Volume 5, Issue 1, Pages 87-98 (October 2013) The Organization of Submodality-Specific Touch Afferent Inputs in the Vibrissa Column  Katsuyasu Sakurai, Masahiro Akiyama, Bin Cai, Alexandra Scott, Bao-Xia Han, Jun Takatoh, Markus Sigrist, Silvia Arber, Fan Wang  Cell Reports  Volume 5, Issue 1, Pages 87-98 (October 2013) DOI: 10.1016/j.celrep.2013.08.051 Copyright © 2013 The Authors Terms and Conditions

Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure 1 Genetic Labeling of RA Longitudinal Lanceolate and SA Merkel-Ending Neurons in the Vibrissa Sensory System (A) Schematic drawing of the types of touch sensory endings in the mouse vibrissa follicle is presented. (B) Representative images of AP staining result from P7 ChodlPLAP/+ mice on TG section (left) and vibrissa follicle sections (right two panels). Longitudinal lanceolate-ending neurons were selectively labeled. (C) Representative images of AP staining result from P7 Pv::Cre; RΦAP mice on TG (left) or vibrissa follicle sections (right two panels). Pv::Cre; RΦAP labeled Merkel-ending (arrows) and a few longitudinal lanceolate-ending (arrowhead) neurons. (D) Representative images of two-color fluorescent in situ hybridization with Chodl (green) and Ret (red) probes on the P7 wild-type (WT) TG sections. A total 96.46% ± 0.27% chodl+ cells coexpressed Ret (arrow). (E) Representative images of two-color fluorescent in situ hybridization on TG sections from P21 Pv::Cre; RΦGFP mice. Top row shows GFP (green) and TrkC (red), middle row presents GFP and Ret, and bottom row shows GFP and TrkA. A total of 62.4% ± 1.5% GFP+ cells (labeled by Pv::Cre) coexpressed with TrkC, 25.6% ± 1.0% GFP+ cells coexpressed with Ret, and 17.3% ± 2.9% GFP+ cells coexpressed with TrkA. (F) Double immunostaining shows sensory axons (green) and Merkel cells (red) on sections from the mystacial pad from Pv::Cre; RΦGFP mice at P7. In the small vibrissa (top row), more than 90% of Merkel cells were innervated by Pv::Cre-labeled axons; in contrast, in the large vibrissa (bottom row images), Pv::Cre only labeled subsets of axons innervating Merkel cells. (G) Representative TG sections from Pv::Cre; RΦtomato; ChodlPLAP/+ triple-transgenic mice (P7) are shown. A total of 22.3% ± 0.8% Chodl+ (AP stained) neurons exhibit tomato fluorescence (PLAP and tomato double positive). Conversely, 24.3% ± 1.3% tomato+ neurons exhibit PLAP activity (Chodl+). Scale bars, 100 μm. See also Figure S1. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure 2 Axonal Projections and Presynaptic Termini Distribution Maps of RA Longitudinal Lanceolate and SA Merkel-Ending Neurons within Individual Barrelette Columns in SpI and PrV Nuclei (A and C) Left panel is a schematic drawing of the brainstem TG complex (sagittal view) with the arrow indicating the SpI (A) or PrV (C) nucleus. Right four panels are representative images of immunostaining of vGluT1 (gray or blue), PLAP (green), and tomato (red) on the sections of SpI (A) or PrV (C) nucleus from Pv::Cre; RΦtomato; ChodlPLAP mice (P7). White circle indicates the D6 barrelette. White arrows indicate examples of dense patches of afferent termini. All images are of C view. (B and D) Maps of afferent presynaptic termini inside D6 barrelette in SpI (B) or PrV (D) nucleus from three different samples (numbered 1–3) are presented. Left panels show representative results of presynaptic sites extracted from confocal images of individual serial sections through each D6 barrelette in Spl (B) or PrV (D). Blue indicates vGluT1+ loci, green indicates presynaptic termini of RA afferents (PLAP+ ∩ vGluT1+), and red indicates presynaptic termini of SA afferents ((tomato+ ∩ vGluT1+) − (PLAP+ ∩ vGluT1+)). Right panels show the heatmaps of RA and SA synaptic densities in each sample; 2D projections of the 3D data from each barrelette column onto three different planes are shown. The color on the heatmaps represents relative synaptic density in each sample. The highest density is shown as red. The lowest density is shown as blue. Scale bars, 100 μm (A and C) and 20 μm (B and D). See also Figures. S1, S2, and S3, and Movies S1 and S2. