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. 2017 Jul 5;37(27):6527-6538.
doi: 10.1523/JNEUROSCI.3250-16.2017. Epub 2017 Jun 2.

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility

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Free PMC article

Reduced Slc6a15 in Nucleus Accumbens D2-Neurons Underlies Stress Susceptibility

Ramesh Chandra et al. J Neurosci. .
Free PMC article

Abstract

Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility.SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The reduction of Slc6a15 occurs selectively in the NAc D2-neurons. Genetic reduction of Slc6a15 induces susceptibility to a subthreshold stress, while genetic overexpression in D2-neurons prevents social avoidance after chronic social defeat stress.

Keywords: Slc6a15; depression; medium spiny neurons; nucleus accumbens; social defeat stress.

Figures

Figure 1.
Figure 1.
Slc6a15 is reduced in the stress-susceptible NAc and postmortem in NAc of individuals with major depressive disorder. A, Illustration of the CSDS experimental paradigm. Subject mice underwent 10 d of physical interaction with a novel aggressive CD1 mouse for 10 min each day. This is followed by sensory contact, on the other side of a perforated divider, for 24 h after each physical interaction. On day 11, mice are tested in a social interaction test with a novel mouse. B, Susceptible mice display decreased time (in seconds) in the social interaction zone, interacting with the novel mouse, compared with control or resilient mice (n = 12 control mice; n = 11 susceptible mice; and n = 10 resilient mice). Heat map of the mouse video tracking control, susceptible, and resilient mice (right), which demonstrates increased time in the social interaction zone for control and resilient mice. In contrast, susceptible mice spent less time in this zone. C, Slc6a15 mRNA is reduced in NAc, 24 h after CSDS, of susceptible mice compared with resilient and nonstressed control mice (n = 12 control mice; n = 11 susceptible mice; and n = 10 resilient mice). D, Slc6a15 mRNA is decreased postmortem in NAc of individuals with MDD who committed suicide who were either ON or OFF antidepressant treatment (n = 10 control subjects; n = 11 individuals with MDD who were receiving medication; n = 12 individuals with MDD who were receiving medication). The table (top) indicates the number of subjects and the sex in each group. *p < 0.05. Error bars indicate SEM.
Figure 2.
Figure 2.
Slc6a15 mRNA and protein are enriched in NAc D2-neurons but reduced in NAc D2-neurons of CSDS-susceptible mice. A, Slc6a15 mRNA is enriched in NAc D2-neurons compared with D1-neurons and input (nonimmunoprecipitated mRNA) from each group (n = 8 input; n = 4 D1-Cre-RT mice; and n = 4 D2-Cre-RT mice). B, Representative D2-GFP NAc images after IHC with anti-NeuN (blue), anti-GFP (green) and anti-Slc6a15 (red). Arrows demonstrated colocalization of Slc6a15 with D2-GFP(+) neurons. Scale bar, 50 μm. C, Quantification of Slc6a15 neurons colocalized with D2-GFP(+) and D2-GFP(−). NAc neurons demonstrated that Slc6a15 colocalizes significantly more with D2-GFP(+) neurons (n = 3 per group). D, Heat map of the video tracking in control, susceptible, and resilient D2-Cre-RT and D1-Cre-RT mice during SI with a novel social target. Quantification of time spent (in seconds) in the interaction zone with the novel social target by controls, resilient mice, and susceptible mice. Susceptible mice display reduced time in the interaction zone (n = 5 controls; n = 5 susceptible D2-Cre-RT mice; and n = 4 resilient D2-Cre-RT mice; n = 5 controls; n = 6 susceptible D1-Cre-RT mice; and n = 3 resilient D1-Cre-RT mice). E, Cell-type-specific expression of Slc6a15 in NAc D2- and D1-neurons after CSDS demonstrates decreased Slc6a15 ribosome-associated mRNA in NAc D2-neurons of susceptible mice compared with resilient and control mice. However, Slc6a15 levels remain unchanged in NAc D1-neurons (n = 5 controls; n = 5 susceptible D2-Cre-RT mice; and n = 4 resilient D2-Cre-RT mice; n = 5 controls; n = 6 susceptible D1-Cre-RT mice; and n = 3 resilient D1-Cre-RT mice). F, Representative D2-GFP NAc images of CSDS control, susceptible, and resilient mice after IHC with anti-NeuN (blue), anti-GFP (green), and anti-Slc6a15 (red). Scale bar, 50 μm. G, Quantification of Slc6a15 colocalized neurons with D2-GFP(+) and D2-GFP(−) NAc neurons demonstrates reduced Slc6a15 colocalization with D2-GFP(+) neurons in susceptible mice compared with control and resilient mice. No change is observed in D2-GFP(−) neurons among the three groups. (n = 3 controls; n = 4 susceptible; and n = 4 resilient). *p < 0.05. Error bars indicate SEM.
