Serotonin receptor 3A controls interneuron migration into the neocortex

Abstract

Neuronal excitability has been shown to control the migration and cortical integration of reelin-expressing cortical interneurons (INs) arising from the caudal ganglionic eminence (CGE), supporting the possibility that neurotransmitters could regulate this process. Here we show that the ionotropic serotonin receptor 3A (5-HT(3A)R) is specifically expressed in CGE-derived migrating interneurons and upregulated while they invade the developing cortex. Functional investigations using calcium imaging, electrophysiological recordings and migration assays indicate that CGE-derived INs increase their response to 5-HT(3A)R activation during the late phase of cortical plate invasion. Using genetic loss-of-function approaches and in vivo grafts, we further demonstrate that the 5-HT(3A)R is cell autonomously required for the migration and proper positioning of reelin-expressing CGE-derived INs in the neocortex. Our findings reveal a requirement for a serotonin receptor in controlling the migration and laminar positioning of a specific subtype of cortical IN.

Figure 1. 5-HT_3AR is specifically expressed and upregulated in caudal ganglionic eminence (CGE)-derived interneurons (INs) during the phase of cortical invasion.

(a) Microdissection of cortical tissue (dotted lines) containing GAD65-GFP⁺ INs was performed at three developmental time points corresponding to the phase of tangential migration (E14.5), cortical invasion (E18.5) and termination of migration (P2.5). GAD65-GFP⁺ INs were isolated using fluorescence-assisted cell sorting (FACS) and gene expression analysis was performed using microarrays. (b) Microarrays revealed that Htr3a mRNA expression increases during cortical invasion in FACS-isolated GAD65-GFP⁺ INs (n=3 replicates at each time point). (c) Genetic fate mapping indicates that Htr3a-GFP⁺ INs only rarely (<%1) overlap with Nkx2.1-Cre; TOM⁺ cells and preferentially populate superficial cortical layers at P21 (n=4,636 Htr3a-GFP⁺ cells and 7,588 TOM⁺ cells in four brains). (d) The majority of Htr3a-GFP⁺ INs located in the mantle zone of the CGE at E14.5. CGE cells are immunolabelled for the CGE-enriched transcription factors COUP-TFII and/or SP8 (n=3,451 Htr3a-GFP⁺ cells in three brains). (e) Htr3a-GFP⁺ INs populate the CGE but not the medial ganglionic eminence (MGE) and migrate tangentially to reach the dorsal pallium through the subventricular zone (SVZ) stream (arrowheads). (f) In situ hybridization showing that the expression pattern of the Htr3a mRNA at E17.5 is similar to Htr3a-GFP⁺ mouse. Error bars are means±s.e.m. COUP-TFII, chicken ovalbumin upstream promoter transcription factor 2; Hst, Hoechst; LGE, lateral ganglionic eminence; VZ, ventricular zone. Scale bars: (a) 200 μm; (c) 50 μm; (d) 50 μm (low magnification), 10 μm (high magnification); (e,f) 200 μm.

Figure 2. 5-HT_3AR activation increases the migratory speed of CGE-derived interneurons (INs) during the phase of cortical plate invasion.

(a) Microdissection of cortical tissue (dotted lines) containing GAD65-GFP⁺ INs was performed at E14.5 corresponding to the phase of tangential migration or at E17.5 during the phase of cortical invasion. GAD65-GFP⁺ INs were platted in culture and time-lapse imaging was performed at day in vitro 1 (+DIV1). (b) Illustrative time-lapse sequence showing that GAD65-GFP⁺ INs (arrowheads) at E17.5 (+DIV1) increase their migration after exposure to the 5-HT_3AR agonist SR57227 (100 nM). Cells were tracked during a control period and a drug period of 360 min each. (c, d) Quantification revealed that 5-HT_3AR activation significantly increases the migratory speed and decreases the pausing time of GAD65-GFP⁺ INs at E17.5 (+DIV1; n=156 cells in three independent experiments; c) but not at E14.5 (+DIV1; n=140 cells in three independent experiments; d). ***P<0.001, paired Student’s t-test. Error bars are means±s.e.m. Scale bars: (a) 150 μm; (b) 10 μm.

Figure 3. The 5-HT_3AR specifically regulates growth cone dynamics of interneurons (INs) derived from the CGE but not the MGE.

