Behavioral consequences of a bifacial map in the mouse ...

Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2017 the authors This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Behavioral/Cognitive Behavioral consequences of a bifacial map in the mouse somatosensory cortex Vassiliy Tsytsarev 1 , Hiroyuki Arakawa 1 , Shuxin Zhao 1 , Alain Chédotal 2 and Reha S. Erzurumlu 1 1 Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201 2 Centre de Recherche Institut de la Vision INSERM UMRS968, 75012 Paris, France DOI: 10.1523/JNEUROSCI.0598-17.2017 Received: 3 March 2017 Revised: 14 June 2017 Accepted: 19 June 2017 Published: 29 June 2017 Author contributions: V.T., H.A., A.C., and R.S.E. designed research; V.T., H.A., and S.Z. performed research; V.T., H.A., and R.S.E. analyzed data; V.T., H.A., and R.S.E. wrote the paper; A.C. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. We thank P. Gaspar for her critical comments on the manuscript, K. Arakawa for help with the colony management and genotyping, B. Okura, K. Tsubokura and T. Sakuraba for their help with imaging., Grant support: Agence Nationale de la Recherche (ANR) (ANR-14-CE13-0004-01) to AC, NIH/NINDS R01NS084818 to RSE. Corresponding author: R.S. Erzurumlu, Dept. Anatomy and Neurobiology, 20 Penn Street, HSFII- S251, University of Maryland School of Medicine, Baltimore, MD. 21210, Tel 410 706-7401, e-mail: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0598-17.2017 Alerts: Sign up at to receive customized email alerts when the fully formatted version of this article is published.

Transcript of Behavioral consequences of a bifacial map in the mouse ...

Page 1: Behavioral consequences of a bifacial map in the mouse ...

Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreadingprocess.

Copyright © 2017 the authors

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version.

Research Articles: Behavioral/Cognitive

Behavioral consequences of a bifacial map in the mouse somatosensorycortex

Vassiliy Tsytsarev1, Hiroyuki Arakawa1, Shuxin Zhao1, Alain Chédotal2 and Reha S. Erzurumlu1

1Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland212012Centre de Recherche Institut de la Vision INSERM UMRS968, 75012 Paris, France

DOI: 10.1523/JNEUROSCI.0598-17.2017

Received: 3 March 2017

Revised: 14 June 2017

Accepted: 19 June 2017

Published: 29 June 2017

Author contributions: V.T., H.A., A.C., and R.S.E. designed research; V.T., H.A., and S.Z. performed research;V.T., H.A., and R.S.E. analyzed data; V.T., H.A., and R.S.E. wrote the paper; A.C. contributed unpublishedreagents/analytic tools.

Conflict of Interest: The authors declare no competing financial interests.

We thank P. Gaspar for her critical comments on the manuscript, K. Arakawa for help with the colonymanagement and genotyping, B. Okura, K. Tsubokura and T. Sakuraba for their help with imaging., Grantsupport: Agence Nationale de la Recherche (ANR) (ANR-14-CE13-0004-01) to AC, NIH/NINDS R01NS084818to RSE.

Corresponding author: R.S. Erzurumlu, Dept. Anatomy and Neurobiology, 20 Penn Street, HSFII-S251, University of Maryland School of Medicine, Baltimore, MD. 21210, Tel 410 706-7401, e-mail:[email protected]

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.0598-17.2017

Alerts: Sign up at to receive customized email alerts when the fully formattedversion of this article is published.

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Behavioral consequences of a bifacial map in the mouse somatosensory cortex 1

Abbreviated title: Bilateral whisker maps and behavior 2 3 Vassiliy Tsytsarev (1)*, Hiroyuki Arakawa (1)*, Shuxin Zhao (1), Alain Chédotal (2), 4 Reha S. Erzurumlu (1) 5 1Department of Anatomy and Neurobiology, University of Maryland School of Medicine, 6 Baltimore, Maryland 21201 7 2 Centre de Recherche Institut de la Vision INSERM UMRS968, 75012 Paris, France 8 9

* Equal contribution. 10

Present address: Dr. H. Arakawa, Director, Rodent Behavior Core, School of Medicine, 11 Case Western Reserve University, Cleveland, OH, 44106 12 13

Corresponding author: R.S. Erzurumlu, Dept. Anatomy and Neurobiology, 20 Penn 14

Street, HSFII-S251, University of Maryland School of Medicine, Baltimore, MD. 21210 15

Tel 410 706-7401, e-mail: [email protected] 16

Number of pages: 35 17

Number of Figures: 3 (color: 2) Number of Tables: 0 18

Number of words: Abstract: 254, Introduction: 681, Discussion: 1398 19

Conflict of interest: The authors declare no competing financial interests. 20 21

Acknowledgements. We thank P. Gaspar for her critical comments on the manuscript, K. 22

Arakawa for help with the colony management and genotyping, B. Okura, K. Tsubokura 23

and T. Sakuraba for their help with imaging. 24

Grant support: Agence Nationale de la Recherche (ANR) (ANR-14-CE13-0004-01) to 25

AC, NIH/NINDS R01NS084818 to RSE. 26




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The whisker system is an important sensory organ with extensive neural representations 31

in the brain of the mouse. Patterned neural modules (barrelettes) in the ipsilateral 32

principal sensory nucleus of the trigeminal nerve (PrV) correspond to the whiskers. 33

Axons of the PrV barrelette neurons cross the midline, and confer the whisker-related 34

patterning to the contralateral ventroposteromedial (VPM) nucleus of the thalamus, and 35

subsequently to the cortex. In this way, specific neural modules called barreloids and 36

barrels in the contralateral thalamus and cortex represent each whisker. Partial midline 37

crossing of the PrV axons, in a conditional Robo3 mutant (Robo3R3-5cKO) mouse line, 38

leads to the formation of bilateral whisker maps in the VPM, as well as the barrel cortex. 39

