In this study we have identified and characterized a tripartite MBγ>M6>aSP13 recurrent circuit that is essential for courtship memory in Drosophila. Our behavioral and physiological data suggest the following model for the function of this feedback loop in short-term courtship memory. When a naïve male courts a mated female, the aSP13 and MBγ neurons may both be activated, perhaps in response to behavioral rejection and olfactory stimuli presented by the female, respectively. Dopamine released by aSP13 neurons potentiates transmission from MBγ to M6 neurons, which in turn provide a recurrent excitatory glutamatergic input back onto aSP13 neurons. Upon activation by M6, aSP13 activity persists for several minutes, providing a short time window during which continued MBγ activity can further drive M6 and aSP13. Thus sustained, aSP13 activity can lead to a longer-lasting accumulation of dopamine in the γ5 compartment, facilitating MBγ>M6 neurotransmission for up to 2–3 hr.

The timescales for these physiological processes in ex vivo brain preparations broadly match the dynamics of courtship training and short-term memory formation. In our standard training paradigm, the male typically courts the female over several minutes, during which he performs a series of courtship bouts, each lasting for several seconds. As a result, a behavioral memory forms that lasts for several hours (Keleman et al., 2012). Memory formation during training requires both M6 and aSP13, consistent with the notion that it reflects activation of the recurrent circuit (Figure 4 and [Keleman et al., 2012]). Memory readout requires M6 but not aSP13 (Figure 4 and [Keleman et al., 2012]), and so evidently does not involve the recurrent circuit. We infer that M6 suppresses courtship through other, aSP13-independent, pathways, and that its ability to do so is independent of experience. The consequence of training is to provide MBγ neurons with access to this M6-dependent courtship suppression pathway (Figure 1).

Two important open questions are, first, what mechanism underlies the persistent calcium response in aSP13, and second, how does potentiation of MBγ>M6 synapses result in enhanced sensitivity to cVA, the hallmark of courtship memory (Keleman et al., 2012). The persistent response in aSP13 is evidently not an intrinsic property of aSP13, as it is not induced when aSP13 neurons themselves are activated. This observation would also likely exclude reciprocal excitation between aSP13 and other DANs (Plaçais et al., 2012). Persistent aSP13 activity is induced in response to transient M6 activation, and is not associated with any persistent activity of M6 neurons themselves. Thus, it is also unlikely to involve feedback from aSP13 and M6, although aSP13 >M6 synapses likely do exist (Eichler et al., 2017; Lin et al., 2007). One possibility is that aSP13 persistence reflects unusually prolonged activation of the glutamatergic M6 >aSP13 synapses, or perhaps lies within interposed but still unidentified circuit elements.

Given that M6 neurons activate a courtship suppression pathway, the potentiation of MBγ>M6 neurotransmission may explain why MBγ activation suppresses courtship in trained but not naïve flies. But MBγ neurons likely do not specifically respond to cVA (Caron et al., 2013; Gruntman and Turner, 2013), so this change alone cannot account for the enhanced sensitivity of trained flies to cVA. A small and variable subset of MB γneurons do receive input from the olfactory pathway that processes cVA, but cVA is not required during training (Keleman et al., 2012) and it is difficult to envision any other mechanism by which aSP13-dependent plasticity could be specifically restricted to the cVA-responsive MBγ neurons. It is formally possible that, despite the broad potentiation of MBγ output synapses upon training, it is only the contribution of the cVA-responsive MBγ neurons that drives courtship suppression when the male subsequently encounters as mated female. Alternatively, it has been suggested that M6 neurons encode a generic aversive signal (Aso et al., 2014b), and so specificity to cVA might instead arise in downstream circuits that selectively integrate M6 output with the innate cVA-processing pathway from the lateral horn. In this regard, it is interesting to note that other MBONs have been implicated in courtship learning (Montague and Baker, 2016) or general aversion (Aso et al., 2014b), but M6 is the only MBON common to both.

Late activation of the same aSP13 neurons in the time window of 8–10 hr after training is both necessary and sufficient to consolidate STM to LTM (Krüttner et al., 2015). Thus, in the time window when STM would otherwise decay (Keleman et al., 2007), reactivation of the same MBγ>M6>aSP13 recurrent circuit may instead consolidate it into LTM. The mechanism by which aSP13 neurons are reactivated is unknown, but is evidently dependent upon their activation within the MBγ>M6>aSP13 recurrent circuit during training. It will be interesting to find out how this late aSP13 reactivation mechanism might relate to the mechanism that underlies persistent aSP13 activity during training.

In summary, our data suggest that a brief persistent activity of aSP13 neurons represents a neural correlate of courtship working memory, while the prolonged potentiation of MBγ>M6 synapses corresponds to STM. We propose that persistent activity of the dopaminergic neurons in the MBγ>M6>aSP13 feedback loop lays the foundation for formation of short-term courtship memory in Drosophila, and that later reactivation of the same recurrent circuit consolidates STM into LTM. Thus, in contrast to the prevailing view of memory progression in the Drosophila MB that distinct memory phases are located in different compartments or lobes (Aso and Rubin, 2016; Davis, 2011; Pascual and Préat, 2001), our data suggest that in the context of courtship conditioning, working memory, STM, and LTM all reside in the same γ5 compartment. Our conclusions do not preclude however, the involvement of other MB neurons in courtship memory (Montague and Baker, 2016) as it is conceivable that modulation, potentially of the opposite sign, of the appetitive memory pathways could be critical for courtship learning (Perisse et al., 2016). We therefore envision that distinct courtship memory types are not located in distinct circuits, but rather mediated by distinct processes within a common circuit. Encoding distinct memory phases within a common circuit may be an efficient mechanism for encoding memories for which the behavioral consequence is largely independent of timing and context (Fusi et al., 2005).