Mice with compromised 5-HTT function lack phosphotyrosine-mediated inhibitory control over prefrontal 5-HT responses

Abstract

The activity of the prefrontal cortex is essential for normal emotional processing and is strongly modulated by serotonin (5-HT). Yet, little is known about the regulatory mechanisms that control the activity of the prefrontal 5-HT receptors. Here, we found and characterized a deregulation of prefrontal 5-HT receptor electrophysiological signaling in mouse models of disrupted serotonin transporter (5-HTT) function, a risk factor for emotional and cognitive disturbances. We identified a novel tyrosine kinase-dependent mechanism that regulates 5-HT-mediated inhibition of prefrontal pyramidal neurons. We report that mice with compromised 5-HTT, resulting from either genetic deletion or brief treatment with selective serotonin reuptake inhibitors during development, have amplified 5-HT1A receptor-mediated currents in adulthood. These greater inhibitory effects of 5-HT are accompanied by enhanced downstream coupling to Kir3 channels. Notably, in normal wild-type mice, we found that these larger 5-HT1A responses can be mimicked through inhibition of Src family tyrosine kinases. By comparison, in our 5-HTT mouse models, the larger 5-HT1A responses were rapidly reduced through inhibition of tyrosine phosphatases. Our findings implicate tyrosine phosphorylation in regulating the electrophysiological effects of prefrontal 5-HT1A receptors with implications for neuropsychiatric diseases associated with emotional dysfunction, such as anxiety and depressive disorders.

Keywords: 5-HT transporter; 5-HT1a receptor; Kir3.1; prefrontal cortex; serotonin; tyrosine phosphorylation.

Figure 1.

Constitutive or developmental 5-HTT disruption leads to amplified, inhibitory 5-HT responses in prefrontal cortex. Averaged recordings (A) and quantification (B) of the outward currents elicited by 5-HT (10 μm) in neurons from 5-HTT WT (n = 21), HET (n = 30), and KO (n = 12) mice. Representative immunohistochemical staining (C) and quantification (D) of eYFP-labeled 5-HT fibers in the medial prefrontal cortex. Scale bar, 25 μm. E, The neuronal 5-HT currents in the absence (baseline, n = 18) or the acute presence of SSRIs (fluoxetine, 10 μm, n = 7; citalopram, 10 μm, n = 6) on WT brain slices (p > 0.05). F, The 5-HT currents in neurons from group-housed (WT, n = 20; HET, n = 22) or single-housed (WT, n = 21; HET, n = 21) mice. G, Graph summarizing the 5-HT currents from adult mice treated chronically during the postnatal period with the SSRI fluoxetine (PN-Flx, n = 15), vehicle (PN-Veh, n = 15), or control mice (PN-Ctl, n = 11). *p < 0.05. **p < 0.01.

Figure 2.

The 5-HT_1A receptor–Kir3 channel pathway mediates the amplified 5-HT electrophysiological response in mice with 5-HTT disruption. Representative recordings (A) and scatterplot (B) of the 5-HT response before and after application of the 5-HT_1A antagonist, WAY100635 (30 nm) in within-cell paired recordings from 5-HTT HET (n = 5) and PN-Flx (n = 7) neurons. C, Representative Western blot (top) and quantification (bottom) showing prefrontal protein levels of 5-HT_1A receptor (n = 4 each group). D, The 5-HT_1A current before and after application of the Kir3 channel blocker tertiapin Q (0.1 μm) and washout (10–20 min) in neurons from WT (n = 6) and HET mice (n = 5). Representative Western blots (top) and quantification (bottom) showing prefrontal protein levels of Kir3.1 (E), Kir3.2 (F), and Kir3.4 (G) in WT (n = 8 or 9) and HET mice (n = 8–10). *p < 0.05. **p < 0.01.

Figure 3.

Inhibition of 5-HT_1A electrophysiological responses by tyrosine kinase activity is inefficient, but not lost, in mice with 5-HTT disruption. A, The 5-HT_1A responses from WT neurons in the absence (combined baseline, n = 51) or presence of either tyrosine kinase inhibitors lavendustin A (10 μm, Lav A, n = 19) or PP2 (10 μm, n = 41). Averaged recordings (B) and quantification (C) of the 5-HT_1A response in the absence or presence of Src family tyrosine kinase inhibitor PP2 in neurons from WT (baseline, n = 22; PP2, n = 17), HET (baseline, n = 18; PP2, n = 18), and PN-FLX (baseline, n = 13; PP2, n = 19) mice. In graph, baseline-normalized data are shown to demonstrate that the effect of PP2 differs significantly across groups. Independent replications of group differences in 5-HT response: WT versus HET or PN-FLX (p < 0.01). Averaged recordings (D) and quantification (E) of the 5-HT_1A responses in the absence or presence of tyrosine phosphatase inhibitor OVD (1 mm) from neurons of littermate WT (baseline, n = 25; OVD, n = 20) and HET (baseline, n = 13; PP2, n = 14) mice. In graph, baseline-normalized data are shown to demonstrate that the effect of OVD differs significantly between groups. Independent replication of group difference in 5-HT response: WT versus HET (p = 0.0001). F, Schematic illustrating the working model of the relationship between prefrontal 5-HT_1A responses and tyrosine phosphorylation in 5-HTT WT and HET mice. *p < 0.05. **p < 0.01.