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. 2019 May;24(5):726-745.
doi: 10.1038/s41380-018-0260-9. Epub 2018 Oct 2.

SSRIs Target Prefrontal to Raphe Circuits During Development Modulating Synaptic Connectivity and Emotional Behavior

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Free PMC article

SSRIs Target Prefrontal to Raphe Circuits During Development Modulating Synaptic Connectivity and Emotional Behavior

M Soiza-Reilly et al. Mol Psychiatry. .
Free PMC article

Erratum in

Abstract

Antidepressants that block the serotonin transporter, (Slc6a4/SERT), selective serotonin reuptake inhibitors (SSRIs) improve mood in adults but have paradoxical long-term effects when administered during perinatal periods, increasing the risk to develop anxiety and depression. The basis for this developmental effect is not known. Here, we show that during an early postnatal period in mice (P0-P10), Slc6a4/SERT is transiently expressed in a subset of layer 5-6 pyramidal neurons of the prefrontal cortex (PFC). PFC-SERT+ neurons establish glutamatergic synapses with subcortical targets, including the serotonin (5-HT) and GABA neurons of the dorsal raphe nucleus (DRN). PFC-to-DRN circuits develop postnatally, coinciding with the period of PFC Slc6a4/SERT expression. Complete or cortex-specific ablation of SERT increases the number of functional PFC glutamate synapses on both 5-HT and GABA neurons in the DRN. This PFC-to-DRN hyperinnervation is replicated by early-life exposure to the SSRI, fluoxetine (from P2 to P14), that also causes anxiety/depressive-like symptoms. We show that pharmacogenetic manipulation of PFC-SERT+ neuron activity bidirectionally modulates these symptoms, suggesting that PFC hypofunctionality has a causal role in these altered responses to stress. Overall, our data identify specific PFC descending circuits that are targets of antidepressant drugs during development. We demonstrate that developmental expression of SERT in this subset of PFC neurons controls synaptic maturation of PFC-to-DRN circuits, and that remodeling of these circuits in early life modulates behavioral responses to stress in adulthood.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Molecular identity of SERT+ neurons in the prefrontal cortex (PFC). a Transient SERT expression in the PFC during postnatal development revealed by in situ hybridization on coronal sections through the frontal pole (postnatal ages (P): 4, 7, 10, and 14). b Immunolabeling against Ctip2 (layer 5), Foxp2 (layer 6), and GFP (SERTCre/+) in the PFC. b’ SERT-GFP neurons often colocalize with Ctip2 (arrowheads), Foxp2 (white arrows), or both (yellow arrows). c GFP-expressing neurons from the PFC of SERTCre/+:RCE mice were dissected and subsequently isolated using FACS (in c', individual cells indicated by arrows). d Expression levels of monoamine-related transcripts in isolated PFC SERT-GFP neurons from (c') represented by the normalized read counts obtained after deep transcriptome sequencing. e Expression levels of cortical layer-specific molecular markers after transcriptome analysis indicating an enrichment of deep layer markers (layers 5 and 6) in PFC SERT-GFP neurons
Fig. 2
Fig. 2