We used voltage-sensitive dye optical imaging (VSDi) and somatosensory and motor 40

behavioral tests to characterize the consequences of bifacial maps in the thalamocortical 41

system. VSDi verified functional, bilateral whisker representation in the barrel cortex and 42

activation of distinct cortical loci following ipsi- and contralateral stimulation of the 43

specific whiskers. The mutant animals were comparable to the control animals in 44

sensorimotor tests. However, they showed noticeable deficits in all of the whisker-45

dependent or -related tests, including Y-maze exploration, horizontal surface approach, 46

bridge crossing, gap crossing, texture discrimination, floating in water, and whisking 47

laterality. Our results indicate that bifacial maps along the thalamocortical system do not 48

offer a functional advantage. Instead, they lead to impairments, possibly due to the 49

smaller size of the whisker-related modules and interference between the ipsi- and 50

contralateral whisker representations in the same thalamus and cortex. 51



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Significance Statement 55

The whisker sensory system plays a quintessentially important role in exploratory 56

behavior of mice and other nocturnal rodents. Here, we studied a novel mutant mouse 57

line, in which the projections from the brainstem to the thalamus are disrupted. This led 58

to formation of bilateral whisker maps in both the thalamus and the cortex. The two 59

whisker maps crowd in a space normally devoted to the contralateral map alone and in a 60

nonoverlapping fashion. Stimulation of the whiskers on either side activates the 61

corresponding region of the map. Mice with bilateral whisker maps perform well in 62

general sensorimotor tasks but show poor performance in specific tests that require 63

whisker-dependent tactile discrimination. These observations indicate that, contralateral 64

instead of bilateral representation of the sensory space plays a critical role in acuity and 65

fine discrimination during somesthesis 66


Keywords: barrel cortex; barreloids; optical imaging; Robo3; midline crossing defects; 68

whisker-sensitive behaviors 69









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Introduction 78

A conspicuous feature of the primary sensory brain areas is the formation of a neural 79

map of the sensory periphery. Sensory maps of the retina, cochlea, and the somatic 80

sensory space develop early in perinatal development and show critical period plasticity. 81

Understanding the mechanisms of wiring and plasticity of these maps in both neonatal 82

and adult states is a major endeavor in neuroscience. Experimental manipulations of 83

developing sensory pathways and genetic mutations reveal the mechanisms underlying 84

map formation and map plasticity. 85

The mouse whisker system is an exquisite tactile organ, essential to construction of 86

the perceptual world and exploratory behaviors of this species. At various levels of the 87

mouse brain, discrete neural modules represent the orderly array of whisker follicles on 88

the snout. Trigeminal ganglion axons, bridging the sensory periphery to the brainstem, 89

convey whisker-specific patterns to the ventral portion of the trigeminal principal sensory 90

nucleus (PrV). Here, neuronal aggregates, termed barrelettes, pattern after the spatial 91

arrangement of the whiskers on the snout (Ma and Woolsey, 1984). The two caudally 92

located subnuclei of the spinal trigeminal nucleus also develop whisker barrelettes. 93

However, they do not contribute to the whisker-specific patterning downstream of the 94

trigeminal brainstem (Killackey and Fleming, 1985; Erzurumlu and Ebner, 1988; Chiaia 95

et al., 1991; Iwasato et al., 1997). 96

PrV projection neurons send their axons across the midline and reach the 97

contralateral ventroposteromedial nucleus (VPM) of the thalamus. Here too whisker-98

specific neural modules develop, and they are called barreloids (Van der Loos, 1976). 99

Barreloid neurons, in turn, convey the pattern template to layer IV of the primary 100

somatosensory, or the barrel cortex (Woolsey and Van der Loos, 1970, Erzurumlu and 101

Jhaveri, 1990). Thus, due to midline crossing of the PrV trigeminothalamic projections, 102

the patterned maps in the thalamus and cortex represent contralateral whiskers. 103

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During embryonic development, attractive and repulsive axon guidance cues 104

govern the contralaterality of the trigeminothalamic projections. Midline guidance cues, 105

such as netrin and slits, play major roles in temporally regulating axon attraction and 106

repulsion (Chédotal, 2011; Nawabi and Castellani, 2011; Izzi and Charron, 2011; 107

Friocourt and Chédotal, 2016). The PrV axons express receptors for slits and netrins; 108

mutations that affect their expression lead to aberrant development of their projections 109

(Mirza et al., 2013). 110

The PrV barrelette neurons are largely derived from rhombomere 3 (r3) (Oury et al., 111

2006; Bechara et al., 2015; reviewed in Erzurumlu et al., 2010). The Krox20/Egr2 gene, 112

which encodes a zinc finger transcription factor required for the development of r3 and r5 113

(Schneider-Maunoury et al., 1993). Conditional mutation of the Robo3 receptor, using 114

the Krox20 promoter in mice (Krox20Cre;Robo3lox/lox, named Robo3R3-5cKO) results in 115

disrupted midline crossing of the PrV trigeminothalamic axons. This midline crossing 116

defect leads to the formation of bilateral whisker maps in the VPM and in the barrel 117

cortex (Renier et al., 2017). Barrelette forming parts of the spinal trigeminal nucleus and 118

the dorsal column nuclei are caudal to r3-5, and their efferents are not affected. 119

In Robo3R3-5cKO mice, bilateral whisker inputs segregate into well-defined 120

territories, both in the thalamus and in the cortex. Interestingly, the area devoted to the 121

face map does not alter in size or orientation, but instead, houses almost double the 122

number of barrels (Renier et al., 2017). The total whisker representation areas in the 123

thalamus and cortex do not change, perhaps because the sensory epithelium in the 124

periphery has not changed and some of the PrV neurons that normally project to the 125

contralateral thalamus now project ipsilaterally. 126

Since whiskers and barrels play major roles in the perceptual and behavioral 127

repertoire of mice, we sought to determine whether having bilateral whisker 128

representations and increased numbers of barrels in each thalamus and cortex would be 129

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advantageous for somesthesis. With this objective in mind, we assessed the general 130

sensorimotor abilities, overall somatic sensory discrimination, whisker-specific sensory 131

discrimination, and social behaviors of the mutant mice. We expected that bilateral 132

whisker representation in a nonoverlapping fashion would increase discriminative 133

capability. Instead, we found that this was not the case, most likely due to the smaller 134

size of the whisker representing modules and potential interference between the two 135

face maps. 136

Materials and Methods 137

Animals. A. Chédotal provided the founders of the Robo3R3-5cKO mice, and we 138

established a breeding colony at the University of Maryland Baltimore, School of 139

Medicine in an AAALAC accredited laboratory facility. In a recent report Renier et al., 140

(2017) described in detail, generation of the mutants, and whisker-related patterns along 141

their trigeminal somatosensory pathway. We determined the genotypes by PCR from 142

tail lysate DNA samples. We used Robo3 lox/lox and Krox20Cre;Robo3 lox/+ -named Robo3 143

