a fly of the opposite sex) rather than more peripheral neurons, which can be activated by more diverse stimuli.
Outside the olfactory system, a number of relatively discrete dimorphisms could alter the processing of external stimuli or the behaviors to which they are eventually connected. However, the analogy with an electronic circuit that can be (developmentally) reconfigured by altering some connections between the same set of elements is incomplete. There are some dimorphic clones that have many more neurons in males, most obviously pMP-e and aSP-a, which have extensive male-specific processes in the MER. The MER is rich in dimorphic fru+
neurons and appears to receive diverse sensory information and to be connected to the VNC (; ; Figure S4
]. Masculinization of pMP-e (P1) in otherwise female flies is strongly correlated with induction of male courtship behavior [20
], so it is proposed to initiate male behavior. Although the circuit logic of the MER is not yet clear, we propose two specific models for these highly dimorphic neurons. First, they may integrate multiple sensory streams and, when activated, directly excite descending neurons that control male motor programs. Alternatively, they may reflect the state of excitation of the male by generating a long-lasting potentiation of connections in the MER between sensory inputs specific to particular descending neurons.
Alterations in the presence and wiring of specific circuit elements according to the logic we have just described could result in sex-specific activation of motor programs that are largely conserved between the sexes. This is consistent with the finding that direct activation of fru+
neurons in the VNC of females can induce courtship song [43
]. However, song is abnormal unless those fru+
neurons are masculinized by fruM
, so there may be similar developmental alterations in motor circuits of the VNC (e.g., Figure S2
D). The logic of sexually dimorphic behavior has strong parallels in mice, in which removal of the pheromone-sensing vomeronasal organ from females results in male behavior [44
]. The resultant hypothesis proposes that circuits for male and female behavior exist in females but that sensory information from the vomeronasal organ normally represses male behavior. This, then, begs the question of what circuit difference alters the behavioral significance of vomeronasal organ activation. Genetic approaches [45
] to identifying and characterizing circuit level differences in mice will find answers in the long run. However, we are now ready to test how dimorphic anatomy can alter neural processing and behavior in Drosophila