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. 2015 Jan 20;10(3):346-358.
doi: 10.1016/j.celrep.2014.12.033. Epub 2015 Jan 15.

Disruption of Transient Serotonin Accumulation by Non-Serotonin-Producing Neurons Impairs Cortical Map Development

Xiaoning Chen  1 , Ran Ye  2 , J Jay Gargus  3 , Randy D Blakely  2 , Kostantin Dobrenis  4 , Ji Ying Sze  5
Affiliations

Disruption of Transient Serotonin Accumulation by Non-Serotonin-Producing Neurons Impairs Cortical Map Development

Xiaoning Chen et al. Cell Rep. .

Abstract

Polymorphisms that alter serotonin transporter SERT expression and functionality increase the risks for autism and psychiatric traits. Here, we investigate how SERT controls serotonin signaling in developing CNS in mice. SERT is transiently expressed in specific sets of glutamatergic neurons and uptakes extrasynaptic serotonin during perinatal CNS development. We show that SERT expression in glutamatergic thalamocortical axons (TCAs) dictates sensory map architecture. Knockout of SERT in TCAs causes lasting alterations in TCA patterning, spatial organizations of cortical neurons, and dendritic arborization in sensory cortex. Pharmacological reduction of serotonin synthesis during the first postnatal week rescues sensory maps in SERTGluΔ mice. Furthermore, knockdown of SERT expression in serotonin-producing neurons does not impair barrel maps. We propose that spatiotemporal SERT expression in non-serotonin-producing neurons represents a determinant in early life genetic programming of cortical circuits. Perturbing this SERT function could be involved in the origin of sensory and cognitive deficits associated with neurodevelopmental disorders.