R3-5-Het) mice as controls. We found that there were no differences between these two 144

genotypes and also when compared to the C57BL/6J 145

(, RRID:IMSR JAX:000664) line that we routinely 146

use in morphological, functional and behavioral assays. 147

We performed all animal procedures were performed according to the regulations 148

of the UMB SOM IACUC, and the National Institute of Health Guide for the Care and 149

Use of Laboratory Animals (ISBN:13:978-0-309-15400-0, revised in 2011.) 150

Histological confirmation of the bifacial maps in the somatosensory thalamus and cortex. 151

We performed VGLUT1 and VGLUT2 immuno- and cytochrome oxidase (CO) 152

histochemistry in aldehyde-fixed brain sections to confirm and visualize the presence of 153

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bilateral whisker maps in Robo3R3-5cKO mice (both sexes, n=12). The immunostaining 154

for glutamate transporters and CO histochemistry are routine markers for whisker-155

specific patterns in the rodent brain. 156

We removed the neocortex from each brain and flattened them between glass 157

slides. We sectioned flattened cortical specimens at 50μm thickness on a vibratome 158

(Leica 1000S). We split the remaining brain tissue at the level of the midbrain. We 159

sectioned the diencephalic pieces at an oblique horizontal plane and the remaining 160

brainstem sections at the coronal plane. After several rinses in phosphate buffer (PB), 161

we incubated the free-floating sections in antibody solutions at 4 °C for 48 hours. We 162

performed immunostaining using guinea pig polyclonal antibodies (Abs) against 163

vesicular glutamate transporter (VGLUT) 1 and 2 from EMD Millipore (Millipore Cat# 164

AB5905 RRID:AB_2301751, Millipore Cat# AB2251 RRID:AB_1587626). We used 165

VGLUT-2 (1:500) for thalamocortical afferent terminals in the barrel cortex, and 166

trigeminothalamic afferents in the thalamus and VGLUT-1 (1:500) for the trigeminal 167

afferents in the PrV. After we washed the primary Abs from the sections, we applied 168

fluorescent secondary Abs (FITC-conjugated donkey anti guinea pig, 1:80, from Jackson 169

Laboratories, Code: 706-545-148) for 1.5 hours. Afterwards, we rinsed the sections in 170

phosphate buffer several times, and then mounted them onto glass slides and 171

coverslipped them. 172

For CO histochemistry, we incubated flattened cortex sections in cytochrome C 173

solution made up with phosphate buffered saline with diaminobenzidine, until the 174

sections turned golden brown in color (Arakawa et al., 2014b). 175

We examined the immunostained sections under epifluorescence and CO-176

stained sections under light field illumination and photographed regions of interest. 177

Voltage-sensitive dye optical imaging. We imaged the barrel cortex of Robo3R3-5cKO (n = 178

6) and control (Robo3 R3-5-Het, n = 6) male and female mice at 4–9 weeks of age using 179

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the MiCAM02 system (Brain Vision). After applying urethane anesthesia (1.15 g/kg body 180

weight), and shaving their heads, we placed individual mice into a stereotaxic frame 181

(Stoelting, USA). We made a 3 x 3 mm cranial opening over the left parietal cortex. We 182

used hemostatic sponges dipped in artificial cerebrospinal fluid (ACSF) to clean the 183

exposed surface. Next, we applied voltage-sensitive dye RH-1691 (Optical Imaging; 2.0 184

mg/ml in ACSF) for 1 hr., and washed afterwards with ACSF for 15 minutes (Tsytsarev 185

et al, 2010; Lo et al, 2013). We covered the exposed area with high-density silicone oil 186

and a 0.1-mm-thick coverslip. 187

We positioned the area to be imaged below the objective of the imaging system 188

with its optical axis perpendicular to the cortical surface. The focusing plane was 189

adjusted to the depth of 300 μm below the dura mater. At the start of each imaging 190

session, we obtained a gray scale image of the recording area. 191

Before the experiment all of the whiskers, except the whiskers to be stimulated, 192

were clipped close to the skin. At the start of each optical imaging session, a gray scale 193

image of the dural surface was obtained and saved as a graphic file. Each experiment 194

consisted of 100 trials, 500 frames per trial, with the stimulus (whisker deflection) 195

presented at the 250th frame, 1 trial per stimulus. The inter trial interval was 12 s. 196

Change in fluorescence was calculated as F/F using the BrainVision Analyzer. 197

For the whisker stimulation, a glass capillary (1.0 mm in diameter) fitted onto an 198

XYZ manipulator was aimed at the single, intact whisker. We applied air-puff of 10-ms 199

duration through Picospritzer pressure valve connected to the glass capillary. The 200

MiCAM02 imaging system controlled the Picospritzer, so that the whisker stimulation 201

and changes in fluorescence signals could be synchronized. 202

After data collection, we generated pseudocolor maps using first frame analysis 203

and then averaged the data for each session (Ferezou et al. 2006; Tsytsarev et al., 204

2010). 205

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Behavioral tests. Our histological and VSDi data verified the presence of bifacial whisker 206

maps at both the morphological level and the functional level. Next, we investigated the 207

behavioral repertoire of the mutant mice in order to assess the consequences of bilateral 208

whisker maps along the thalamocortical system. In this vein, we tested general 209

sensorimotor abilities, overall somatic sensory discrimination, whisker-specific sensory 210

discrimination, and social behaviors. 211

We used 9-12 week-old mice for behavioral testing in comparisons between 212

Robolox/lox as a control and Robo3R3-5cKO. Unlike the morphology and the imaging 213

experiments, we used only male mice in behavioral tests, for the following reasons: 1) 214

We did not find sex differences in several of the somatosensorimotor tests in a 215

developmental study on C57BL/6 mice (Arakawa and Erzurumlu, 2015); 2) To keep in 216

line with our previous behavioral studies in other mutant lines with whisker-barrel pattern 217

defects (Arakawa et al., 2014a, b); and 3) One of the tests (social behavior) depends on 218

the “resident-intruder” paradigm that is based on male behavior. 219

To avoid nonspecific behavioral reduction by general habituation with repeated 220

test sessions, we separated the mice into 2 groups (group1: control and Robo3R3-5cKO, 221

each n=9, and group 2: each n=8). We assigned Group 1 to a set of behavioral tests 222

including open field, social interaction, object recognition, Y-maze, bridge crossing, 223

swimming, adhesive removal, and whisking tests. We assigned Group 2 to the remaining 224

tests. The mice were housed 3-5 animals per cage in standard filter-top cages with 225

access to water and rodent chow at all times, maintained on a 12:12 h light:dark cycle 226

