ORIGINAL RESEARCH ARTICLE
published: 11 October 2013
doi: 10.3389/fncir.2013.00162

NEURAL CIRCUITS

Viral vector-mediated selective and reversible blockade of
the pathway for visual orienting in miceThongchai Sooksawate 1,2*, Kaoru Isa 1 , Ryosuke Matsui 3 , Shigeki Kato 4 , Masaharu Kinoshita 1† ,Kenta Kobayashi 5,6 , Dai Watanabe 3 , Kazuto Kobayashi 4 and Tadashi Isa 1,5,6*
1

Department of Developmental Physiology, National Institute for Physiological Sciences, Okazaki, Japan
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
3
Department of Molecular and Systems Biology, Graduate School of Biostudies, Kyoto University, Kyoto, Japan
4
Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan
5
Section of Viral Vector Development, National Institute for Physiological Sciences, Okazaki, Japan
6
Department of Life Sciences, The Graduate University for Advanced Studies (SOKENDAI), Hayama, Japan
2

Edited by:
Yasuo Kawaguchi, National Institutefor Physiological Sciences, Japan
Reviewed by:Paul J. May, University ofMississippi Medical Center, USA
Masahiko Takada, Kyoto University,
Japan*Correspondence:Thongchai Sooksawate, Departmentof Pharmacology and Physiology,
Faculty of Pharmaceutical Sciences,
Chulalongkorn University, PhayathaiRoad, Patumwan, Bangkok 10330,
Thailande-mail: sthongch@chula.ac.th;
Tadashi Isa, Department ofDevelopmental Physiology, National
Institute for Physiological Sciences,
Myodaiji, Okazaki 444-8585, Japane-mail: tisa@nips.ac.jp† Present address:
Masaharu Kinoshita, Department of
Physiology, Hirosaki University
School of Medicine, Hirosaki, Japan

Recently, by using a combination of two viral vectors, we developed a techniquefor pathway-selective and reversible synaptic transmission blockade, and successfully
induced a behavioral deficit of dexterous hand movements in macaque monkeys byaffecting a population of spinal interneurons. To explore the capacity of this techniqueto work in other pathways and species, and to obtain fundamental methodologicalinformation, we tried to block the crossed tecto-reticular pathway, which is known tocontrol orienting responses to visual targets, in mice. A neuron-specific retrograde genetransfer vector with the gene encoding enhanced tetanus neurotoxin (eTeNT) tagged withenhanced green fluorescent protein (EGFP) under the control of a tetracycline responsive
element was injected into the left medial pontine reticular formation. 7–17 days later,
an adeno-associated viral vector with a highly efficient Tet-ON sequence, rtTAV16, wasinjected into the right superior colliculus. 5–9 weeks later, the daily administration of
doxycycline (Dox) was initiated. Visual orienting responses toward the left side wereimpaired 1–4 days after Dox administration. Anti-GFP immunohistochemistry revealed
that a number of neurons in the intermediate and deep layers of the right superior
colliculus were positively stained, indicating eTeNT expression. After the termination ofDox administration, the anti-GFP staining returned to the baseline level within 28 days.
A second round of Dox administration, starting from 28 days after the termination of thefirst Dox administration, resulted in the reappearance of the behavioral impairment. Thesefindings showed that pathway-selective and reversible blockade of synaptic transmission
also causes behavioral effects in rodents, and that the crossed tecto-reticular pathway
clearly controls visual orienting behaviors.
Keywords: superior colliculus, pontine reticular formation, orienting behavior, viral vector, Tet-ON, tetanus
neurotoxin, mouse

INTRODUCTION
To study structure-function relationships within neural circuits,
it is necessary to manipulate the activity of an “identified” population of neuronal elements in these circuits. Recent advances
in molecular genetic techniques enabled such targeted manipulation by making transgenic animals with cell-specific promoters(Kobayashi et al., 1995; Watanabe et al., 1998). However, suchtechniques have generally been constrained to those animals inwhich the transgenic manipulation of gene expression is possible (i.e., mouse, nematode, Drosophila, etc.) and to neuron types
whose cell-specific promoter has been identified. An alternative
possibility is to use viral vectors to introduce particular genes. To
enable pathway specificity, lentivirus vectors pseudotyped withrabies virus glycoprotein (RVG) were developed as vectors specific for retrograde transport (Kato et al., 2007). However, suchvectors were not sufficiently efficient to affect enough of a specificneuron population to induce behavioral effects, especially inlarger animals such as non-human primates. To overcome such

