Heterotopic Transplantations Reveal Environmental Influences on Interneuron Diversity and Maturation  Giulia Quattrocolo, Gord Fishell, Timothy J. Petros  Cell Reports  Volume 21, Issue 3, Pages 721-731 (October 2017) DOI: 10.1016/j.celrep.2017.09.075 Copyright © 2017 Terms and Conditions

Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 1 Transplanted MGE-Derived Interneurons Migrate and Mature Normally in Host Tissue (A) Schematic depicting the cortical and hippocampal regions isolated from P0–P2 mouse brains. (B) Coronal section through a P1 Nkx2.1-Cre;Ai9 brain showing the regions of cortex (orange) and hippocampus (blue) that were used for transplantations. (C) Flow cytometry dot plots of tomato+ interneurons from Nkx2.1-Cre;Ai9 cortex and hippocampus. (D) Schematic of different transplantation strategies. (E) Examples from P30 WT brains receiving homotopic tomato+ interneuron grafts at P1. (F) Two transplanted interneurons displaying characteristic interneuron morphology and markers of a PV+ basket cell and SST+ Martinotti cell. (G) Histogram depicting the percentage of cortically targeted transplanted tomato+ cells expressing either PV or SST. n = 5 brains for Ctx-to-Ctx and Hip-to-Ctx. Data are represented as mean ± SEM; scale bars, 200 μm in (B) and 50 μm in (E) and (F). Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 2 Homotopically Grafted Interneurons Resemble Endogenous Interneurons (A) Cortex and hippocampus from endogenous Nkx2.1-Cre;Ai9 and grafted brains depicting tomato+ cells immunostained for SST (arrowheads), PV (arrows), and nNOS (open arrowheads). (B) Pie charts depicting the proportion of transplanted neurons expressing PV, SST, or nNOS in endogenous brains and homotopic grafts. n = 3 brains for endogenous cortex and hippocampus, 5 brains for Ctx-to-Ctx, 7 brains for Hip-to-Hip. (C) Pie charts depicting the proportion of transplanted interneurons classified based on their intrinsic firing properties. n = 19 cells Ctx-to-Ctx, 23 cells Hip-to-Hip. (D) Examples of the highest recorded firing frequencies of tomato+ cells in endogenous and homotopic conditions. For late spiking interneurons, the darker traces represent the first step at which firing was observed. Injected current steps for depicted traces: −100 pA and 520 pA (left panels); −100 pA and 520 pA (middle, top panel); −100 pA and 320 pA (middle, bottom panel); −100 pA, 60 pA (darker trace) and 520 pA (right, top panel); −100 pA, 40 pA (darker trace) and 520 pA (right, bottom panel). n = 20 cells endogenous cortex, 25 cells endogenous hippocampus, 18 cells Ctx-to-Ctx, 23 cells Hip-to-Hip. (E) Histograms depicting the highest firing frequency and input resistance in fast spiking interneurons from endogenous and Ctx-to-Ctx tomato+ cells. n = 13 cells endogenous cortex, 13 cells Ctx-to-Ctx. Data are represented as mean ± SEM; ∗∗∗p ≤ 0.005; scale bars, 50 μm. See also Figures S1–S4. Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 3 The Host Region Determines the Class of Interneurons in Heterotopic Transplants (A) Images from heterotopic grafts (Ctx-to-Hip and Hip-to-Ctx) depicting tomato+ cells immunostained for various interneuron markers. Arrowheads, SST+ cells; open arrowheads, SST+/nNOS+ cells. (B) Pie charts depicting the proportion of transplanted neurons expressing PV, SST, or nNOS (left) and their firing properties (right) in heterotopic grafts. Markers, n = 5 brains Hip-to-Ctx, 3 brains Ctx-to-Hip. Ephys: n = 21 cells Hip-to-Ctx, 19 cells Ctx-to-Hip. (C) Examples of highest firing frequency patterns of late spiking cells recorded in heterotopic grafts, darker traces represent the first step at which firing was observed. Injected current steps for depicted traces: −100 pA, 40 pA (darker trace), and 520 pA (left); −100 pA, 60 pA (darker trace), and 400 pA (right). n = 2 cells Hip-to-Ctx, 3 cells Ctx-to-Hip. (D) Examples of sEPSCs recorded in non-fast spiking interneurons in homotopic (red) and heterotopic (pink) transplants. (E) Histograms depicting the frequency of non-fast spiking sEPSC. n = 4 cells Ctx-to-Ctx, 10 cells Hip-to-Ctx, 5 cells Hip-to-Hip, 7 cells Ctx-to-Hip. Data are represented as mean ± SEM. Scale bar, 50 μm. See also Figures S1–S3. Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 4 Transplanted Interneurons Target Endogenous Pyramidal Cells Representative section from brain transplanted with Nkx2.1-Cre;Ai32 cells (YFP), with GABAergic postsynaptic currents evoked by blue light pulses recorded in adjacent pyramidal cells (red, biocytin). In black, average traces; in red, response recorded in the presence of Gabazine. n = 10 cells Ctx-to-Ctx, 5 cells Hip-to-Ctx. Scale bars, 50 μm. Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 5 The Subtype of Transplanted nNOS+ Cell Correlates with the Donor Brain Region (A) Nkx2.1-Cre;Ai9 endogenous cortex and Ctx-to-Ctx transplants stained with nNOS (arrowheads). Note that the targeted Ctx-to-Ctx injection was deep and some cells settled in the hippocampus, allowing for direct comparison of homotopic (Ctx-to-Ctx) and heterotopic (Ctx-to-Hip) nNOS+ cells in the same brain. (B) Histogram depicts the proportion of type I and type II cells in the endogenous cortex and hippocampus, and hippocampus targeted transplants. n = 3 brains endogenous cortex and hippocampus, 4 brains Ctx-to-Hip and Hip-to-Hip. Data are represented as mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01; scale bars, 50 μm. See also Figures S5 and S6. Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions

Figure 6 Cortex-Targeted Transplantations Are Characterized by a Larger Percentage of SST+/CR+ Grafted Interneurons (A) Calretinin expression (arrowheads) in endogenous and transplanted brains. (B) Histograms depicting the proportion of SST+ cells that express CR in endogenous and transplanted brains. n = 3 brains for all conditions except n = 4 brains for Hip-to-Hip. Data are represented as mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.005; scale bars, 50 μm. See also Figures S5 and S6. Cell Reports 2017 21, 721-731DOI: (10.1016/j.celrep.2017.09.075) Copyright © 2017 Terms and Conditions