Volume 149, Issue 5, Pages 1152-1163 (May 2012) Identification of the Cortical Neurons that Mediate Antidepressant Responses  Eric F. Schmidt, Jennifer L. Warner-Schmidt, Benjamin G. Otopalik, Sarah B. Pickett, Paul Greengard, Nathaniel Heintz  Cell  Volume 149, Issue 5, Pages 1152-1163 (May 2012) DOI: 10.1016/j.cell.2012.03.038 Copyright © 2012 Elsevier Inc. Terms and Conditions

Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 The S100a10 bacTRAP Mouse Line Labels a Subpopulation of Pyramidal Cells in the Cerebral Cortex (A) Schematic depicting the modification of the BAC vector containing the S100a10 gene by RecA-mediated homologous recombination in bacteria. Exons 1–3 of the S100a10 gene are represented by black bars. (A) and (A′) refer to the homology region used for recombination. Upstream and downstream transcriptional regulatory elements are represented by white bars on the BAC vector. The red box following the EGFP-L10a transgene represents the polyA site. (B) Low magnification anti-EGFP IF image of EGFP-L10a transgene expression in the brain of S100a10 bacTRAP mouse. Scale bar represents 2 mm. See also Figure S1. (C) EGFP-L10a expression in multiple regions of neocortex. EGFP (green) and NeuN (red) staining are shown. Layers are indicated. Scale bar represents 500 μm. (D) High magnification image of cell bodies of EGFP+ cells. Apical dendrites are indicated by double arrowheads (where visible). In cases where the apical dendrite is out of the plane of section, single arrowheads indicate the base. Scale bar, 25 μm. (E) Double-labeling with anti-EGFP and anti-p11 antibodies in WT (top row) and constitutive p11 KO (bottom row) in sensorimotor cortex of S100a10 bacTRAP mice. Scale bar, 50 μm. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Retrograde Tracing Reveals Axonal Targets of S100a10-Expressing Cells (A) Representative images from CTβ injections into dorsal striatum (CPu; top row), nucleus accumbens (NAc, middle row), and contralateral cortex (bottom row). Left panels are low magnification images of cortex with the injection site shown in inset, and right three panels are higher magnification IF images of double-labeling with anti-EGFP and anti-CTβ antibodies. Arrows indicate double-labeled cells; asterisks indicate CTβ+ cells that are not colabeled with EGFP. (B) Histogram of the depth of the cell bodies of EGFP+ cells (EGFP), and CTβ+ cells projecting to CPu or spinal cord (SpC) in motor cortex (left), and CTβ+ commissural projection neurons (Cortex) in sensory cortex (right). (C) Quantification (mean ± SEM) of the number of CTβ+ cells that were colabeled with EGFP following injections into CPu, NAcc, and contralateral cortex. (D) Schematic demonstrating the projection targets of S100a10-expressing cortical projection neurons. Brain diagram was modified from Franklin and Paxinos (2008). See also Figure S2. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 S100a10 and Glt25d2 Are Molecularly Distinct Cell Populations (A) Scatter plot comparing normalized expression values of neuronal genes (see Figure S4 and Table S1) in S100a10 IP (x axis) or whole cortex input (y axis). Blue dots represent individual probe sets. Outer gray lines represent 1.5-fold enrichment in either sample. Probe sets representing the S100a10 gene are circled. (B) Quantification (mean ± SEM) of expression of control genes (Aldh1l1, Cnp, Gad1, and Slc17a7) and previously reported BAC driver genes (Etv1 and Ntsr1, gray bars) by qRT-PCR in S100a10 IP versus whole cortex (UB) RNA. Dotted lines mark 2-fold regulation. ∗p < 0.05; ∗∗p < 0.01. (C) Scatter plot comparing normalized expression values of CST (red dots) or CPN (blue dots) markers (see Figure S3 and Table S2) in Glt25d2 (x axis) or S100a10 (y axis) IP samples. Outer gray lines represent 1.5-fold enrichment in either cell population. Probe sets representing S100a10 and Glt25d2 are indicated. (D) qRT-PCR quantification (mean ± SEM) of the expression of cortex layer 5 genes in S100a10 IP compared to either Glt25d2 IP (white bars) or whole cortex (UB, gray bars). Positive values indicate enrichment in S100a10 IP whereas negative values indicate enrichment in UB or Glt25d2 IP. Dotted lines mark 2-fold regulation. p < 0.05 for all genes shown. (E–J) In situ hybridizations showing expression patterns in cortex of genes enriched in S100a10 (E–I) or Glt25d2 (J) cells. All images are from the Allen Brain Atlas. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Cell Type-Specific Molecular Responses to SSRI Treatment (A) The level of p11 mRNA was quantified by ISH and expressed as percent of control (mean ± SEM). Representative images are shown below the graph. ∗p < 0.05. (B) Scatter plot comparing normalized expression values of FLX- (x axis) or VEH (y axis)-treated cortex IP samples from S100a10 (left) or Glt25d2 (right) bacTRAP mice. Dots represent probe sets that are significantly increased (red) or decreased (green) >1.4-fold by drug treatment (see Table S3). Outer gray lines represent 1.4-fold enrichment in either sample. (C) Quantification of Htr expression following chronic FLX in S100a10 and Glt25d2 cells by qRT-PCR (mean ± SEM). Positive values indicate upregulation and negative values indicate downregulation by FLX. Dotted lines show a 2-fold change in either direction. ∗p < 0.05. (D) Quantification of Htr expression following chronic FLX in whole cortex by qRT-PCR (mean ± SEM). See also Figure S4. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 Molecular Changes Are Dependent on p11 Expression (A) Anti-EGFP IF image of EGFP-L10a expression in constitutive p11 KO mice crossed to the S100a10 bacTRAP line. See Figure S5 for further anatomical analysis. (B) Quantification of Htr expression in IP RNA from S100a10 bacTRAP/p11 KO relative to WT cortex by qRT-PCR (mean ± SEM). Dotted line indicates a 2-fold change. Negative values indicate downregulation in KO compared to WT. ∗p < 0.05, ∗∗p < 0.01. (C) Scatter plot comparing normalized expression values of FLX- (x axis) or VEH (y axis)-treated cortex IP samples from S100a10 bacTRAP/p11 KO mice. Dots represent probe sets that are significantly increased (red) or decreased (green) >1.4-fold by drug treatment in WT cortex (see Figure 4B and Table S3). Outer gray lines represent 1.4-fold enrichment in either sample. (D) Quantification of Htr expression following chronic FLX in S100a10 bacTRAP/p11 KO cells by qRT-PCR (mean ± SEM). Positive values indicate upregulation and negative values indicate downregulation by FLX treatment. Dotted lines indicate a 2-fold change in either direction. ∗p < 0.05. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Cortical Expression of p11 Is Required for Behavioral Responses to Chronic SSRI Administration (A) Anti-p11 IF in brain sections from floxed p11 mice (WT, top row) and Emx1/p11KO mice (bottom row) from layer 5 of sensorimotor cortex (left panels), striatum (center), or dentate gyrus (right). Inset shows anti-NeuN staining from the same field. Asterisks label p11-positive cells. (B) Bar graph representing the effect of chronic FLX treatment on the latency to feed in WT and Emx1/p11 KO (Cortex KO) mice in the novelty NSF paradigm (mean ± SEM). ∗∗p < 0.01. (C) Bar graph representing the effect of chronic FLX treatment on immobility in WT and Emx1/p11 KO (Cortex KO) mice in TST (mean ± SEM). ∗p < 0.05. (D) Bar graph representing thigmotaxis in an open field in WT or Emx1/p11 KO (Cortex KO) mice (mean ± SEM). (E) Bar graph representing sucrose preference in WT or Emx1/p11 KO (Cortex KO) mice (mean ± SEM). Dotted line indicates no preference. See also Figure S5. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S1 EGFP-L10a Transgene Expression Matches Endogenous S100a10 Expression throughout Brain of S100a10 bacTRAP Transgenic Mouse Line, Related to Figure 1 (A) A comparison of S100a10 in situ hybridization (left side) and anti-EGFP immunohistochemistry (right side) in three representative brain sections that include frontal, motor, and sensory cortex. (B–H) Side-by-side comparison of high resolution S100a10 in situ hybridization images (left) and anti-EGFP immunocytochemistry (right) in a variety of sub-cortical structures including: granule cell layer of the olfactory bulb (B), large cell body interneurons in the striatum (C), colliculus, midbrain, and pons (D), thalamus, hypothalamus, and globus pallidus (E), superior and brainstem motor nuclei (F and G), and cervical spinal cord (H). All in situ hybridization images of brain in B–G and spinal cord in H were obtained from the Allen Brain Atlas (http://www.brain-map.org) image series 70560277 and 100013736, respectively. In situ image in C is from the Allen Brain Atlas “expression” view. Scale bars are 500 μm (B, C, and H) and 1 mm (D–G). 3n, oculomotor nucleus; 7n, facial nucleus; 10n, dorsal motor nucleus of vagus; 12n, hypoglossal nucleus; ad, anterior dorsal thalamic nucleus; am, nucleus ambiguus; gr, gigantocellular reticular nucleus; gcl, granule cell layer; gp, globus pallidus; io, inferior olive; is, inferior salivatory nucleus; lp, lateral paraventricular hypothalamic nucleus; lr, lateral reticular nucleus; lv, lateral vestibular nucleus; rn, red nucleus; sc, superior colliculus; sn, substantia nigra; so, solitary nucleus; th, thalamus. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S2 Retrograde Labeling in S100a10 bacTRAP Line, Related to Figure 2 (A) Representative low magnification immunofluorescent images of injection sites for CPu (left), NAcc (center), and contralateral cortex (right). These are larger versions of the images shown in Figure 2. Sections were stained with anti-CTβ (red) and anti-NeuN (blue) antibodies. White arrowheads indicate CTβ-labeled fibers. ac, anterior commissure; cc, corpus collosum; ctx, cortical plate; ec, external capsule; hip, hippocampus; p, pial surface. (B) Immunofluorescent images of double-labeling with anti-EGFP and anti-CTβ antibodies following CTβ injections into spinal cord. (C) Low magnification immunofluorescent image of the spinal cord injection site. Dcst, dorsal corticospinal tract; vh, ventral horn. (D) Immunofluorescent images demonstrating examples of double-labeling of EGFP and CTβ in multiple cortical areas following CTβ injections into CPu. Top panels show low magnification images of sections stained with anti-EGFP (green), anti-CTβ (red), and anti-NeuN (blue) from ventrolateral prefrontal cortex (vlPFC), auditory cortex (Aud), and primary visual cortex (V1). Bottom panels are higher magnification images of boxes in top panel. Arrows indicate double-labeled cells. hip, hippocampus; ob, olfactory bulb; p, pial surface; str, striatum. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S3 S100a10 and Glt25d2 Label Morphologically Distinct Cell Populations, Related to Figure 3 (A) Representative images of anti-EGFP immunohistochemistry of cortex from S100a10 and Glt25d2 bacTRAP mouse lines. (B) Histogram of the quantification of cell depths of EGFP+ cells in S100a10 (black lines) and Glt25d2 (gray lines) bacTRAP mice in motor (left) and somatosensory (right) cortex. (C) Quantification of cell area (Mean ± SEM) of EGFP-positive cells in motor and somatosensory cortex of S100a10 and Glt25d2 bacTRAP mice. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S4 EGFP-L10a Expression in Nonneuronal Cells and Filtering for Neuronal Expressed Genes, Related to Figures 3 and 4 (A and B) Immunofluorescent images showing EGFP expression in the pia mater and some blood vessels in the S100a10 bacTRAP mouse brain. Sections were labeled with anti-EGFP (green) and DAPI (blue). Arrows indicate the outer pial surface and arrowheads indicate the midline between two cortical hemispheres. Scale bars, 50 μm. (C and D) Immunofluorescent images showing EGFP expression in the lining of a subset of blood vessels (bv) in the cortex. Pyramidal cells are indicated by asterisks. Scale bars, 25 μm. (E) Anti-EGFP immunohistochemistry in cortex of Snap25 bacTRAP mouse brain. Inset is a low magnification image with the region shown indicated by the box. Cortical layers are indicated on right. (F) Scatter plot view comparing normalized expression values of Snap25 IP samples to whole cortex (unbound). Red dots are genes that were scored as being expressed in blood vessels or pia on the Allen Brain Atlas. Outer green lines represent 2-fold enrichment in either sample. (G) Schematic depicting filtering of microarray data sets using the specificity index. The left column represents various cell types that comprise the cortex and the right column lists the TRAP RNA samples. Arrows show which cell types are represented by each TRAP data set. (H) Quantification of scoring the expression patterns of the top 200 probe sets enriched in S100a10 bacTRAP IP over whole cortex unbound before (gray bars) and after (white bars) filtering for neuronal-expressed genes using the Snap25 ranking analysis. The percent of unscoreable in situs from both conditions is shown on the right. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure S5 Further Anatomical and Behavioral Characerization of the Emx1/p11 KO Mice, Related to Figure 6 (A) Representative images showing double-labeling with anti-EGFP and anti-CTβ antibodies following CTβ injections into dorsal striatum (CPu; top row) or contralateral cortex (bottom row) of S100a10 bacTRAP/p11 KO mice. Arrows indicate double-labeled cells. (B) Quantification of the total distance traveled in an open field for WT and Emx1/p11 KO (Cortex KO) mice. The difference in the first 10 min is not statistically significant. (C) Bar graph representing the effect of chronic FLX treatment on immobility in the FST of WT and Emx1/p11 KO mice. While there is a slight trend for FLX to reduce immobility in WT mice (#, p = 0.08), there is no difference between VEH and FLX in the Cortex KO mice. Cell 2012 149, 1152-1163DOI: (10.1016/j.cell.2012.03.038) Copyright © 2012 Elsevier Inc. Terms and Conditions