Abstract
The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] modulates many key brain functions including those subserving sensation, emotion, reward, and cognition. Efficient clearance of 5-HT after release is achieved by the antidepressant-sensitive 5-HT transporter (SERT, SLC6A4). To identify novel SERT regulators, we pursued a proteomic analysis of mouse midbrain SERT complexes, evaluating findings in the context of prior studies that established a SERT-linked transcriptome. Remarkably, both efforts converged on a relationship of SERT with the synaptic adhesion protein neuroligin 2 (NLGN2), a post-synaptic partner for presynaptic neurexins, and a protein well-known to organize inhibitory GABAergic synapses. Western blots of midbrain reciprocal immunoprecipitations confirmed SERT/NLGN2 associations, and also extended to other NLGN2 associated proteins [e.g., α-neurexin (NRXN), gephyrin]. Midbrain SERT/NLGN2 interactions were found to be Ca2+-independent, supporting cis vs. trans-synaptic interactions, and were absent in hippocampal preparations, consistent with interactions arising in somatodendritic compartments. Dual color in situ hybridization confirmed co-expression of Tph2 and Nlgn2 mRNA in the dorsal raphe, with immunocytochemical studies confirming SERT:NLGN2 co-localization in raphe cell bodies but not axons. Consistent with correlative mRNA expression studies, loss of NLGN2 expression in Nlgn2 null mice produced significant reductions in midbrain and hippocampal SERT expression and function. Additionally, dorsal raphe 5-HT neurons from Nlgn2 null mice exhibit reduced excitability, a loss of GABAA receptor-mediated IPSCs, and increased 5-HT1A autoreceptor sensitivity. Finally, Nlgn2 null mice display significant changes in behaviors known to be responsive to SERT and/or 5-HT receptor manipulations. We discuss our findings in relation to the possible coordination of intrinsic and extrinsic regulation afforded by somatodendritic SERT:NLGN2 complexes.
Highlights
In the mammalian central nervous system (CNS), serotonin [5-hydroxytryptophan (5-HT)] acts as an essential modulatory neurotransmitter, regulating a wide variety of brain functions that range from sensory perception and pain responses to emotion and cognition, among many others (Azmitia, 2007)
We found that strain variation in Nlgn2 mRNA levels exhibited a significant, positive correlation with strain variation of SERT (Slc6a4) and Tph2 mRNA levels, suggesting that Nlgn2 expression may be tightly coordinated with multiple dimensions of 5-HT homeostasis
From the 853 proteins identified in MudPIT analyses, 45 proteins were identified in WT but not SERT null samples, with an additional 80 proteins showing higher than two-fold enrichment in WT compare to SERT null samples (Supplementary File 1)
Summary
In the mammalian central nervous system (CNS), serotonin [5-hydroxytryptophan (5-HT)] acts as an essential modulatory neurotransmitter, regulating a wide variety of brain functions that range from sensory perception and pain responses to emotion and cognition, among many others (Azmitia, 2007). CNS serotonergic innervation originates from small clusters of mesencephalic and brainstem dorsal raphe neurons, with forebrain projections derived largely from dorsal and median raphe nuclei (Abrams et al, 2004; Gaspar and Lillesaar, 2012). In both somatodendritic and synaptic compartments, the availability of 5-HT is actively constrained by the high-affinity, antidepressant-sensitive 5-HT transporter (SERT, SLC6A4; Bunin and Wightman, 1998; Montanez et al, 2003; Mathews et al, 2004; Jennings et al, 2006). We found that strain variation in Nlgn mRNA levels exhibited a significant, positive correlation with strain variation of SERT (Slc6a4) and Tph mRNA levels, suggesting that Nlgn expression may be tightly coordinated with multiple dimensions of 5-HT homeostasis
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