Subcortical brain targets of PFC-SERT+ neurons and their large contribution to the PFC-to-DRN synaptic circuit. a Schematic illustration of the injection site in the PFC. The AAV2/1-CAG-LSL-EGFP-bGH virus was used for conditional anterograde tracing in SERTCre/+ mice. Injection was done at P4–5 and histology 3 weeks after. b Effective recombination is visible in PFC neurons that express EGFP (indicated by arrows). c-e Main brain targets of PFC-SERT+ neuron axons: c Thalamic nuclei including the paraventricular (PV), mediodorsal (MD), rhomboid (Rh), ventromedial (VM), and ventrolateral (VL) nuclei; (d) the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA); (e) the dorsal raphe nucleus (DRN) and periaqueductal gray (PAG). Laterodorsal nucleus (LD), aqueduct (Aq), substantia nigra pars reticulata (SNpr). f AAV1-CAG.FLEX-TdTomato-WPRE-bGH was bilaterally injected in the PFC of P4-P5 SERTCre/+ mice used for the array tomographic anterograde tracing analysis in the DRN. g Array tomography image of seven serial-ultrathin 70-nm sections immunolabeled against synapsin (green), VGLUT1 (blue), and TdTomato (red), after anterograde viral tracing from the PFC. Asterisks indicate the same PFC TdTomato-positive axon terminal across the multiple serial sections often colabeled for VGLUT1 and synapsin. The arrow indicates a cortical axon bouton negative for TdTomato. The pie chart shows the percentages of cortical axon boutons present in the DRN that exclusively originated from PFC-SERT neurons (67 ± 8 %, red) in comparison with those arising from other cortical neurons (33 ± 8 %, blue)
Fig. 3
Fig. 3
Cortical deletion of SERT results in synaptic hyperinnervation of the DRN. a Diagram summarizing the excitatory glutamate and inhibitory GABAergic synaptic inputs received by the dorsal raphe nucleus (DRN) neurons. Synaptic inputs can be selectively identified in array tomography by the presence of specific synaptic markers including the vesicular glutamate transporter type 1 and 2 (VGLUT1 and VGLUT2, respectively) and the enzyme responsible for GABA synthesis, the glutamate decarboxylase 2 (GAD2). The prefrontal cortex (PFC), lateral habenula (LHb), laterodorsal tegmental nucleus (LDTg), ventral tegmental area (VTA), substantia nigra (SN), rostromedial tegmental nucleus (RMTg), periaqueductal gray (PAG), and hypothalamus (Hyp), have been noted as the main synaptic inputs to the DRN [50, 53, 54]. b Immunolabeling against the 5-HT biosynthetic enzyme tryptophan hydroxylase (TPH) illustrating the distribution of 5-HT neurons in the midbrain DRN. The bracketed area shows the sampling region in the midline DRN used for array tomography quantitative analyses (at P28). c Array tomography projection image of three serial-ultrathin 70-nm-thick sections of the DRN immunolabeled against VGLUT1 (green) and synapsin (red) to specifically identify cortical synaptic boutons. The arrows indicate double-labeled boutons in control and SERT-KO mice. d-e Quantitative analysis of cortical glutamate synaptic boutons (VGLUT1+) (d), and subcortical glutamate (VGLUT2+) and GABAergic (GAD2+) synaptic boutons (e) in the DRN of control and SERT-KO mice (4 mice/genotype; for VGLUT1/Synapsin pairs: F1,6 = 36.45, *p < 0.001; for VGLUT2/Synapsin pairs: F1,6 = 0.24, p = 0.64; and for GAD2/Synapsin pairs: F1,6 = 0.34, p = 0.58). f Analysis of cortical synaptic boutons in the DRN after fluoxetine-treatment (FLX) during the postnatal critical period (P2-14) (5 mice/group; F1,8 = 9.94, *p < 0.02). g-h Density of cortical synaptic boutons in the DRN after conditional SERT invalidation using Emx1bCre/+:Sertfl/fl mice (SERT-KOCTX) (g) and Pet1Cre:Sertfl/fl mice (SERT-KORaphe) (h). (g): 5 mice/genotype (Welch’s statistic = 11.20, *p < 0.03); (h): 3–4 mice/genotype (F1,5 = 3.76, p = 0.11). i Cortical synaptic boutons in the mediodorsal thalamic nucleus (MD) of control and SERT-KO mice (3 mice/genotype; F1,4 = 18.16, *p < 0.02). One-way ANOVA (d, e, f, h, i), and Welch’s t test (g). Error bars represent SEM