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Figures

Figure 1
Figure 1. Generation of Transgenic Mice with Selective Knockout of SERT in Vglut2-Expressing Neurons (SERTGluΔ) or Raphe 5-HT-Producing Neurons (SERTRapheΔ)
(A) Schematic illustration of the construction of SERT conditional knockout mice. Two loxP cassettes flank exons 3 and 4 of the SERT gene Slc6a4. The reading frame is disrupted when exon 2 splices to exon 5. Rectangles denote exons, and lines denote introns. Transgenic mice carrying the floxed SERT gene (SERTfl/fl) were crossed with mice expressing Cre in either Vglut2+ glutamatergic neurons (SERTGluΔ) or ePet1+ raphe 5-HT-producing neurons (SERTRapheΔ). Transient SERT expression in Vglut2+ TCAs projecting to control sensory cortices is shown in Figures S1 and S2, and Vglut2-Cre-induced recombination in thalamic neurons is shown in Figure S3. (B) qPCR analysis of SERT mRNA levels in brainstem and VB nuclei of P7 mice. Results are average of five animals/genotype. Values from SERTfl/fl littermates of SERTGluΔ and SERTRapheΔ mice were pooled and compared to that of WT, SERTGluΔ, and SERTRapheΔ mice (mean ± SEM, ***p < 0.001, one-way ANOVA). (C–E) In situ hybridization analysis of SERT mRNA in P7 mice. (C) Representative images showing SERT mRNA expression in thalamic VB and DLG neurons diminished in SERTGluΔ, but not in SERTRapheΔ mice. Notably, SERT mRNA in hippocampal neurons was also diminished in SERTGluΔ mice, consistent with Vglut2-Cre activity in hippocampal neurons shown in Figure S3. (D) Images of serial coronal sections of brainstem, showing SERT mRNA expression in raphe neurons diminished in SERTRapheΔ, but not in SERTGluΔ mice. (E) Quantification of SERT+ signals in dorsal raphe nuclei (mean ± SEM, ***p < 0.001, Student’s t test). NS, not significantly different.
Figure 2
Figure 2. Vglut2-Cre Eliminates SERT in TCAs but Not in Raphe Neurons
(A) Vglut2 and SERT double immunolabeling of TCAs projecting to visual (v), auditory (a), and somatosensory barrel (s) cortices on tangential sections of P7 mutant and control littermate mice. SERT staining in the Vglut2+ TCAs was diminished in SERTGluΔ, SERTGluΔ/−, and SERTNull but not SERTRapheΔ mice. SERT immunohistochemistry on serial tangential sections through the PMBSF from a control mouse is shown in detail in Figure S2. (B) SERT and 5-HT immunostaining of adjacent coronal sections of the raphe nuclei from P7 mice. SERT staining was abolished in SERTNull, diminished in SERTRapheΔ, but unaffected in SERTGluΔ mice. Aq, aqueduct. (C) Western blots of SERT protein relative to actin in somatosensory barrel cortex and brainstem collected from individual P7 mice. SERTfl/fl mice were littermates of SERTGluΔ (one) and SERTRapheΔ (two) mice.
Figure 3
Figure 3. Requirement of SERT Expression in TCAs for 5-HT Uptake in the Barrel Cortex
(A) Double immunohistochemistry for Vglut2 and 5-HT on coronal sections of the somatosensory cortex of P7 mice. The positions of cortical layers are indicated. Third row images show an enlarged view of 5-HT immunohistochemistry in areas outlined by red boxes on images in the second row. Note two types of 5-HT+ fibers in WT and control mice: fibers that colocalized with Vglut2+ TCA patches at layer IV (L4, strongly) and layer VI (L6 weakly), and Vglut2-negative fibers, which were thicker, highly punctated, and randomly distributed. 5-HT staining in Vglut2+ TCAs, not the thicker fibers, was abolished in SERTGluΔ and SERTGluΔ/ and SERTNull mice. 5-HT staining in Vglut2+ TCAs in SERTRapheΔ mice was indistinguishable from the control littermate and WT mice. Cortical sections from mutant and control littermate mice were always stained in parallel. For each of the brain samples, alternate sections were analyzed by double immunostaining of Vglut2 and SERT, and data are presented in Figure S4. (B) HPLC analysis of 5-HT concentrations in brain tissues collected from P7 mice. Values from SERTfl/fl littermates of SERTGluΔ and SERTRapheΔ mice were pooled and compared to that of WT, SERTGluΔ, and SERTRapheΔ mice (n = 4, mean ± SEM, **p < 0.01, ***p < 0.001, one-way ANOVA). SERT protein abundance in each of the brain samples was analyzed by western blotting shown in Figure 2C. (C) Model for SERT expressed in TCAs and raphe afferents during barrel map development: Left, in SERTRapheΔ mice, TCA-expressed SERT clears 5-HT from the sensory cortex for barrel map development. Right, in SERTGluΔ mice, trophic 5-HT accumulates around growing TCAs despite SERT expression in raphe afferents and impairs barrel map formation. Yellow lines denote TCAs, blue/yellow lines denote raphe neuron axons, and red dots denote 5-HT.
Figure 4
Figure 4. SERT Expressed in TCAs Is Essential for Barrel Map Development