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure 3 Axon Collaterals of Single-Labeled RA Lanceolate and SA Merkel Neurons inside the Barrelette Unit (A) Schematic representation of the strategy used to sparsely label longitudinal lanceolate and Merkel-ending neurons is shown. (B) Representative images of the peripheral ending of single-labeled Merkel or lanceolate neuron are shown. Axon is shown in red; DAPI is in blue. (C) Representative C views and L views of 3D reconstructed collaterals from single-labeled Merkel and single-labeled lanceolate neuron in the SpI and PrV at P8 and P28, respectively. Axon is shown in red; outlines of the barrelette structure are in yellow. (D) Quantification of relative volumes occupied by axon collaterals of single-labeled Merkel or lanceolate neurons normalized with respect to the volume of each corresponding barrelette column. All comparisons between P8 and P28 are not statistically different (p > 0.05, t test). (E) Quantification of relative synaptic densities of single-labeled Merkel or lanceolate neurons normalized with respect to the total volume of the collaterals. All comparisons between P8 and P28 are not statistically different (p > 0.05, t test). Mer, Merkel-ending neuron; Lan, lanceolate-ending neuron. Scale bars, 20 μm. See also Figure S4, and Movies S3, S4, S5, and S6. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure 4 Convergent RA and SA Inputs onto Brainstem PNs (A and C) Representative low-magnification images show the four-color labeling experiments in SpI (A) or PrV (C) nucleus: vGluT1+ synaptic sites (synapses are in gray or blue), PNs (green), RA afferents (PLAP is in magenta), and SA afferents (tomato is in red), from Pv::Cre; RΦtomato; ChodlPLAP triple transgenic mice (P7) with ΔG-GFP rabies virus injected into the thalamus. (B and D) Two representative high-magnification images show the four-color labeling experiments in SpI (B) or PrV (D) nucleus. White dashed lines encircle the barrelette structure. Rightmost panels show synaptic terminals of SA Merkel and RA lanceolate-ending neurons onto the labeled PNs. Red dots indicate SA synapses. Green dots indicate RA synapses. Insets are the zoom-in of the yellow boxed region. Scale bars, 100 μm (A and C) and 20 μm (B and D). See also Figures S5 and S6. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure 5 Hypothetical Model Involving Temporal Coding of Tactile Information in the First Relay Station of the Vibrissa Sensory Pathway (A) A PN receives convergent SA and RA inputs and thus shows mixed RA and SA responses. (B) Hypothetical model is presented. The sensory circuit from a single vibrissa to the brain has the “one-to-many and many-to-one” connectivity architecture. The individual neuronal spike and adaption patterns elicited by a touch stimulus are hypothetical on the model. We speculate that the spike timing differences in sensory neurons in response to the touch stimulus and coincident detection of transiently synchronous spikes by PNs can result in a temporal code from the vibrissa column to encode the nature of tactile stimulus. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S1 Characterization of ChodlhPLAP Mice and vGluT1 Staining, Related to Figures 1 and 2 (A–C) Generation of ChodlhPLAP mice. (A) Schematic representation of the genetic insertion. hPLAP gene with ACN cassette was inserted at the translational start codon of Chondrolectin gene. Exons are represented as white boxes. (B) Southern blot analysis of genomic DNA from wild-type and heterozygous mice. The 9.3 and 5.0 kb fragments indicate wild-type and mutant allele, respectively. (C) Genomic PCR analysis of the progeny. Wild-type and mutant alleles produced 300 and 700 bp fragments, respectively. (D) Representative TG sections from P7 ChodlPLAP/+ mice. 90.5% Chodl+ (AP stained) neurons were co-labeled with NF200 antibody (PLAP and NF200 double positive). (E) Representative TG section from wild-type mice. 98.7% NF200+ neurons were co-labeled with vGluT1 antibody (NF200 and vGluT1 double positive). Scale bars: 100 μm. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S2 Analyzing Lanceolate- and Merkel-Ending Afferent Inputs into Barrelette Columns, Related to Figure 2 (A) Lanceolate- and Merkel-ending neurons project to all brainstem trigeminal nuclei. Immunostaining of vGluT1, PLAP and tomato on the sections of SpC and SpO nucleus from P7 Pv::Cre; RΦtomato; ChodlPLAP/+ mice. Grey and Blue, vGluT1+ presynaptic sites; Green, ChodlPLAP/+ labeled axon; Red, Pv::Cre; RΦtomato labeled axon. Arrow indicates facial motor neurons. Scale bar: 100 μm. (B–D) Image processing steps for extracting the sites of lanceolate- and Merkel- afferents presynaptic termini. Serial sections of SpI and PrV nuclei from Pv::Cre; RΦtomato; ChodlPLAP/+ mice were immunostained with vGluT1, PLAP and RFP antibody and Z-stack images (at 1 μm step) were taken using confocal microscopy for each serial section. To extract