Fig. 4
Fig. 4
Lack of SERT increases the number of functional PFC-to-DRN synapses. a rAAV-CAG-hChR2(H134R)-mCherry was bilaterally injected into the PFC of P4–P5 control or SERT-KO mice. Photograph showing mCherry expression after the PFC AAV injection (upper left). Optogenetic stimulation and electrophysiological patch clamp recordings were made starting at P28 in coronal sections containing the DRN, as shown by the photograph of the immunolabeling of PFC mCherry+ axons innervating to DRN 5-HT neurons, identified by the presence of the enzyme TPH2 (upper right). b Amplitude of optogenetically evoked EPSCs (oEPSCs) at synapses from PFC terminals onto DRN putative 5-HT neurons (left) and non-5-HT neurons (right) at various light stimulation intensities. In control (SERTCre/+) (5-HT: n = 10 cells/5 animals; non-5-HT: n = 7 cells/4 animals); in SERT-KO (SERTCre/Cre) (5-HT: n = 10 cells/3 animals; non-5-HT: n = 6 cells/3 animals). Top: example traces at 9.8 mW (black/gray) and at 2 mW (red) stimulation); Bottom: input/output curves. Two-way ANOVA on 9.8 mW intensity: genotype x cell-type interaction (F1,29 = 0.003, p = 0.95); Genotype main effect (F1,29 = 9.32, *p < 0.01); Cell-type main effect (F1,29 = 0.51, p = 0.48). c AMPAR/NMDAR ratios at synapses from PFC-to-DRN 5-HT neurons (left) and non-5-HT neurons (right) in control (5-HT: n = 10 cells/4 animals; non-5-HT: n = 7 cells/3 animals), and SERT-KO (5-HT: n = 11 cells/3 animals; non-5-HT: n = 6 cells; 3 animals). The AMPAR responses were calculated at the peak of −50 mV, whereas NMDAR responses were determined at + 40 mV, 50 ms after stimulation. Top: example traces; bottom: bar graphs. Two-ways ANOVA: Genotype x Cell-type interaction (F1,30 = 0.007, p = 0.94); Genotype main effect (F1,30 = 0.16, p = 0.69); Cell-type main effect (F1,30 = 4.51, p < 0.05). Blue bars indicate blue light stimulation. Error bars represent SEM
Fig. 5
Fig. 5
Bidirectional modulation of developmental SSRI-induced emotional alterations by PFC-SERT+ neurons. a For chemogenetic manipulation of PFC glutamate projection-neurons, AAV5-CaMKIIa-hM4D(Gi)-mCherry (c-e) or AAV8-CaMKIIa-hM3D(Gq)-mCherry (i-k) was bilaterally injected into the PFC of P5 wild-type C57BL/6 mice. For conditional expression in PFC-SERT neurons the AAV5-hSYN-DIO-hM4D(Gi)-mCherry (f-h) and AAV8-hSYN-DIO-hM3D(Gq)-mCherry (l-n) were used in SERTCre/+ mice. Mice were treated with fluoxetine (FLX) (10 mg/kg/day in 5% sucrose) or with 5% sucrose (SUC), from P2 to P14. Behavioral analyses were started at P80, starting with the novelty-suppressed feeding test (NSF), the forced swim test (FST), and locomotor activity spaced by 7 days intervals. b Immobility time in the FST during the first day “drug-free” session. Control group: 25 mice (15 males, 10 females); FLX group: 23 mice (10 males, 13 females). Two-ways ANOVA: gender x treatment interaction (F1,44 = 1.855, p = 0.18); gender main effect (F1,44 = 6.407, p < 0.02); treatment main effect (F1,44 = 5.228, *p < 0.03). c: Immobility time in the FST (day 2); d: latency to feed in the NSF; e: locomotor activity, after a single i.p. injection of saline (NaCl 0.9%) or CNO (1 mg/kg) 30 min. before the