Sexually Dimorphic Regulation of Norepinephrine Projection Neurons: Transcriptional Profiling of Mouse Locus Coeruleus

Background: A number of neuropsychiatric diseases demonstrate sex-biased incidence, including ADHD and autism. The locus coeruleus (LC), which sends noradrenergic projections throughout the brain, is known to demonstrate sex-specific responses to stress-related molecules like corticotropin-releasing factor at both cellular and behavioral levels (Curtis et al 06, Bangasser et al 16). Moreover, the engrailed-2 (En2) knockout mouse—an animal model for autism spectrum disorders—has been demonstrated to have sex specific deficiencies in norepinephrine signaling (Genestine et al 2015). However, the full extent and clinical significance of sex differences in the LC have not been defined. We employed Translating Ribosome Affinity Purification (TRAP) to selectively profile the transcriptome of mouse LC, providing a new look into the breadth of—and cellular functions implicated by—sex-specific gene expression in the LC.

Objectives:

1. Identify genes whose expression is unique to the LC (among hindbrain cells).
2. Identify genes expressed in a sex-specific manner within the mouse LC.
3. Verify expression patterns using existing resources, as well as immunohistochemistry (IHC) and in-situ hybridization (ISH).
4. Identify functional consequences of sex-specific gene expression.

Methods: TRAP utilizes transgenic mice expressing a GFP-tagged ribosomal subunit under the control of a cell-type specific promoter (in this case, the Slc6a2, aka NET, the norepeniphrine transporter). Affinity purification of GFP-conjugated ribosomes from brain homogenate allows for collection of mRNAs being translated at the time of collection in the LC, and subsequent quantification—for analysis of cell-type specific gene expression and comparison of cell-type specific expression between the sexes. Using the Allen Brain Atlas, IHC, and ISH, and electrophysiology, we validate our findings of LC-specific gene expression.

Results: We identified 188 transcripts enriched in the LC compared to the rest of the hindbrain, and validated these with independent methods. Moreover, we identified 84 transcripts significantly enriched in female LC, and 75 enriched in male LC, suggesting broad mechanisms of sex-specific gene expression and cellular function. In contrast, we find almost no (≤10 total) genes with sex-specific expression in TRAP of serotonergic neurons, indicating the LC is strikingly dimorphic by comparison. Among the genes we found to be both LC-specific and differentially expressed (>2-fold enriched in females) was a prostaglandin receptor, Ptger3. Electrophysiology of LC neurons in the presence of a Ptger3 agonist, sulprostone, revealed a robust response of female LC neurons compared to male LC neurons.

Conclusions: A comprehensive analysis of gene utilization has not been performed on the LC. Here, we identify transcripts uniquely expressed in LC and demonstrate that the LC has robust of sex differences, which may underlie sexually dimorphic behaviors in LC related functions such as response to stress, novelty, and attention. Moreover, this work raises the question of whether dimorphic regulation of the norepinephrine signaling in the brain might affect dosing and efficacy of noradrenergic drugs in the treatment of neurodevelopmental and psychiatric diseases. Finally, we have discovered a receptor that may be used to alter noradrenergic signaling in a sex specific manner.