(A) Barrel maps on tangential sections of the somatosensory cortex visualized by CO staining. In SERTGluΔ and SERT KO mice, but not in SERTRapheΔ mice, barrel structures corresponding to anterior snout (AS), lower lip (LL), forepaw (FP), and hindpaw (HP) were largely absent, but barrels in PMBSF representing whiskers were preserved. Images of CO staining of additional SERT mutant and relative control mice are presented in Figure S5A. (B) TCA patterning at layer IV visualized by Vglut2 immunostaining of tangential sections through the somatosensory cortex of P7 mice. In SERTGluΔ/−, SERTGluΔ, and SERT KO mice, Vglut2+ patches in the PMBSF were discernible but more blurred suggestive of poorer segregation, as compared to corresponding control littermate and WT mice, and Vglut2+ TCA projections to the other barrel subfields failed to display a discernable patterning. Two SERTfl/fl mice each represent control littermates for SERTGluΔ and SERTRapheΔ mice as shown. (C and D) Evaluation of Vglut2+ TCA patterning in P7 PMBSF and AS, by calculating BAI-Vglut2 (C) and BFI-Vglut2 (D). Mutant and control littermate mouse samples were stained in parallel (n = 5–6, mean ± SEM, *p < 0.05, **p < 0.01, Student’s t test). The differences between WT, SERTRapheΔ, and control groups and the differences between SERT KO, SERTGluΔ, and SERTGluΔ/− groups were not statistically significant (p = 1, one-way ANOVA followed by Bonferroni test). The methods for quantifying BAI-Vglut2 and BFI-Vglut2 are illustrated in Figure S6.
Figure 5
Figure 5. Inhibition of 5-HT Synthesis Rescues TCA Patterning in SERT Mutants
(A) Inhibition of 5-HT synthesis by daily PCPA treatment from P1 to P7 rescued Vglut2+ TCA patterning in SERTGluΔ/− mice compared to untreated littermates. Daily injection of PCPA at the dosage of 100 mg/kg (n = 6) or 300 mg/kg (n = 4) produced similar effects. Results following 300 mg/kg PCPA treatments are shown (mean ± SEM, *p < 0.05, Student’s t test). (B) Knockout of Tph1 partially improved Vglut2+ TCA patterning in the barrel cortex of SERT KO mice. Each dot represents one mouse (*p < 0.05, Student’s t test).
Figure 6
Figure 6. SERT Expressed in TCAs Governs Multiple Spatial Organizations of Satb2+ Neurons in the Somatosensory Cortex
(A) Serial sections through the PMBSF of P7 mice labeled by double immunostaining of Vglut2+ TCAs and Satb2+ cell nuclei, with DAPI counterstain. The distance from the pial surface is indicated. In control littermates (SERTfl/−), Satb2+ cells, as well as DAPI-labeled nuclei, displayed two distinct patterns: stripes (arrowhead) located ~250 μm from the pial surface and barrels (arrow) located below the stripe structure. Both Satb2+ patterns were largely absent in SERTGluΔ/− mice. Impaired spatial organization of other neuronal markers in P7 and mature barrel cortex of SERTGluΔ/− mice is shown in Figure S7. (B) Evaluation of Satb2 barrel structure by calculating BFI of Satb2 immunohistochemistry in P7 PMBSF (n = 5, mean ± SEM, *p < 0.05, Student’s t test).
Figure 7
Figure 7. Knockout of SERT Function in TCAs Causes Mistargeting of GABA Dendrites in the Barrel Cortex
(A) Immunohistochemistry for GABA on tangential sections through the PMBSF of P7 mice. In control littermates (SERTfl/−), GABA+ cells showed an organization of barrel structures. In the SERTGluΔ/− mice, GABA+ cells failed to form a discernable pattern, even though rudiment ring-like cell arrangements were evident with DAPI counterstain. (B) Distribution of immunolabeled GABA+ cells in barrel walls delineated by densely packed DAPI-labeled nuclei relative to that in the center of barrel hollows (n = 4–6, mean ± SEM, *p < 0.05, Student’s t test). (C) Examples of reconstruction of the dendritic trees of calretinin+ GABAergic neurons located close to the edge of barrel walls in P7 PMBSF. Ci and Cii, low-magnification images show calretinin+ cells (green) at barrel walls delineated by the high density of DAPI-stained nuclei (blue) on tangential sections through the PMBSF. Calretinin immunostaining colocalized with a subset of GABAergic neurons located in the barrel cortex. In three P7 control mice examined, 102 out of 127 calretinin+ neurons at layer IV colabeled with GABA immunostaining. Ciii and Civ representative higher-magnification confocal images show neurons that, labeled with both calretinin (green) and GABA (red), were selected for dendritic reconstruction. Cv and Cvi, higher-magnification confocal images show calretinin+ dendritic profiles. Cvii and Cviii, reconstruction of calretinin+ dendritic trees superimposed over barrel walls from collapsed serial confocal images encompassing 18 μm above and below the center of the cell body. Fourteen to 15 neurons from three mice for each genotype were analyzed. (D) Selective knockout of SERT in TCAs, not in raphe axons, disrupted oriented calretinin+ GABAergic dendrite patterning. Primary barrels were defined as the barrels that house the majority of total dendritic length (mean ± SEM, **p < 0.01, ***p < 0.001, Student’s t test).
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