(a) Schema illustrating that MGE-derived INs (mINs) were labelled using focal electroporation of a tdTOM-expressing plasmid in the MGE, whereas CGE-derived INs (cINs) were labelled using Htr3a-GFP⁺ slices. At E14.5 (+DIV3), confocal time-lapse imaging of the growth cones (GCs) of cINs and mINs was performed in the cortical plate (dotted area) and GC dynamics was assessed at baseline, during and after 5-HT_3AR activation with m-chlorophenylbiguanide (mCPBG 100 μM). (b) Growth cone dynamics was assessed by tracing the GC area in sequential time-lapse images and by calculating the speed of progression of the growth cone. (c) Time-lapse sequence illustrating that 5-HT_3AR activation induces a delayed increase in the GC size of Htr3a-GFP⁺ cINs (green arrowheads) but not TOM⁺ mINs (white arrowheads). (d) Illustrative tracing of growth cone areas of Htr3a-GFP⁺ cIN and TOM⁺ mIN during the baseline period, during exposure to mCPBG and during the two wash periods. Note that GC size of cIN increases during the second wash period but not the GC of mIN. (e) Quantification revealed that the GC area and speed of cINs (n=13 GC in 10 slices) but not of mINs (n=12 GC in 10 slices) significantly increases after 5-HT_3AR activation (***P<0.001, *P<0.05, two-way analysis of variance with Bonferroni’s test). Error bars are means±s.e.m. Scale bars: (a) 200 μm (low magnification), 5 μm (high magnification); (c) 10 μm; (d) 5 μm.

Figure 4. 5-HT_3AR controls the migration and positioning of CGE-derived INs in the cortical plate (CP).

(a) Examples of migratory paths in E17.5 acute cortical slices showing cINs remaining in the intermediate zone (IZ; white tracks) or invading the CP (red tracks). Squares indicate start positions and circles indicate end positions of tracked cells after 8 h imaging. Quantification showing a significant reduction in the percentage of Htr3a-ko; GAD65-GFP⁺ INs (n=124 cells in five slices) migrating from the IZ into the CP in comparison with GAD65-GFP⁺ INs (n=106 cells in five slices). In addition, the migratory speed of Htr3a-ko; GAD65-GFP⁺ INs is significantly decreased, whereas their pausing time significantly increased versus GAD65-GFP⁺ INs (***P<0.001, **P<0.01, unpaired Student’s t-test). (b) In vivo quantification reveals significant decrease in the percentage of Htr3a-ko; GAD65-GFP⁺ INs (n=6144 cells in four brains) in layer 2–4 versus GAD65-GFP⁺ INs (n=6708 cells in four brains; **P<0.01, unpaired Student’s t-test) in the prospective P0.5 somatosensory cortex. (c) Quantification of MGE-derived INs in the prospective P0.5 somatosensory cortex labelled by in situ hybridization for the MGE-specific transcription factor Lhx6 reveals no significant layering differences between Htr3a-ko; GAD65-GFP⁺ mice (n=2,013 cells in three brains) versus control GAD65-GFP⁺ (n=3,731 cells in four brains). (d) In vivo quantification in host E19.0 wild-type (WT) cortex reveals significant misdistribution of isochronic-grafted Htr3a-ko; GAD65-GFP⁺ INs (n=882 cells in five brains) in the developing cortex versus GAD65-GFP⁺ INs (n=736 cells in four brains; *P<0.05, unpaired Student’s t-test). Hst, Hoechst; MZ, marginal zone. Error bars are means±s.e.m. Scale bars: (a) 50 μm; (b–d) 100 μm.

Figure 5. 5-HT_3AR controls the laminar positioning of reelin-expressing CGE-derived INs in the cortex.

(a) At P21, the positioning of Htr3a-ko; GAD65-GFP⁺ INs (n=2,775 cells in six brains) was significantly altered in superficial layers compared with GAD65-GFP⁺ INs (n=2,707 cells in six brains; ***P<0.001, *P<0.05 unpaired Student’s t-test). (b) The percentage of Htr3a-ko; GAD65-GFP⁺ INs expressing reelin (n=706 cells in five brains) was significantly decreased in layer 1 compared with reelin⁺/GAD65-GFP⁺ INs (n=1,376 cells in six brains; **P<0.01 unpaired Student’s t-test). (c,d) The laminar distribution of Htr3a-ko; GAD65-GFP⁺ INs expressing NPY (n=1,093 cells in eight brains) or VIP (n=963 cells in six brains) was not altered compared with NPY⁺ (n=1,127 cells in six brains) or VIP⁺/GAD65-GFP⁺ INs (n=1,219 cells in six brains; unpaired Student’s t-test). Error bars are means±s.e.m. NPY, neuropeptide Y; VIP, vasointestinal peptide. Scale bar, (a) 50 μm.