testing. Two-ways ANOVA of the FST (c): treatment combination x gender interaction (F3,40 = 1.345, p = 0.27); treatment combination main effect (F3,40 = 9.510, p < 0.0001); gender main effect (F1,40 = 3.550, p = 0.07). FLX-CNO vs. Control-Saline (*p < 0.0001), vs. Control-CNO (*p < 0.001) and vs. FLX-Saline (*p < 0.02) by Tukey’s test. Two-ways ANOVA of NSF (d): treatment combination x gender interaction (F3,40 = 0.657, p = 0.58); treatment combination main effect (F3,40 = 6.994, p < 0.001); gender main effect (F1,40 = 0.011, p = 0.916). FLX-CNO vs. Control-Saline (*p < 0.001), vs. Control-CNO (*p < 0.004) and vs. FLX-Saline (*p < 0.04) by Tukey’s test. Two-ways ANOVA of locomotor (e): treatment combination x gender interaction (F3,40 = 0.245, p = 0.865); treatment combination main effect (F3,40 = 0.394, p = 0.758); gender main effect (F1,40 = 3.318, p = 0.08). In (c-e): Control/Saline group: 13 mice (8 males, 5 females); Control/CNO group: 12 mice (7 males, 5 females); FLX/Saline group: 12 mice (6 males, 6 females); FLX/CNO group: 11 mice (4 males, 7 females). f-h Conditional inhibition of PFC-SERT neurons in SERTCre/+ mice treated or not with fluoxetine (FLX) from P2 to 14) (a). Immobility in the FST (day 2) (f), latency to feed in the NSF (g) and locomotor activity (h) after an i.p. injection of either CNO (1 mg/kg) or NaCl 0.9% saline (SAL), 30 min. before testing. In (f): ANOVA: treatment main effect (F1,9 = 5.982, p < 0.04), in (g): ANOVA: treatment main effect (F1,9 = 6.287, p < 0.04), in (h): ANOVA: treatment main effect (F1,9 = 2.712, p = 0.134). In (f-h): FLX/Saline group: 5 males; FLX/CNO group: 6 males. Immobility time in the FST (day 2) (i), latency to feed in the NSF (j) and locomotor activity (k), after a single i.p. injection of either CNO (1 mg/kg) or NaCl 0.9% saline, 30 min. before testing. Two-ways ANOVA in (i): treatment combination x gender interaction (F3,43 = 0.612, p = 0.61); treatment combination main effect (F3,43 = 4.177, p < 0.02); gender main effect (F1,43 = 0.315, p = 0.58). FLX-Saline vs. control-saline (*p < 0.05), vs. FLX-CNO (*p < 0.01) by Tukey’s test. Two-ways ANOVA in (j): treatment combination x gender interaction (F3,43 = 0.490, p = 0.69); treatment combination main effect (F3,43 = 9.532, p < 0.0001); gender main effect (F1,43 = 3.218, p = 0.08). FLX-Saline vs. Control-Saline (*p < 0.0002), vs. Control-CNO (*p < 0.0001) and vs. FLX-CNO (*p < 0.01) by Tukey’s test. Two-ways ANOVA in (k): treatment combination x gender interaction (F3,43 = 0.815, p = 0.492); treatment combination main effect (F3,43 = 0.521, p = 0.670); gender main effect (F1,43 = 0.137, p = 0.713). In (i-k): Control/Saline group: 16 mice (8 males, 8 females); Control/CNO group: 12 mice (8 males, 4 females); FLX/Saline group: 12 mice (6 males, 6 females); FLX/CNO group: 11 mice (4 males, 7 females). l-n Conditional activation of PFC-SERT neurons in SERTCre/+ mice treated or not with fluoxetine (FLX) from P2 to P14). a Immobility in the FST (day 2) (l), latency to feed in the NSF (m) and locomotor activity (n) after an i.p. injection of either CNO (1 mg/kg) or NaCl 0.9% saline (SAL), 30 min. before the testing. In (l): ANOVA: treatment main effect (F1,13 = 5.100, p < 0.05), in (m): ANOVA: treatment main effect (F1,13 = 13.792, p < 0.01), in (n): ANOVA: treatment main effect (F1,13 = 0.0006, p = 0.981). In (l-n): FLX/Saline group: 8 males; FLX/CNO group: 7 males

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