Figure 3.
Figure 3.
Genetic reduction of Slc6a15 in D2-neurons induces susceptibility to a subthreshold social defeat stress. A, Mouse Slc6a15 shRNA sequences and their targets on the Slc6a15 gene. B, qRT-PCR analysis of Slc6a15 demonstrates sufficient Slc6a15 shRNA knockdown in Neuro2a cells. C, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Slc6a15miR-mCitrine. D, Illustration of bilateral infusion of AAVs into NAc. E, D2-Cre mice receiving AAV-DIO-Slc6a15miR-mCitrine to NAc display reduced Slc6a15 mRNA in NAc (n = 4 per group). F, IHC in Slc6a15miR and SSmiR injected NAc in D2-Cre mice, mCitrine (green) and Slc6a15 (red), demonstrates reduced Slc6a15 in Slc6a15miR-mCitrine expressing D2-neurons. Scale bar, 50μm. G, Illustration of the SSDS experimental paradigm. Subject mice underwent 1 day of 3 physical interactions with a novel aggressive CD1 for 2 min each day. This is followed by sensory contact, on the other side of a perforated divider, for 15 min after each physical interaction. On day 2, mice are tested in a social interaction test with a novel mouse. H, D2-Cre mice receiving AAV-DIO-Slc6a15miR-mCitrine to NAc display reduced time interacting with a novel mouse (Target) compared to mice receiving AAV-DIO-SSmiR-mCitrine or non-stressed control mice after SSDS (n = 9 control SSmiR, n = 9 defeat SSmiR, n = 5 control Slc6a15miR, n = 9 defeat Slc6a15miR). Heat density map of video tracking of SSDS Slc6a15miR injected or SSmiR injected D2-Cre mice (right panel). I, Time spent in the interaction zone without a novel mouse (No Target) is unchanged between all groups. *p < 0.05, Error bars indicate SEM.
Figure 4.
Figure 4.
Overexpression of Slc6a15 in D2-neurons prevents social avoidance after chronic social defeat stress. A, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Slc6a15-tGFP. B, Illustration of bilateral infusion of virus into NAc. C, D2-Cre mice expressing AAV-DIO-Slc6a15-tGFP in NAc display increased the Slc6a15 mRNA compared with AAV-DIO-EYFP controls (n = 5 EYFP; n = 4 Slc6a15-tGFP). D, Immunohistochemistry with tGFP or GFP (green) and Slc6a15 (red) in NAc of D2-Cre mice demonstrates high colocalization of Slc6a15 (red) with Slc6a15-tGFP (green). Scale bar, 50 μm. E, D2-Cre mice with Slc6a15-tGFP in D2-neurons that underwent CSDS display a time spent in the interaction zone (in seconds), with a social target present, that is comparable to nonstressed control conditions, while D2-Cre mice expressing EYFP in D2-neurons display reduced time in the interaction zone (n = 11 control EYFP mice; n = 11 defeat EYFP mice; n = 6 control Slc6a15-tGFP mice; and n = 8 defeat Slc6a15-tGFP mice). Heat-density maps of video tracking in D2-Cre CSDS Slc6a15-tGFP and EYFP mice during SI (right). F, Time spent in the interaction zone without a novel mouse (No Target) is unchanged among all groups. *p < 0.05. Error bars indicate SEM.
Figure 5.
Figure 5.
Slc6a15 is unaltered in ChAT interneurons and Slc6a15 reduction selectively in D2-MSNs induces susceptibility to SSDS. A, IHC in D2-GFP NAc, after CSDS, shows anti-Slc6a15 (red) colocalization with ChAT (blue) and D2-GFP (green) neurons in control, susceptible, and resilient mice. Scale bar, 10 μm. B, Slc6a15 colocalization is unaltered in ChAT interneurons in CSDS-susceptible or CSDS-resilient mice compared with nonstressed controls (n = 3 controls; n = 4 CSDS-susceptible mice; and n = 4 CSDS-resilient mice). C, Schematic of the double-floxed, inverted, open reading frame Cre-dependent AAV vector expressing Slc6a15miR-mCitrine. Illustration of bilateral infusion of AAVs into NAc. D, IHC in Slc6a15miR- and SSmiR-injected NAc in A2A-Cre mice, mCitrine (green), and Slc6a15 (red) demonstrates reduced Slc6a15 in Slc6a15miR-mCitrine expressing D2-neurons. Scale bar, 50 μm. E, A2A-Cre mice receiving AAV-DIO-Slc6a15-miR-mCitrine to NAc display reduced time in the interaction zone with the novel social target present (Target) after SSDS, compared with SSmiR controls (n = 6 control SSmiR mice; n = 7 defeat SSmiR mice; n = 9 control Slc6a15miR mice; and n = 8 defeat Slc6a15miR). Heat density map of video tracking of Slc6a15-miR- and SSmiR-injected A2A-Cre mice in the presence of a novel social target (right). F, AAV-Slc6a15-miR expression in NAc D2-MSNs of A2A-Cre mice that underwent SSDS does not alter the time spent in the interaction zone when the target is absent (No Target). *p < 0.05. Error bars indicate SEM.

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