Behind the paper: Cell-type and sex-specific rhythmic gene expression in the nucleus accumbens

Published in Neuroscience

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Circadian rhythms play a critical role in physiology and behavior, but the role of rhythms in gene expression in limbic brain regions is vastly understudied. Circadian rhythms are critical for human health and are highly conserved across species, controlling physiological processes across the light/dark cycle. The core molecular clock is found in almost every cell in the brain and body and is strongly implicated in these processes. In addition to regulating basic physiological processes and brain function, disruptions in these rhythms are linked to many diseases, including altered mood and substance use (SU) disorder vulnerability across species. Our lab has been investigating the intersection between circadian rhythms and psychiatric disease for decades and, along with others, have found that behavioral changes are often mediated by the function of circadian genes in reward-related brain regions, such as the nucleus accumbens (NAc).

The role of the NAc in physiology and behavior is largely cell-type specific. Gene expression rhythms have been thoroughly studied across cell types in the central pacemaker controlling circadian rhythms, the suprachiasmatic nucleus (SCN). However, even though rhythms in transcripts expression are abundant and widespread in the NAc, little is known about rhythmicity across cell type and the function of these cell-type specific rhythms.

In this study we identified and characterized, for the first time, the rhythmic translatome specifically in dopamine D1 and D2 receptor expressing medium spiny neurons (D1, D2 MSNs) and compared rhythmicity results to homogenate as well as astrocyte samples taken from the NAc. Since sex differences are prominent in circadian rhythms and reward-related behavior, we measured sex differences by collecting samples from male and female mice in order to determine whether gene expression rhythms differ by cell type and sex. Lastly, we chose to focus on actively translated mRNA expression by leveraging Ribotag mouse lines crossed to cre lines. In this context, rhythms in expression are more likely to be functionally relevant at the protein level. 

We identified diurnal rhythms in all cell types, with astrocytes having the most rhythmic transcripts and D1 cells having the least. Top rhythmic transcripts are largely core clock genes, which peak at approximately the same time of day in each cell type and sex. While cell signaling and signal transduction related processes are most commonly enriched in MSNs, protein regulation pathways are enriched across cell type. These processes and their related transcripts also tend to peak during the dark, or active, phase in rodents when protein synthesis, folding, modification and degradation would be critical to cell function in the brain, as well as behavior. Protein regulation may be more quiescent during the light phase because animals are asleep.

This work also allowed us to confirm that the relationship between rhythms in MSNs and astrocytes are different in the NAc compared to the SCN. We found that core clock genes peak at roughly the same time of day in the NAc, but peak times are about 6-h later in neurons compared to astrocytes in the SCN. This is driven by divergent rhythms in neurons, where transcripts tend to peak several hours later in the SCN compared to the NAc. This suggests the relationship between astrocytes and neurons is different in these regions, especially for circadian regulation. In this paper, we hypothesize that while rhythmic glutamatergic synthesis and release from astrocytes suppresses neuronal activity in the SCN, resulting in an anti-phasic relationship, rhythms in glutamate reuptake in NAc astrocytes might be causing astrocytes to fall into phase with neurons.

We also identified a critically important difference in peak time for rhythmic transcripts across sex. Understanding peak time is critical to determining how transcripts work together. Shared rhythmic transcripts tend to reach their peak in expression about 2-h later in females, suggesting diurnal rhythms in reward may be delayed in females. This is particularly true for D1 MSNs and less so for D2 MSNs and astrocytes. If so, this could indicate that reward-related behavior should be measured, and therapeutic approaches might be more beneficial if delivered at later clock times in females. We have shown that mutations in circadian genes increase SU to a greater extent in females. Perhaps this delay in rhythms in MSNs could make females more vulnerable to circadian disruptions, especially in the context of SU. Future studies will be critical to determine both why rhythms are delayed in females and what the functional implications of those delays are for animal behavior.

Lastly, we used these novel data to investigate whether any classically used cell-type specific markers in the NAc are rhythmic. This issue is critical since neuroscience research is focusing more and more on investigating cell-type-specific changes in the brain. The use of rhythmic markers could lead to over- or under-enrichment of targeted cell types depending on peak and sampling times. This could be particularly problematic because each marker has a different peak/trough time. We confirmed that several classically used cell-type markers are rhythmic in the NAc and identified novel markers that do not have rhythms in gene expression over the 24 hour period. It will be crucial for rigorous experimental design to consider marker rhythmicity moving forward.

This study greatly expands our knowledge of how individual cell types contribute to rhythms in the NAc. Understanding rhythms at baseline will help us determine how the NAc regulates rhythms in reward and how disruptions to these rhythms might lead to altered behavior. Most importantly, this study indicates that gene expression rhythms in MSNs might play an important role in neuronal activity. Future studies will be imperative to understanding how these rhythms in MSNs or astrocytes drive activity in the NAc and impact diurnal rhythms in reward.

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