PMC

Spatial and temporal origin of main hippocampal CA1 interneuron subtypes. Schematic diagram summarizing the findings reported in the present study. The scheme is completed based on previous reports showing overlapping expression of PV/SOM and VIP/CR/nNOS (; ; ).

Inducible genetic fate mapping of CGE-derived interneurons. A–F, Left, Representative examples of M2R (A), CCK (B), VIP (C), CoupTFII (D), reelin (E), and CR (F) expression in fate-mapped interneurons in mature hippocampus of Mash1CreER:RCE mice treated with tamoxifen at E12.5 (for M2R), E15.5 (for VIP and CR), or E16.5 (for CCK, CoupTFII, and reelin). Scale bar: 25 μm. Right, Contribution of M2R+ (A), CCK+ (B), VIP+ (C), CoupTFII+ (D), reelin+ (E), and CR+ (F) interneurons to the cohort arising from CGE between E12.5 and E16.5 (for M2R, n = 168, 188, 236, 290, and 185, respectively; for CCK, n = 167, 225, 234, 209, and 203, respectively; for VIP, n = 187, 211, 205, 312, and 193, respectively; for CoupTFII, n = 168, 201, 217, 296, and 217, respectively; for reelin, n = 142, 294, 161, 303, and 262, respectively; for CR, n = 164, 266, 248, 350, and 233, respectively). Error bars indicate SEM.

Inducible genetic fate mapping of MGE-derived interneurons. A–C, Left, Representative examples of PV (A), SOM (B), and nNOS (C) expression in fate-mapped interneurons in the mature hippocampus of Olig2CreER:ZEG mice treated with tamoxifen at E9.5 (for PV and SOM) or E10.5 (for nNOS). Scale bar: 25 μm. Right, Contribution of PV+ (A), SOM+ (B), and nNOS+ (C) interneurons to the cohort arising from MGE between E9.5 and E13.5 [for PV, n = 179, 141, 121, 177, and 50, respectively; for SOM, n = 89, 104, 73, 97, and 37; for nNOS, n = 79, 79, 124, 79, and 39; group data for GFP+/nNOS+ cells were previously reported by ]. Error bars indicate SEM.

Immunohistochemical markers primarily associated with MGE-derived interneurons. A, B, Representative images illustrating the coexpression of GFP with PV (i), SOM (ii), nNOS (iii), and reelin (iv) in the Nkx2-1Cre:RCE (A) and GAD65-GFP (B) lines. The filled arrowheads indicate interneurons coexpressing GFP and the indicated marker. C, Histogram showing the contribution of GFP+ cells from Nkx2-1Cre:RCE (warm colors) and GAD65-GFP (cool colors) lines to the populations of PV, SOM, nNOS, and reelin immunolabeled interneurons in CA1 [n = 135, 150, 990, and 295, respectively, in the Nkx2-1Cre:RCE. Note that group data reported for GFP+/nNOS+ cells in Nkx2-1:RCE were previously reported by ; n = 236, 505, 385, and 901, respectively, in the GAD65-GFP line]. D, Number of cells coexpressing GFP with PV, SOM, nNOS, and reelin in the Nkx2-1Cre:RCE line presented as a percentage of the total number of GFP+ cells (n = 395, 417, 1992, and 504, respectively, in the Nkx2-1Cre:RCE). Scale bar: 25 μm.

Immunohistochemical markers primarily associated with CGE-derived interneurons. A, B, Representative images illustrating the coexpression of GFP with M2R (i), CoupTFII (ii), CCK (iii), VIP (iv), and CR (v) in the Nkx2-1Cre:RCE (A) and GAD65-GFP (B) lines. The filled arrowheads indicate interneurons coexpressing GFP and the indicated marker. The open arrowheads indicate cells expressing the indicated marker but not GFP. C, Histogram showing the contribution of GFP+ cells from Nkx2-1Cre:RCE (warm colors) and GAD65-GFP (cool colors) lines to the populations of M2R-, CR-, CCK-, VIP-, and CoupTFII-immunolabeled interneurons in CA1 (n = 76, 151, 242, 144, and 216, respectively, in the Nkx2-1Cre:RCE; n = 133, 854, 281, 163, and 1376, respectively, in the GAD65-GFP). D, Number of cells coexpressing GFP with M2R, CR, CCK, VIP, and CoupTFII in the GAD65-GFP line presented as a percentage of the total number of GFP+ cells [n = 867, 798, 545, 556, 767, respectively, in the GAD65-GFP; note that group data concerning GFP+/VIP+ cells in GAD65-GFP mice includes counts previously reported as supplemental data in ]. Scale bar: 25 μm.

