Fig 1.
The ProMMT algorithm split CA1 GABAergic neurons into 49 clusters.
Dendrogram (left) shows a hierarchical cluster analysis of these classes. Table shows class names (chosen hierarchically according to strongly expressed genes), number of cells per class, isolation metric of each class (higher for distinct classes), the mean value of latent variable analysis for cells in this class, and the biological cell type identified from its gene expression pattern. Asterisks indicate hypothesized novel classes. Right, bar chart showing log expression of 25 selected genes for all cells in the class. Note the expression pattern of Lhx6, which suggests a developmental origin in medial ganglionic eminence for clusters Cacna2d1.Lhx6.Vwa5a and above. CGE, caudal ganglionic eminence; I-S/IS, interneuron-selective; MGE, medial ganglionic eminence; NGF, neurogliaform cell; O-Bi, Oriens-Bistratified; O-LM, oriens/lacunosum-moleculare; ProMMT, Probabilistic Mixture Modeling for Transcriptomics; slm, stratum lacunosum-moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum.
Fig 2.
Two-dimensional visualization of expression patterns using nbtSNE algorithm, which places cells of similar expression close together.
Each symbol represents a cell, with different color/glyph combinations representing different cell classes (legend, right). Grey boxes and numbers refer to the “continents” mentioned in the text and subsequent figures. nbtSNE, negative binomial t-stochastic neighbor embedding; O-LM, oriens/lacunosum-moleculare.
Fig 3.
Expression levels of 25 selected genes that together allow identification of major cell classes.
Each subplot shows an nbtSNE map of all cells, with marker size indicating log-expression level of the gene named above the plot. Similar maps for all genes can be found online at http://linnarssonlab.org/ca1/.
Fig 4.
Inferred circuit diagram of identified GABAergic cell types.
The identification of transcriptomic clusters with known cell classes is described in full in S1 Text. Laminar locations and connections between each class are derived from previous literature. CGE, caudal ganglionic eminence; IS, interneuron-selective MGE, medial ganglionic eminence; NGF, neurogliaform cell; O-Bi, Oriens-Bistratified; O-LM, oriens/lacunosum-moleculare; R-LM, Radiatum/lacunosum-moleculare.
Fig 5.
Analysis of discrete versus continuous variation by negative binomial discriminant analysis.
(A) Histogram of log-likelihood ratios for three example cluster pairs, measuring how much better each cell’s whole-genome expression pattern is explained by one or the other cluster. The top histogram (basket versus axo-axonic cells) is clearly bimodal, indicating discrete separation. The bottom two histograms (ivy versus MGE-neurogliaform cells; two subclasses of Cck/Cxcl14 cells) show substantial overlap, indicating continuous variation between clusters. The degree of bimodality is captured by the d’ statistic above each plot. (B) Pseudocolor matrix showing continuity of each pair of clusters, as assessed by d’ statistic. White means strongly bimodal; darker colors indicate continuity.
Fig 6.
Latent factor analysis reveals a common mode of continuous variation that is consistent across cell types and correlates with axon target location.
(A) Latent factor analysis assigns a single number to each cell via a search for the factor values that best predict expression of multiple genes. Mean factor values within each cluster differ systematically in a way that correlates with the identified cell class’s axon target location. Each point represents a cell, with the x-coordinate showing latent factor value and y-coordinate showing cluster, sorted by mean latent factor value. (B) Continuous gradient of latent factor values across continent 8 (top; symbol size denotes latent factor value). Largest values are found in western Slc17a8-expressing neurons identified with soma-targeting Cck basket cells; smallest values are found in eastern Calb1-expressing neurons identified with dendrite-targeting Cck cells. (C) Correlation of latent factor values with expression of six example genes. Symbols as in (A); blue, continent 2 (basket/bistratified), green, continent 8 (Cck/Cxcl14). (D) Correlations of genes with the latent factor are preserved across cell classes. X-axis: correlation of gene with latent factor in continent 2; y-axis, correlation in continent 8; Spearman’s ρ = 0.58, p < 10−100. Inset, Spearman ρ values for all pairs of continents, p < 10−100 in each case. AU, arbitrary units.
Fig 7.