(06:00-18:00 h lighting) at 21°C, and relative humidity of 50-60%. For each session, we 227

moved the mice to the behavioral testing room and left them undisturbed for half an hour 228

before the tests. We tested small groups of mice in tandem (Crawley, 2008) and ran 229

three cohorts of mice at three separate times. Each test for individual mice took place 230

with at least one-hour intervals. Notably, we carried out the testing and subsequent 231

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analyses in an investigator blind fashion with respect to the mouse genotypes. Finally, 232

our sample size for behavioral studies has a 90% power of detecting genotype 233

differences at a 5% two-tailed significance level. 234

Previously, we used most of the aforementioned tests to assess the behavioral 235

consequences of genetic deletion of NMDA receptor and Adenylate cyclase type I 236

function in mice (Arakawa et al., 2014a, b), and in developing and adult mice after 237

whisker clipping (Arakawa and Erzurumlu, 2015). In this study, we conducted the 238

following behavioral tests: 239

Wire grip test. This test is used as a measure of forelimb strength and 240

coordination of movements to support the body weight (Crawley, 1999; Deacon, 2013). 241

Instead of a horizontal bar, commonly used in this test, we used the wire top lid of a 242

standard mouse cage. Because of this, we taped the hindpaws of the mice so that they 243

only use their forelimbs. The mice used their forepaws to suspend their body from a 244

single wire of the wire-top cage lid. We recorded the mean time (in seconds) of 245

suspension (or the latency of dropping to the cage floor), in three trials per session. A 246

maximum cut-off latency of 60 sec was recorded if the mouse did not drop to the cage. 247

We took the average time in three trials as a representative value for each mouse. 248

Adhesive removal test. We selected the adhesive removal test (Komotar et al., 249

2007; Bouet et al., 2009) to assess dorsal column-lemniscal sensitivity. We placed an 250

adhesive-backed label (0.5 cm square) on the palmar side of the hind paw and recorded 251

the latency of the first reaction to the stimulus (sniffing, paw lifting, biting and removal of 252

the label). We performed the test in the home cage. 253

Y maze spontaneous alternation test. This test is based on the innate preference 254

of mice to alternate arms of a Y or T-maze while exploring this new environment. The 255

test is most commonly used as a test for hippocampal, prefrontal cortex functions (Wolf 256

et al., 2016). Since whisker sensation is a major perceptual device for rodents in 257

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exploring their environment, we used this test here to compare the behaviors of Robo3R3-258

5cKO mice to that of the controls. The animal is introduced to one arm of the Y-shaped 259

maze (arm length: 35 cm, width: 8cm, height: 15 cm) and the test lasts until the mouse 260

makes 20 arm choices. We then recorded the number of entries and the sequence of 261

entries to calculate the percentage of alternation. 262

Open field test. In general, this test is used to evaluate locomotion, exploratory 263

behavior, anxiety, and general activity levels of rodent subjects, especially following 264

various experimental manipulations or drug treatments (Mothes et al., 1996; Saitoh et 265

al., 2014). We used this test as whisking is an essential component of the mouse 266

exploratory behavior. The open-field apparatus was a clear Plexiglas arena (28 x 26 x 18 267

(height) cm), illuminated by a small red lamp. The floor of the chamber was line-marked 268

into equal size quadrants. After placing the individual mice in the center of the field, we 269

recorded the freely moving mice for 5 minutes to measure the number of line crossings. 270

We also scored stretch-attend postures as an additional exploratory behavior (Rodgers 271

and Dalvi, 1997; Blanchard et al., 2003). 272

Social behavior test: The resident-intruder paradigm is a standard test for 273

measuring social investigation and live performance for social behaviors, including inter-274

male aggression and social defeat in rodents (Koolhaas et al., 2013). Here, we used it 275

as a metric for social behavior, without priming the resident for aggression or introducing 276

an aggressive intruder. We conducted this test following the open-field test. We kept the 277

mice in the chamber after the open-field test and introduced an “intruder” male mouse 278

(C57/Bl6 strain) into the chamber. The behaviors of the mice, videotaped during a 10-279

minute session, included the duration of the contacts when the subject mouse 280

approached the intruder and events of flight responses to the approaches. We 281

calculated the ratio of appearances for flight responses to the intruder’s approaches from 282

the front or back of the body. 283

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Horizontal surface approach test. This test is based on the postural reflex and 284

limb placement when mice are suspended by their tail and are approaching a surface 285

that they can reach with their forepaws (Gerlai et al., 2000). We picked up individual 286

mice by the tail and slowly moved horizontally toward the edge of a desk with 1 cm/5 sec 287

speed. Mice used their whiskers to detect and touch the surface edge as fast as they 288

could. We repeated the procedure four times. We measured the distance (in 289

centimeters) of the body position from the edge of the platform when the mice were able 290

to reach it with the forepaws. We calculated the average of the distances in four trials 291

was calculated as a representative value. 292

Bridge crossing test. This test (a.k.a. raised beam test) is generally used for 293

testing motor coordination (Brooks and Dunnett, 2009). Mice are placed on an end of a 294

bridge (4 cm width, 25 cm length) leading to a box platform in a dark container under 295

infrared lighting. The animals cross the bridge to reach the platform by using whiskers to 296

sense the bridge edges. We performed three trials per mouse and the maximum cut-off 297

time was 90 sec. We used the traverse time (in seconds) as measurements. 298

Swimming test. Mice use their whiskers to maintain balance while floating in 299

water. When placed in water, whisker-clipped mice and “barrelless,” Adenylate cyclase 300

Type 1 deficient mice show similar levels of struggling and paddling, when compared to 301

wild type mice, but their floating ratio decreases significantly (Arakawa et al., 2014a). 302

We placed individual mice in a 2-L Pyrex glass beaker containing 1.8 L of water 303

(maintained at 24±1 °C) for 5 min. We videotaped their performances and analyzed 304

swimming, including paddling and floating without paddling. 305

Measures of whisking behavior. Videotaping of whisking behavior (with a high-306

speed camera, EX-FH100, Casio, at 120 fps), as mice freely move or approach objects 307

provides measures of exploratory and active whisking behaviors. We placed a small, 308

clear glass cup in the cage sideways and allowed the mouse to explore inside and 309

