Brief disruption of activity in a subset of dopaminergic neurons during consolidation impairs long-term memory by fragmenting sleep

Reviewer #1 (Public review):

Summary:

The authors aim to use state-of-the art behaviour, imaging, and connectome techniques to identify the neural interaction between sleep and long-term memory consolidation in the PAM-DPM circuits, a well-known dopaminergic pathway within Drosophila Mushroom Body.

Strengths:

From a Drosophila sleep researcher's perspective, the investigation follows a clear and logical strategy to collect a huge dataset of sleep, appetitive memory, and live imaging. The authors clearly identified and showed that activation of a PAM subset: alpha-1 reduces sleep quality and memory consolidation in a starvation-dependent manner. The authors also convincingly demonstrated the corresponding neuronal responses of DPM neurons following PAM alpha-1 activation, and the positive role of DPM neural activity in sleep and memory consolidation. Moreover, the authors applied a new way of sleep statistics to demonstrate hour-by-hour changes between treatment and genotypes. Importantly, the authors demonstrated that memory loss derived from PAM alpha 1 activation can be partly restored by ectopic sleep enhancement via feeding THIP during the memory consolidation period after training.

Weaknesses:

Two investigatory gaps relate to the misalignment between circuital activity and behaviours, due to the nature of large circuital functional analysis like this. Firstly, the central observation of the study indicates that PAM alpha1 activation causes DPM inhibition which disrupts sleep and memory consolidation. Therefore one would expect a reduced PAMalpha1 and increased DPM activities after memory training, but the authors found that the endogenous CRTC::GFP reported neuronal activity for PAMalpha1 and DPM are both increased after memory training (Figure 9). This can be due to the difficult functional demarcation among the 14 PAMalpha1 projections. Secondly, the authors acknowledged the contradicting finding that memory defect is detected in PAMalpha1 inactivation (Figure 7C), yet suggested a tight link between sleep and memory consolidation; it is clear loss of PAM subset activity can disrupt memory consolidation without affecting sleep (cf Figure 7C and 7I).

Reviewer #2 (Public review):

Summary:

Sleep plays a critical role in memory consolidation, but the neural mechanisms underlying this relationship remain poorly understood. The authors present novel findings implicating two small neuronal groups with inhibitory connections, PAM-a1 to DPM, in sleep regulation and LTM consolidation. However, whether the PAM-a1 to DPM microcircuit promotes LTM consolidation through sleep regulation requires further investigation.

Strengths:

The authors report several novel findings. Brief activation or inhibition of PAM-a1 neurons, or brief inhibition of DPM neurons during the first few hours after training, impairs 24-hour LTM. Notably, these brief manipulations disrupt sleep for many hours afterward, particularly at night. Interestingly, disruption of PAM-a1 and DPM neurons impairs sleep and appetitive memory consolidation only under starvation conditions, and pharmacological induction of sleep during the night rescues the LTM defects. These findings suggest that PAM-a1 and DPM neurons are involved in sleep regulation and LTM consolidation under starvation. These are important findings that advance our understanding of the link between sleep and memory consolidation.

Weaknesses:

Some claims lack sufficient evidence or clarity:

(1) All sleep experiments are conducted under the "training" (temperature-change) condition. While genotypic controls are helpful, additional no-training controls are required to confirm that the observed differences are due to training rather than unknown genotype-related factors. The fact that experimental genotypes exhibit significantly altered sleep even before "training" (e.g., Figs. 7H, J, K, 8A, B, D) highlights the necessity of these controls.

(2) Previous studies on disrupted memory due to sleep reduction have primarily examined conditions with severe sleep deprivation. In contrast, this report claims that relatively small decreases in total sleep accompanied by sleep fragmentation are responsible for impaired memory consolidation. It remains unclear whether sleep fragmentation at this level is truly critical for memory consolidation. The authors should cause sleep loss and fragmentation of similar magnitude through other means and determine whether it can impair LTM.

(3) The authors employed a neural activity reporter to show that starvation increases the basal activity of PAM-a1 but not DPM neurons in untrained flies (Figures 9C-E). They observed small increases in the activity of both neuron groups immediately after training but not one hour later. Given the inhibitory connection from PAM-a1 to DPM, it is unclear why both neuron groups show increased activity after training. Additionally, as the authors acknowledge, it is puzzling how the inactivation of PAM-a1 produces similar effects on sleep and memory as DPM inhibition and PAM-a1 activation. Further experiments are needed to clarify these findings, such as manipulating PAM-a1 activity during the one-hour post-training period and evaluating the effect on DPM activity. Including data from training under fed conditions would provide a more comprehensive understanding of state-dependent neural activity. Even if certain experiments are not feasible, these issues warrant further discussion. It is also important to clarify that the term "synchronized" does not imply single-spike-level synchrony.

(4) The authors considered that PAM-a1 and DPM might function in parallel, independent pathways for sleep and LTM. They rejected this possibility based on the lack of additive effects when both neuronal groups were simultaneously inactivated. However, they found that MB299B-labelled neurons exert stronger memory effects than MB043B-labelled neurons, while MB043B neurons have stronger sleep effects. If sleep is a primary driver of memory consolidation, a stronger correlation between memory and sleep effects would be expected. This observation merits further discussion.

(5) Given prior knowledge that PAM neurons are heterogeneous and that the R58E02 driver is broadly expressed, data in Figures 1-5 concerning PAM are outdated. The use of more restricted PAM-a1 drivers from the outset would make the manuscript easier to read and interpret.

(6) Some figures lack relevant data, certain experiments are missing necessary controls, and anomalies are present in some data sets.

Reviewer #3 (Public review):

Summary:

Understanding the neural circuits that link sleep and memory remains a fundamental challenge in neuroscience. In this study, Lin Yan and colleagues investigate how dopamine signaling in Drosophila regulates long-term memory (LTM) formation in the context of sleep. They identify a specific microcircuit between protocerebral anterior medial dopamine neurons (PAM-DANs) and dorsal paired medial (GABAergic DPM) neurons that modulates memory consolidation. Their findings suggest that disrupting the basal activity of PAM-α1 neurons during early consolidation impairs LTM, with particularly pronounced effects under starvation conditions. Notably, sleep fragmentation caused by this disruption can be pharmacologically rescued, restoring LTM. These results provide compelling evidence that dopamine signaling plays a crucial role in linking sleep and memory, offering new insights into the underlying mechanisms.

Strengths:

This study presents a well-executed investigation into sleep-memory interactions, utilizing a combination of connectomics, behavioral assays, functional imaging, and pharmacological manipulations. The authors convincingly demonstrate that the PAM-α1 and DPM circuits interact, highlighting a potential mechanism by which sleep influences memory consolidation. The anatomical and functional dissection of this circuit is of high interest to the field, and the study's integration of sleep and memory processes contributes significantly to our understanding of dopamine's role in cognitive functions.

Weaknesses:

While the study is well-designed and presents compelling findings, some aspects require further clarification. The interpretation of dopamine receptor signaling remains incomplete, particularly regarding inhibitory pathways. The role of DPM in memory consolidation is not entirely conclusive, as different genetic approaches yield variable results. Additionally, some inconsistencies in neuronal activity patterns and experimental variability, especially regarding sleep patterns or pharmacological rescue, should be addressed to strengthen the mechanistic framework.

Conclusion:

Overall, this study provides valuable new insights into how sleep and dopamine circuits interact to regulate memory consolidation. While the findings are compelling, addressing the points above-particularly receptor signaling and the specific role of DPM and its activity patterns within the microcircuit would further solidify the study's conclusions.