Migration of MGE- and CGE-derived interneurons into the hippocampus during development. A–E, Representative images illustrating the migration of GFP-labeled MGE-derived interneurons in the Nkx2-1Cre:RCE line between E13.5 and P30. F–J, Representative images illustrating the migration of GFP-labeled CGE-derived interneurons in the GAD65-GFP line between E13.5 and P30. All sections were counterstained with DAPI (blue). Interneurons of both MGE and CGE origin follow similar routes of tangential migration along the marginal zone (MZ) and intermediate/subventricular zone (SVZ) from the ganglionic eminences to the hippocampus (HP). Scale bar: (in J) 200 μm (all panels). K, L, Histograms illustrating the density of MGE- and CGE-derived GFP+ interneurons within the hippocampus in the Nkx2-1Cre:RCE and GAD65-GFP lines, respectively, at the times indicated. Total number of cells counted (from left to right) were as follows: for the Nkx2-1Cre:RCE, n = 0, 24, 7493, 8428, 16,256, 14,912, 14,513, 15,610, 10,011, 7810, and 6177; and for GAD65-GFP, n = 0, 40, 406, 2292, 3048, 4284, 9995, 5611, 4591, 2403, and 1776. Error bars indicate SEM.

Representative MGE-derived hippocampal interneurons. A–K, Neurolucida reconstructions of GFP+ interneurons recorded in slices from P15–P30 Nkx2-1Cre:RCE pups (dendrites and soma in black; axon in red). Scale bar: 100 μm. The dashed lines indicate the approximate boundaries of s.o., s.p., s.r., and s.l.m. Under each drawing is shown the molecular profile obtained from single-cell PCR analysis for the recorded cell with filled boxes indicating transcripts detected. Also shown are the electrophysiological responses of the cells to the indicated square wave current pulses (bottom) from a resting potential near −60 mV. Depolarizing current pulses and corresponding responses are for near threshold and 2× threshold stimulation (scale bars shown in K are for all traces). Phase plots of the APs arising from 2× threshold stimulation are shown at right, with the first AP phase plot colored red and subsequent APs progressing from warm to cool colors ending in violet. L, Histogram summarizing the frequency of occurrence for the 16 transcripts probed by scPCR among the MGE cohort of recorded cells.

Unsupervised cluster analyses of hippocampal GABAergic interneurons based on developmental, electrophysiological, and molecular properties. A, Ward's clustering applied to the sample of 142 recorded MGE- and CGE-derived interneurons. In this dendrogram, the x-axis represents individual cells, and the y-axis represents the average euclidean within-cluster linkage distance. B, Comparison of Ward and K-means clustering algorithms. The clustering generated by the K-means algorithm is mostly consistent with the Ward clustering when considering six clusters as revealed by this matching table describing the intersectional relations between K-means and Ward clusters. The numbers at the bottoms and ends of columns and rows, respectively, display the numbers of cells within the corresponding cluster. Entries of the table indicate how many cells of a K-means cluster are contained within a given Ward cluster. C, Histogram summarizing the frequency of occurrence of each of the 16 transcripts probed by scPCR within each cluster obtained with the K-means clustering (K = 6). See D for cluster color code. D, Silhouette plot resulting from the K-means clustering with K = 6 clusters. Within each cluster along the horizontal axis, cells were ranked in decreasing order of their silhouette values. The vertical axis represents the silhouette values S(i) for each individual data point (see Materials and Methods).

Representative CGE-derived hippocampal interneurons. A–K, Neurolucida reconstructions of GFP+ interneurons recorded in slices from P15–P30 GAD65-GFP pups (dendrites and soma in black; axon in red). Scale bar: 100 μm. The dashed lines indicate the approximate boundaries of strata oriens (s.o.), pyramidale (s.p.), radiatum (s.r.), lacunosum moleculare (s.l.m.), and lucidum (s.lu.). Under each drawing is shown the molecular profile obtained from single-cell PCR analysis for the recorded cell with filled boxes indicating transcripts detected. Also shown are the electrophysiological responses of the cells to the indicated square wave current pulses (bottom) from a resting potential near −60 mV. Depolarizing current pulses and corresponding responses are for near-threshold and 2× threshold stimulation (scale bars shown in K are for all traces). Phase plots of the APs arising from 2× threshold stimulation are shown at right, with the first AP phase plot colored red and subsequent APs progressing from warm to cool colors ending in violet. L, Histogram summarizing the frequency of occurrence for the 16 transcripts probed by scPCR among the CGE cohort of recorded cells.