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around the cup. Next, we placed other small objects (e.g., a tooth brush or a plastic cup) 310

in the cage and recorded the whisking behavior of the animal. We analyzed the 311

frequencies, and the types of whisking toward the objects. 312

Gap-crossing test. The gap-crossing test was developed to test cortical whisker 313

barrel function (Hutson and Masterton, 1986; Barnéoud et al., 1991). The test consists of 314

a series of trials requiring the mouse to cross a gap in a bridge with variable distances. 315

We placed the subjects in the center of an elevated lane (4 cm diameter) connected to a 316

platform. The gap distance between the lane and the platform varied from 0 to 8 cm by 317

1 cm increments. We measured the gap distance (in centimeters) that the mice were 318

able to cross to get to the safe platform. We performed the trials under infrared lighting 319

so that the mice relied on tactile rather than visual cues. 320

Object discrimination test. Mice have an innate preference to spend more time 321

exploring the novel objects that they encounter. This particular test relies on this 322

preference. We conducted 3 trials in the home cage under low lighting, and mice were 323

able to see the objects in their cage. During the first trial, we placed two identical objects 324

(plastic Easter eggs) at opposite corners of the cage, and the mouse was allowed to 325

explore the objects for 5 min. After an hour interval, we ran the 2nd trial, in which a new 326

object was placed in the cage along with one of the familiar objects from the first trial in 327

the same location. The new object was a small pot, glass cup, toothbrush, or D-type 328

battery. After another hour interval, in the 3rd trial, we placed one familiar object and a 329

new object in the cage. The order of the objects was counter-balanced for each animal. 330

We measured the time spent in investigating each object and calculated the preference 331

for novel objects as a discrimination measure. 332

Texture discrimination test. We conducted this test in the same way as the object 333

discrimination test but in the dark (without visual cues); also, the objects that we placed 334

in the cage had surfaces made of different textures. The objects were plastic cups filled 335

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with materials of different textures, such as sponge, metal mesh, terry cloth, cardboard, 336

etc.) 337

Whisking laterality test. This test is specifically developed for the Robo3R3-5cKO 338

mice in our laboratory to examine left-right whisking preference at objects encountered 339

on the left or the right side of the snout. The test apparatus is a bridge between two 340

platforms. Along the sides of the bridge six stainless steel thin rods, 3 on each side are 341

vertically positioned 3cm away from the center of the bridge. The test is conducted under 342

infrared light. We position the mouse on one platform and the mouse crosses to the 343

other platform through the bridge encountering the rods with its whiskers along the way. 344

When the animal’s whiskers contact a rod they orient their snout to the rod to investigate 345

further by active whisking. We videotaped whisking behavior and quantified the 346

occurrence of whisker contact with a rod and orientation of the snout to the rod are. 347

Orientation of the snout with respect to the rod is also noted in three categories: 348

ipsilateral, when the mouse whisks at the rod with the whiskers on the side of the rod; 349

medial, when the snout is oriented to the rod and the animal whisks with whiskers on 350

both sides of the snout; and contralateral, when the animal turns its head and whisks 351

with whiskers on the side contralateral to the position of the rod (i.e., if the rod is on the 352

left side of the mouse and the animal whisks the right snout). 353

Statistical analysis. Our experimental design was random. We used one-way or 354

two-way analysis of the variance (ANOVA) for the statistical analysis. We used the 355

Bonferroni correction for the post hoc comparisons when necessary. For pair-356

comparison in behavioral tests (Robolox/lox vs. Robo3R3-5cKO), we used the unpaired 357

Student’s t-test when data sets with equal variances, while the Welch’s t-test was used 358

with unequal variances. For all tests, differences were accepted as significant at p<0.05. 359

All data are expressed as mean ± S.E.M. 360

Results 361

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Immunohistochemistry for glutamate transporters and CO histochemistry 362

confirmed the presence of bilateral whisker representations in the VPM and the barrel 363

cortex of Robo3R3-5cKO mice (Figure 1). Trigeminal afferent patterns corresponding to 364

the barrelettes in the PrV were indistinguishable from those seen in the control mice. 365

The usual numbers of whisker follicles, and their patterned arrangement were also 366

present on the snout. In contrast, there were duplicate barreloids and barrel patterns in 367

the VPM and cortex, respectively, as shown recently (Renier et al., 2017). Selective 368

labeling with lipophilic tracers and activity-dependent immediate early gene (c-Fos) 369

expression identified ipsi- and contralateral individual whisker or whisker row 370

representations in the barrel cortex (Renier et al., 2017). The present 371

immunohistochemistry and histological assays confirmed the thalamic and cortical 372

pattern defects. 373

Voltage-sensitive dye imaging (VSDi) is a convenient approach to map cortical 374

activity following sensory stimulation. We applied voltage-sensitive dye RH-1691 to the 375

barrel cortex and stimulated the second whisker in row C (C2) on the snout. 376

Thereinafter, we imaged the cortical activity. In control animals only contralateral whisker 377

stimulation elicited VSD signals in a given cortex, while ipsilateral stimulation did not 378

evoke any notable activity during the imaging time frame (Figure 2). In contrast to the 379

control mice, in all imaged mutant animals, there were responses to stimulation of both 380

the ipsi- and contralateral C2 whiskers (Figure 2). The distance between centers of 381

activity patterns, elicited by ipsi- and contralateral stimulation, was on the average 0.4 ± 382

0.1 mm, which is in line with the morphological data using immunohistochemistry or CO 383

histochemistry. 384

As seen in Figure 2, activated cortical areas show a shift in time and are larger 385

from those seen with histochemical markers. That is in part due to the imaging 386

approach, and in part spread of activity from layer IV to supragranular layers over time. It 387

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is noteworthy that the time courses of responses to ipsi- and contralateral whisker 388

stimulation were not different. 389

Following confirmation of morphological and functional representations of the 390

whiskers bilaterally in the thalamocortical pathway, we used a battery of sensorimotor 391

tests to determine how bifacial maps in the forebrain would affect the behavior of the 392

animal. We found that the general physical strength of the Robo3R3-5cKO mice appeared 393

normal in the grip test: (t (16)= 0.022, p=0.9827). 394

General paw tactile sensation also appeared normal in Robo3R3-5cKO mice. The 395

latencies of licking the hindpaw to rid it of the adhesive paper were similar in the 396

experimental and control groups (t (16)= 0.4954, p=0.627). This observation indicates 397

that functionally, the dorsal column lemniscal pathway is not affected, as expected from 398

unaltered patterning and size of the paw maps in the brainstem, thalamus and cortex. 399

