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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 www.jneurosci.org/cgi/alerts 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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ABSTRACT 30
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
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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
5
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
6
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
(http://jaxmice.jax.org/strain/013636.html, 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
7
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
10
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
14
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
15
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
16
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
17
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
18
(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
19
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
20
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
21
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
22
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
23
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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FIGURE LEGENDS 839 840
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
34
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
35
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