Influence of developmental, molecular, and electrophysiological properties on the quality of the K-means clustering. A, B, Histograms illustrating the changes in global silhouette value (S′) for the entire dataset following randomization of individual molecular (A) and electrophysiological (B) parameters. The dashed lines indicate S′ for the intact dataset without any randomization. C, Histogram illustrating the effect of randomizing certain combinations of parameters on S′. Scrambled combinations included origin with transcription factors (origin, Lhx6, CoupTFII, Npas1 and 3) with or without 5-HT3; the five most influential interneuron markers that yielded the largest silhouette decrease when individually randomized (PV, SOM, NPY, CCK, nNOS); all frequently used interneuron markers (GADs, CR, PV, SOM, NPY, CCK, VIP, nNOS); all mRNAs probed; and all electrophysiological properties examined. Error bars of the scrambled silhouette width are evaluated by SD over 10 independent randomizations. D, Progressive change in S′ observed upon cumulatively increasing the number of scrambled parameters. On the “random” curve (triangles), scrambled parameters were randomly picked among the list of 37 parameters used in the clustering. On the “sorted” curve (squares), scrambled parameters were chosen in an orderly manner, first randomizing the parameter that least altered S′ (VIP) and continuing with parameters exhibiting increasing discriminative power (last one: SOM).

Time course of MGE- and CGE-derived interneuron migration to the hippocampus from the ganglionic eminences. A–F, Representative images illustrating the migration of GFP+ CGE-derived interneurons generated on E12.5 in the Mash1CreER:RCE line. The time points indicate the duration between tamoxifen administration (E12.5 for all panels) and when tissue was collected. Sections were counterstained with DAPI (blue). The yellow arrowhead indicates the hippocampal anlage (HP). The migration from their site of genesis on E12.5 in the CGE to hippocampus lasts ∼72 h. Scale bar: (in D) 200 μm (all panels). G, H, Histograms illustrating the normalized density of CGE- and MGE-derived hippocampal interneurons from the Mash1CreER:RCE and Olig2CreER:ZEG lines for tamoxifen administrations between E10.5 and E16.5 as indicated. Data are normalized to the peak density observed for all tamoxifen injection time points within each mouse line (E14.5 + 96 h for Mash1CreER:RCE and E12.5 + 48 h for Olig2CrER:ZEG). The duration of migration for interneurons generated later in development is shorter than those born early despite the fact that the enlarging brain has resulted in a longer path of migration. Total number of cells counted (from left to right) were as follows: for the Mash1CreER:RCE, n = 0, 2248, 2713, 2419, 1746, 988, 3516, 3016, 5144, 3402, 1888, 1462, 2282, 2896, 2340, 1455, 1072, 583; and for the Olig2CreER:ZEG, n = 0, 0, 146, 151, 73, 44, 211, 174, 156, 94, 73, 59.

Quantitative scPCR comparisons between PV+ fast-spiking basket cells and CCK+ SCA/non-fast-spiking basket cells. A, Amplification plots for PV (red), CCK (green), GAD65 (blue), VGluT1 (yellow), and actin (black) for harvests from a representative pyramidal cell (left), fast-spiking basket cell (middle), and non-fast-spiking basket cell (right). Relative fluorescence intensities (ΔR_n) were plotted against PCR cycle numbers on a logarithmic scale. B, C, Electrophysiological (left) and morphological (right) profiles of the fast-spiking (B) and non-fast-spiking (C) basket cells that were recorded for the quantitative scPCR analysis in A. D, Pairwise comparisons of the relative abundance of PV, CCK, GAD65, and VGluT1 in individual pyramidal (PYR), fast-spiking basket (FS), and non-fast-spiking basket/SCA (CCK) neurons (n = 5, 4, and 5, respectively). Cells were tested in pairs and plotted versus each other on the same graph. Data were normalized to actin mRNA levels by plotting ΔC_t(probed transcript) = C_t(actin) − C_t(probed transcript). Each point represents the ΔC_t for the indicated transcript of a cell pair processed in parallel. The dashed line represents same ΔC_t value for the two cell types tested such that data points above the dashed line indicate higher abundance in the cell whose ΔC_t is plotted on the y-axis, while points below the line indicate higher expression within the cell whose ΔC_t is plotted along the x-axis. At left, two CCK cells (y-axis) are plotted against two FS cells (x-axis); the middle panel shows the data for two PYR (y-axis) FS (x-axis) pairs processed in parallel; the right panel illustrates findings for three PYR (y-axis)/CCK (x-axis) cell pairs. When the transcript was not detected, ΔC_t value was set arbitrarily at −10. E, Histograms of mean target mRNA abundance relative to actin mRNA calculated from the ΔC_t values plotted in D (see Materials and Methods) for PYR (n = 5), FS-BC (n = 4), and CCK-BC (n = 5) harvests. Error bars indicate SEM.