Spontaneous alternation ratio in a Y-maze was significantly lower in Robo3R3-400

5cKO mice (51.11% ± 3.98) than in Robolox/lox mice (70.56% ± 2.42) (t (16)= 4.17585, 401

p=0.0007). The test is generally used as a measure of rodent cognitive function. The 402

animals have to remember and recognize the shape of the Y-maze, and which arm was 403

visited last, and they will naturally enter the most number of arms in a given trial. Since 404

whiskers are very important in exploratory behavior of mice, we used this test to see 405

whether bifacial barrel maps would affect spatial discrimination. Indeed, a lower 406

performance in Robo3R3-5cKO mice indicates that bilateral whisker representation in the 407

thalamocortical pathway impairs spatial discrimination and possibly cognitive functions. 408

In the open field test, we recorded the behavior of the mice to assess their 409

exploratory behavior, rather than their general activity level. In this test, Robo3R3-5cKO 410

mice showed a significant increase in locomotion (118.67 ± 22.53) when compared to 411

that of controls (16.44 ± 2.20) (t (8)= 4.516, p=0.002). But, we noted no differences in 412

“risk assessment behaviors,” such as stretch-attend postures (t (11)= 0.264, p=0.7966). 413

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We also tested social behaviors in the open field chamber where the subject mouse was 414

confronted with an unfamiliar (intruder) male mouse. We recorded social contacts and 415

flight responses. There were no significant differences in duration of social contacts (t 416

(11)=0.1602, p=0.8755) or flight response (to a frontal approach; t (8)= 1.4944, 417

p=0.1734, and to a back approach; t (12)= 0.695, p=0.5001). 418

The horizontal surface approach test can be used for measuring whisker sense 419

and body coordination. In this test, Robo3R3-5cKO mice exhibited significantly shorter 420

distance (3.77 ± 0.26 cm) to reach the edge surface than control mice (4.58 ± 0.26 cm) t 421

(16)= 2.1829, p=0.0433, which indicates impairment in whisker sensation. A similar 422

impairment in Robo3R3-5cKO mice was found in other tests that require whisker 423

sensation. In the bridge crossing test, Robo3R3-5cKO mice took longer time to cross the 424

bridge on the first trial compared to control mice (58.00 ± 11.07 seconds (sec) versus 425

22.78 ± 4.90 sec) [Gene: F (1,16)= 5.64, p<0.05, Trial: F (2,32)= 15.71, p<0.01, and 426

Gene x Trial: F (2,32)= 6.54, p<0.01, trial 1 F (1,16)= 8.47, p<0.05]. In the swimming 427

test, Robo3R3-5cKO mice exhibited significantly shorter floating time (5.59 ±1.51 sec) 428

compared to control mice (31.67 ± 2.75 sec), t (16)= 8.31, p<0.0001, while they 429

exhibited a similar level of struggling time (32.85 ± 5.30 sec) as control mice (22.67 ± 430

2.14 sec), t (11)= 1.78, p=0.102. 431

The Robo3R3-5cKO mice have full sets of whiskers and show whisking behavior. 432

We recorded the whisking behaviors with a high-speed camera (120 fps). We measured 433

the frequencies and types of whisking when mice freely whisked in the air and at an 434

object they encountered. We found that mice tended to show symmetry whisking 435

(horizontal movement in all directions) when they do not contact an object, and active 436

whisking (bundled and forward oriented movement with high frequency) toward an 437

object. Robo3R3-5cKO mice show a lower ratio of active whisking (0.6307 ± 0.0419) 438

during whisking surfaces of objects compared to that of the controls (0.8394 ± 0.0472) (t 439

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(16)= 3.3076, p=0.0044). Control mice displayed faster frequencies in active whisking 440

than symmetry whisking, while Robo3R3-5cKO mice showed similar lower frequencies of 441

whisking in both symmetry and active whisking. 442

The gap-crossing test is one of the few established tests for whisker-barrel 443

function in rodents, and it is conducted under infrared lighting to eliminate visual cues. 444

We found that Robo3R3-5cKO mice crossed shorter (3.00 ± 0.24 cm) gap distances than 445

controls (4.11 ± 0.35 cm), t (16)= 4.718, p=0.0002. We further investigated sensory 446

performance with an object shape recognition test, which relies in part on vision, and a 447

texture discrimination test, which relies on whisker sensation. In both of these tests 448

(Figure 3), two identical objects were presented in the home cage on trial 1. On trials 2 449

and 3, one of these objects was removed and a novel object was introduced. If mice 450

recognize the difference between the objects they spend more time exploring the novel 451

one. Thus, the score of novel object preference would be higher than the 50% chance 452

level. Mice were able to see the objects visually in the object shape recognition test, but 453

they are not allowed to see the shape in the texture discrimination test, as this test was 454

conducted under infrared light. Both Robo3R3-5cKO mice and control mice displayed a 455

clear discrimination on trial 2 and 3 in the shape recognition test [Gene: F (1,16)= 0.003, 456

n.s., Trial: F (2,32)= 37.44, p<0.01, Gene x trial: F (2,32)= 1.36, n.s.]. On the other 457

hand, Robo3R3-5cKO mice did not show preference on trial 2 and 3 in the texture 458

recognition test, while control mice exhibited clear signs of discrimination [Gene: F 459

(1,16)= 20.24, p<0.01, Trial: F (2,32)= 8.52, p<0.01, Gene x trial: F (2,32)= 3.76, p<0.05. 460

T1: F (1,16)= 0.48, n.s., T2: F (1,16)= 12.18, p<0.01, T3: F (1,16)= 9.62, p<0.01] (Figure 3A). 461

These data clearly indicate that Robo3R3-5cKO mice distinguish object shape novelty 462

using visual cues, but not object texture by whisker-related tactile cues. 463

In a whisking laterality test, Robo3R3-5cKO mice detected the rods along the 464

bridge similar to the control animals, t (7)= 0.09827, p=0.9245 (Figure 3B). Upon 465

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encountering a rod, control mice showed active whisking with a medial approach or on 466

the ipsilateral side with respect to the rod, while Robo3R3-5cKO mice mostly whisked the 467

contralateral side to investigate the rod [ipsilateral: t (14)=0.701158, p=0.4947; medial: t 468