lanceolate and Merkel synapses, all Z-stack images are processed by Fiji and Imaris software. (B) PLAP (ChodlPLAP/+) and vGluT1 co-localized loci were extracted and pseudo-colored “green” to represent lanceolate afferents presynaptic termini. (C) Tomato (Pv::Cre; RΦtomato) and vGluT1 co-localized sites were extracted and pseudo-colored “magenta” to indicate Merkel- plus a subset of lanceolate-ending presynaptic termini. (D) From the tomato (Pv::Cre; RΦtomato) and vGluT1 co-localized sites (magenta), those that overlapped with PLAP (ChodlPLAP/+) and vGluT1 co-localized sites were removed, the remain loci, representing Merkel-ending afferent presynaptic termini, were pseudo-colored “red.” Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S3 Axonal Projections and Presynaptic Termini Distribution Maps of RA Longitudinal Lanceolate and SA Merkel-Ending Neurons within Representative Large Barrelettes in SpI and PrV Nucleus, Related to Figure 2 (A) Schematic drawing of projected 2D heatmaps of the synaptic densities onto coronal (C), lateral/sagittal (L), and horizontal (H) planes. The color represents relative synaptic density in each sample for each type of afferent. The highest density is shown as red. The lowest density is shown as blue. (B) Cross-correlation analysis of distributions of the RA-lanceolate and SA-Merkel synapses inside D6 barrelette in SpI and PrV nuclei from four samples. Color scale indicates correlation coefficient values (−0.1-1). (C and D) Presynaptic density maps of the large C3 and B2 barrelettes in SpI (C) abd PrV (D) from 2 different samples. Left panels shows presynaptic sites extracted from each Z-stacks of the serial sections through each of the C3 or B2 barrelette. Blue: all vGluT1+ loci. Green: presynaptic termini of RA-longitudinal lanceolate afferents (PLAP+ ∩ vGluT1+). Red: presynaptic termini of SA-Merkel afferents ((tomato+ ∩ vGluT1+) – (PLAP+ ∩ vGluT1+)). Right panels shows the synaptic density heatmaps of RA-afferents in each samples on three 2D planes. Scale bars: 20 μm. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S4 Additional Examples of Axon Collaterals from Single-Labeled RA Lanceolate or SA Merkel Neurons inside the Barrelette Column, Related to Figure 3 (A) Representative low magnification images of co-immunostaining of vGluT1 and tomato on the serial sections through the entire SpI in which a single Merkel-ending neuron innervating C2-vibrissa (the ventrally located axons), and another single-neuron innervating a small barrelette (dorsally located axons) were shown. Abbreviations are as follows: A, anterior; P, posterior. Scale bar: 100 μm. (B) Representative coronal and lateral views of the 3D reconstructed collaterals from single-labeled Merkel and single-labeled lanceolate neuron collaterals in the SpI and PrV at P8 and P28, respectively. Red, axon. Yellow, outlines of the barrelette structures. Scale bar: 20 μm. (C) Lateral views of 3D-reconstructed collaterals and identified presynaptic sites (vGluT1+ spots) from a single-labeled Merkel (upper panels) and a single-labeled lanceolate neuron (lower panels) in the SpI and PrV. Red, axon; Green, barrelette structure; Yellow, presynaptic sites. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S5 Retrograde Labeling of Brainstem Projection Neurons that Send Axons into Thalamus, Related to Figure 4 (A) Schematic of the retrograde labeling of projection neurons with deficient rabies virus (ΔG-GFP-RV). (B) Representative images of ΔG-GFP-RV retrogradely labeled projection neurons on the sections of PrV, SpO, SpI-r (rostral SpI) and SpI-c (caudal SpI) nucleus from P7 Pv::Cre; RΦtomato; ChodlPLAP mice. Blue, vGluT1; Green, projection neurons; Red, tomato; Magenta, PLAP. Note that SpI-c contains barrelettes, whereas SpI-r does not. Scale bar: 100 μm. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions

Figure S6 Convergence of SA and RA Afferent Inputs onto Projection Neurons, Related to Figure 4 Representative high magnification images of sections from SpI nucleus (A) or PrV nucleus (B) from the 4-color labeling experiments (i.e., ΔG-GFP-RV injected into Pv::Cre; RΦtomato; ChodlPLAP mice at P7). Rightmost panels show synaptic termini of SA-Merkel-ending or RA-lanceolate-ending neurons onto the projection neurons. Red dots: SA synapses; Green dots: RA synapses. White dash lines encircle the barrelette structure. Insets: zoom of the yellow boxed region. Scale bars: 20 μm. Cell Reports 2013 5, 87-98DOI: (10.1016/j.celrep.2013.08.051) Copyright © 2013 The Authors Terms and Conditions