Immunohistochemical characterization of intensely NOS1-positive neurons.
(A) A large multipolar neuron in stratum pyramidale is strongly SST and NPY positive in the somatic Golgi apparatus and weakly positive for CHRM2 in the somatodendritic plasma membrane (maximum intensity projection, z stack, height 11 μm; inset, maximum intensity projection of three optical slices, z stack height 2 μm). A smaller, more weakly NOS1-positive cell (double arrow) in the lower left is immunonegative for the other molecules; a second NPY-positive cell (arrow) adjoining the NOS1+ neuron is immunonegative for the other three molecules. (B) A NOS1-positive cell and another NOS1-immunonegative cell (asterisk) at the border of stratum radiatum and lacunosum-moleculare are both positive for GRM1 in the plasma membrane and PENK in the Golgi apparatus and in granules, but only the NOS1+ cell is immunopositive for CHRM2 (maximum intensity projection, z stack, height 10 μm). (C) An intensely NOS1-positive cell in stratum radiatum is also positive for PCP4 in the cytoplasm and nucleus and for PENK in the Golgi apparatus and in granules (maximum intensity projection, z stack, height 15 μm).
Fig 8.
Confirmation of predicted colocalization of NPY and pro-CCK.
(A) Interneurons at the sr/slm border immunopositive for both NPY and pro-CCK (cells 1 and 2), one of which (cell 1) is also immunopositive for CALB1. A third neuron is positive only for pro-CCK and CALB1 (cell 3). (B) Interneurons at the sr/slm border immunopositive for NPY (cells 1–3), pro-CCK (cells 2 and 4), and SLC17A8 (VGLUT3, cell 2). Note SLC17A8-positive terminals targeting unlabeled cells (arrows). (A, B) Both NPY and pro-CCK are detected in the Golgi apparatus and endoplasmic reticulum surrounding cell nuclei; in addition, some axons are also immunopositive for NPY (see arrow in (A); average intensity projections, z stacks, height 6.3 μm and 10.4 μm, respectively). (C) Combined double in situ hybridization and immunohistochemistry shows that nearly all Slc17a8-expressing cells also express Npy and are immunopositive for pro-CCK (arrows), but some Npy/pro-CCK cells do not express Slc17a8 (arrowheads). Scale bars: 10 μm (A, B), 50 μm (C). sr/slm, stratum radiatum and stratum lacunosum-moleculare.
Fig 9.
Analysis of Cxcl14 co-expression patterns confirms predicted properties Cck.Cxcl14 cells.
(A) Cxcl14-expressing cells are CGE derived: in situ hybridization for Cxcl14 combined with immunohistochemistry for YFP in the Lhx6-Cre/R26R-YFP mouse yields no double labeling. (B) Double in situ hybridization for Cxcl14 and Reln marks a population of neurons located primarily at the sr/slm border. Note Reln expression without Cxcl14 in so and slm, likely reflecting O-LM and neurogliaform cells. (C–E) Subsets of the Cxcl14-positive neurons are positive for pro-CCK or CALB1 (in situ hybridization plus immunohistochemistry), or Npy (double in situ hybridization). (F, G) No overlap was seen of Cxcl14 with Nos1 or Kit. In all panels, arrowheads indicate double-expressing neurons. Scale bars: 200 μm (A), 100 μm (B–G). b, sr/slm border region; O-LM, oriens/lacunosum-moleculare; slm, stratum lacunosum-moleculare; sm, stratum moleculare of the dentate gyrus; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; sr/slm, stratum radiatum and stratum lacunosum-moleculare; YFP, yellow fluorescent protein.
Fig 10.
The class Cck.Cxcl14.Vip represented a puzzle: Vip/Cck cells had previously been reported in sp, but Cxcl14 is detected primarily at the sr/slm border, although exceptional cells can be detected in sp also. (A) Double fluorescent in situ hybridization images reveal that the vast majority of cells co-expressing Cxcl14 and Vip were found at the sr/slm border, confirming the location of this novel class. (B) Zoom into rectangles 1 and 2. Arrowheads: double-expressing cells. b, sr/slm border region; slm, stratum lacunosum-moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; sr/slm, stratum radiatum and stratum lacunosum-moleculare.