(14)= 9.0911, p<0.0001; and contralateral: t (7)= 24.677, p<0.0001] (Figure 3B). 469

Discussion 470

Whisker-specific neural patterns depend on an intact sensory periphery during a critical 471

period in development (reviewed in Erzurumlu and Gaspar, 2012). Spontaneous 472

mutation or experimental induction of extra whisker follicles on the snout causes 473

formation of corresponding additional barrels and subcortical whisker neural modules 474

(Welker and Van der Loos, 1986; Ohsaki et al., 2002). At the other end of the spectrum, 475

alterations in molecular signals, intrinsic to the developing neocortex, can also 476

dramatically alter the positioning and size, even leading to the duplication of the barrel 477

fields (Fukuchi-Shimogori and Grove, 2001; Shimogori et al., 2004; Assimacopoulos et 478

al., 2012; Zembrzycki et al., 2013). We recently reported that axon pathfinding errors in 479

subcortical connections, such as those between the PrV and VPM, create pattern 480

deficits in whisker maps as well (Renier et al., 2017). In Robo3R3-5cKO mice, 481

contralateralization defects of the trigeminothalamic pathway lead to crowding of bilateral 482

whisker-related neural modules in the face representation areas of the VPM and the SI 483

cortex. In both areas, the size, orientation, and positioning of the face representations do 484

not seem to be altered. Also, there are no effects on the surrounding paw and body 485

representation areas. Thus, in accordance with the studies cited above, the size, 486

orientation, and positioning of sensory maps are most likely determined by intrinsic 487

molecular cues. Here, we show that the bilateral whisker maps are active and influence 488

sensorimotor behaviors. 489

In the Robo3R3-5cKO cortex, individual barrels are notably smaller and the 490

ipsilateral barrel map inserts itself inside the contralateral barrel map, without any 491

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overlap. The ipsilateral and contralateral whiskers have their distinct barrels that do not 492

neighbor each other. This brings out a distinct contrast to the visual system, where ipsi- 493

and contralateral visual inputs that are in register with respect to the sensory space 494

converge on to the layer IV cells and eventually lead to the perception of depth. The 495

visual system too has a number of mutations that affect contralateralization. Midline 496

crossing defects of the optic nerve lead to significant alterations in retinotopic and eye-497

specific maps, most notably in the visual cortex. In the human visual pathway, nasal 498

retinal axons cross the midline at the chiasm while temporal retinal axons do not, 499

yielding approximately 50% crossing of retinal axons from each eye. In albinism, part of 500

the temporal retinal axons also cross at the chiasm leading to a partially overlaid 501

representation of opposing visual fields in the primary visual cortex (V1). Visual 502

perception remains relatively intact, but this condition of partial map duplication is 503

accompanied by optokinetic nystagmus, reduced visual acuity and binocular vision, 504

among other symptoms (Creel et al., 1990). In an achiasma condition, all retinal axons 505

project to the ipsilateral hemisphere leading to overlay of nasal and temporal 506

hemiretinae maps in V1. Recent imaging studies show that within the overlapping maps, 507

projections from the nasal and temporal retina are interdigitated, much like ocular 508

dominance columns. Binocular interactions are only seen in higher cortical visual fields 509

such as V5 and fusiform gyrus (Hoffman et al., 2012; Davies-Thompson et al., 2013; 510

Olman et al., 2016). As in albinism, despite reduced visual acuity and depth perception, 511

overall visual perception remains intact. This is most likely due to corticocortical 512

compensatory interactions. Several previous studies on albino and achiasmatic animals 513

have described mapping defects and physiological consequences (Guillery, 1971; 514

Guillery and Kaas, 1973; Cucchiaro and Guillery, 1982; Guillery et al., 1984; Williams et 515

al., 1994; Hogan et al., 1999). The present Robo3R3-5cKO somatosensory phenotype 516

introduces a different map layout as a consequence of bilateral trigeminothalamic 517

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projections. Here the two face maps with rows of whisker representing modules are 518

squeezed into separate zones of a normal size face map area. Interestingly, the 519

ipsilateral face map is nestled inside a split contralateral face map. Overall, individual 520

barrels representing the same whisker on both sides are distant from one another. Since 521

the barrels are activated by stimulation of ipsi- or contralateral whiskers, overall cortical 522

whisker perception should be present. However, due to the reduced size of individual 523

whisker barrels and/or activation of the barrel cortex from both sides of the face, sensory 524

acuity, and discrimination are impaired. 525

Sensory maps of the neocortex form close functional relations with the 526

association and motor cortices and, moreover, all this circuitry is in intimate relation with 527

the motivational and cognitive circuitries of the brain. The Robo3R3-5cKO mouse model 528

offers a means to explore how a significant change in a major sensory map in the cortex 529

may affect the rest of the brain circuitry and consequently the behavioral repertoire of the 530

animal. The results of the sensorimotor tests in Robo3R3-5cKO mice do not show notable 531

motor defects, except those that involve whisker sensation. We do not yet know how 532

duplicate maps in the barrel cortex alters other associated cortical circuitry such as the 533

callosal, S1 to M1 and corticocortical involving supragranular layers, or for that matter, 534

even infragranular layers that project to the VPM and brainstem trigeminal centers. An 535

MRI study found that the size and the connectivity of the corpus callosum appeared 536

normal in an achiasma case (Davies-Thompson et al., 2013). If afferent projections to a 537

given target follow “chemoaffinity,” or intrinsic molecular guidance cues, the other 538

somatosensory-related projections in in Robo3R3-5cKO mice should not be affected. 539

Perhaps, the similar organization of the whisker map in the VPM and in the barrel cortex 540

is indicative of intrinsic wiring cues. Nevertheless, we do not know how partial crossing 541

of trigeminothalamic projections lead to an ipsilateral map, carving an ipsilateral territory 542

within the contralateral map. This collateralization defect in the sensory thalamus is quite 543

Page 23: Behavioral consequences of a bifacial map in the mouse ...


unlike that which is seen in retinal projections to the LGN of achiasmatic dogs (Williams 544

et al., 1994; Hogan et al., 1999). 545

Differences in exploratory versus discriminative whisking behaviors of Robo3R3-546

5cKO mice suggest that exploratory, passive whisking is mediated through unaffected 547

brainstem circuits involving trigeminofacial connections between the spinal trigeminal 548

subnucleus caudalis and the facial nucleus. Active, discriminative whisking most likely 549

requires sensorimotor integration at higher centers; bilateral whisker maps and smaller 550

whisker-related neural modules in the thalamus and cortex interfere with this process. 551

Like the somatosensory pathway, the descending motor pathway is largely 552

crossed. Motor commands from the cortex reach the contralateral facial nucleus in the 553

brainstem, the lateral subnuclei of which control facial muscles used in whisking. In its 554

normal operation, when both the ascending tactile sensory and descending motor 555

pathways are crossed, they interact synergistically. When one of these systems has 556

midline crossing defects, the synergistic activity is disrupted. There are examples of 557

such crossing defects in humans as well, such as congenital mirror movement disorder 558

and horizontal gaze palsy with progressive scoliosis (HGPPS). These midline crossing 559

defects have been linked to mutations in DCC and Robo3 receptors respectively (Jen et 560

al., 2004; Nugent et al., 2012, Chédotal, 2014; Meneret et al., 2015; Friocourt and 561

Chédotal, 2016). The peculiar tendency of the Robo3R3-5cKO mice to use contralateral 562

whiskers when they encounter an object on the left or right side of the snout in the 563

laterality test could perhaps could be explained by conflicting sensory and motor activity. 564

In the presence of visual cues, Robo3R3-5cKO mice distinguish object shape 565

novelty, but perform poorly when they are challenged to discriminate object textures 566

relying on their whiskers and without visual cues. Our laboratories have observed 567

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impaired performance in whisker-related tasks in common laboratory mice following 568

clipping of all their whiskers (Arakawa and Erzurumlu, 2015), and in a variety of 569

genetically altered mouse lines that have barrel cortex or thalamic pattern deficits. So 570

far, these have included mice that lack functional thalamus-specific RIM proteins 571

involved in neurotransmitter release at the thalamocortical synapse (Narboux- Nême et 572

al., 2012), Adenylate cyclase Type I gene AC1 and thalamus-specific AC1 null mice that 573

lack barrels (Arakawa et al., 2014a), thalamus-specific Grin 1 null mice that lack thalamic 574

patterns (Arakawa et al., 2014b). Whether whisker clipped, or with absent or aberrant 575

thalamic, cortical axonal/cellular neural patterns, whisker-related behavioral performance 576

is impaired in varying degrees. Robo3R3-5cKO mice differ from these previous mouse 577

phenotypes such that the whisker patterns are not abolished but emerge as bilateral 578

representations. This model offers unique opportunities to investigate how increased 579

numbers of barrels representing both sides of the snout connect to the rest of the brain 580

circuitry, and moreover, whether barrel size is an important factor in sensory acuity. 581

Perhaps selectively silencing inputs from the PrV on one side will allow us to determine 582

similarities and differences between ipsi- and contralateral whisker maps in the cortex. 583

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834 835





Figure 1. Patterning of the whisker-barrel pathway in Robo3R3-5cKO mice. 841

Glutamate transporter immunohistochemistry reveals afferent terminal patterns that 842

correspond to barrelettes in the PrV, barreloids in the VPM nucleus and barrels in the 843

cortex (BCx). Left column photomicrographs show normal patterning in control animals 844

and the right column photomicrographs in Robo3R3-5cKO mice at successive levels of the 845

trigeminal neuraxis. Note that while barrelette patterns in the principal sensory nucleus 846

are normal, there is aberrant patterning in the VPM and in the barrel cortex (BCx). In the 847

immunostained mutant barrel cortex asterisks mark the boundary of the ipsilateral map. 848

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Whisker barrel rows are indicated in the control cortex. CO stained sections shown in the 849

bottom micrographs further illustrate the smaller size of the barrels in the Robo3R3-5cKO 850

cortex. C2 whisker is indicated and the approximate locations of the ipsi (i) and contra 851

(c) C2 whisker barrels in the Robo3R3-5cKO cortex section are marked. Lower panel 852

cartoons illustrate the ipsi and contra whisker representations in the barrel cortex of 853

normal (left) and mutant (right) mice. 854

Figure 2. VSD imaging in barrel cortex after C2 whisker stimulation. 855

A. Pseudocolor map of the responses in the barrel cortex to contralateral C2 stimulation. 856

B. Pseudocolor map of the ipsi (blue) and contra (red) responses in the left hemisphere 857

in a Robo3R3-5cKO mouse. The time after stimulus onset is at the top left corner of each 858

plate. C, D. The time course of the integrated fluorescence signals (ipsi blue, contra red) 859

calculated in the squares (7x7 pixels) in insets at 40ms image in control (C) and 860

Robo3R3-5cKO case (D). To visualize the color-coded squares, pseudocolor coded 861

responses are filtered, the unfiltered images are seen in 40ms response images in A and 862

B. 863

Figure 3. Object and texture recognition and whisking laterality tests. 864

In object recognition test, two identical objects (an Easter egg) were presented in 865

opposite corners of the cage and mice were allowed to freely investigate them during 866

trial 1 (T1). In trial 2 (T2) one of the objects was removed and replaced with a novel 867

object (small flower pot) and in trial 3 (T3) one object from the first trial (Easter egg) and 868

yet another novel object (toothbrush) was presented. Normally mice spend more time 869

sniffing and whisking at the new object. In this experiment mice were allowed to use 870

visual cues. The texture discrimination test was conducted the same way but under 871

infrared lighting and the object shapes were the same but they all had different textures. 872

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Significant differences between the control and Robo3R3-5cKO mice were found on T1 873

and T2 only in the texture discrimination test. 874

In the whisking laterality test, there was no significant difference in rod detection 875

between the control and cKO mice, t(7)= 0.09827, p=0.9245. When mice oriented their 876

snout and left or right set of whiskers to the rod significant differences in laterality were 877

observed. “Ipsilateral” indicates that the mouse whisked the same side of whiskers at a 878

rod located on the same side (e.g., left side rod, left whiskers). “Medial” indicates that 879

the mouse oriented its snout to the rod. “Contralateral” indicates that the mouse 880

oriented the opposite side of whiskers to a rod located at one side (e.g., left side rod, 881

right whiskers). Control mice tended to use the medial approach or ipsilateral whiskers 882

to investigate a rod, while Robo3R3-5cKO mice tended to use the contralateral set of 883

whiskers to explore the rod [lateral: t(14)=0.701158, p=0.4947; medial: t(14)= 9.0911, 884

p<0.0001; and contralateral: t(7)= 24.677, p<0.0001].* Data are expressed as mean ± 885

S.E.M. 886

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