Frontiers in Neural Circuits

difficulties, Kobayashi and colleagues recently enhanced the retrograde transport efficiency of lentivirus vectors by pseudotyping
with a chimera of rabies virus and vesicular stomatitis virus
glycoprotein (VSVG) [highly efficient retrograde gene transfer(HiRet) vector; Kato et al., 2011a]. We incorporated a tetracycline responsive element (TRE) and enhanced tetanus neurotoxin(eTeNT) tagged with enhanced green fluorescent protein (EGFP)
into this retrograde vector (HiRet-TRE-EGFP.eTeNT). We also
incorporated the newly developed efficient Tet-ON sequence
rtTAV16 into an adeno-associated type 2 virus vector (AAV-2)
with a cytomegalovirus promoter (CMV) (AAV2-CMV-rtTAV16)(Kinoshita et al., 2012) as a switch to regulate the first construct. To study the function of a subpopulation of propriospinal
neurons (PNs) whose cell bodies are located in the mid-cervicalsegments and project to hand/arm motor neurons in macaquemonkeys, we injected HiRet-TRE-EGFP.eTeNT into the ventral
horn of the C6–Th1 segments; one week later, we injected AAV2CMV-rtTAV16 into the intermediate zone of the C2–C5 segmentswww.frontiersin.org

October 2013 | Volume 7 | Article 162 | 1

Sooksawate et al.

Selective blockade of orienting pathway

where the PN cell bodies are located. 1–2 months later, we initiated the administration of doxycycline (Dox). We could observe
deficits in reach and grasp movements. Acute electrophysiologicalexperiments clarified that approximately 90% of the transmission through the PNs was blocked. Theoretically, this methodshould be available for universal use in the various pathways of thecentral nervous system without the need to identify cell-specificpromoters or develop genetically modified animals.
At the current stage, we still need to know the fundamental properties of this technique and how to optimize it. For thispurpose, however, non-human primates are not the best animal species because the number of available animals is limited,and behavioral and histological analyses are time consuming.
Therefore, we decided to use mice to establish the fundamental protocol of this technique. Moreover, we wished to selecta pathway whose function is easy to investigate using behavioral observations. We chose the crossed tectoreticular pathway(cTRNs), which originates from the intermediate and deep layers of the midbrain superior colliculus (SC) and terminates in
the medial pontomedullary reticular formation contralateral to
the SC (Grantyn and Berthoz, 1987; Redgrave et al., 1990; Isa
and Sasaki, 2002; Sooksawate et al., 2005, 2008). This pathway
is known to control the orienting response of the eyes, head,and body to visual targets (Wurtz and Albano, 1980; Sparks,
1986; Dean et al., 1989; Isa and Sasaki, 2002; Isa and Hall, 2009),
which is an innate behavior that does not require training. In
this study, we focused on the time course of the effect of pathway
blockade through behavioral and histological examinations after
starting Dox administration and after its offset. We also tested thereversibility of the effects by halting and repeating Dox administration. This technique proved very feasible in mice, suggestingthat it could be used universally as a relatively convenient method
for the pathway-specific manipulation of neural activity and gene
expression.

MATERIALS AND METHODS
The animal experimental procedures in this study were conductedin accordance with the Guidelines of the National Institutes of
Health and the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and were approved by the Institutional
Animal Care and Use Committee of the National Institutes of
Natural Sciences. All attempts were made to minimize the suffering and number of animals used in this study. The methods
and time schedule of the present experiments are summarized in
Figure 1.

type C. In previous studies, it has been shown that pseudotyping the lentiviral vector with the RVG changed the property ofthe vector specific for retrograde gene transfer (Kato et al., 2007).
However, the efficiency of the gene transfer was not enoughto affect the majority of neurons projecting to the injection
site. Later, it was found that replacing RVG with a chimeric
protein of RVG and VSVG domains greatly enhanced the efficiency of retrograde gene transfer (highly efficient retrograde genetransfer vector or HiRet vector) (Kato et al., 2011a). Then, theuse of a different chimeric protein composing RVG and VSVG
(fusion glycoprotein type C) switched the properties to neuronspecific infection (NeuRet) (Figure 1A; Kato et al., 2011b). In thepresent study, the envelope plasmid encoding fusion glycoprotein type C, which was under the control of the cytomegalovirus(CMV) enhancer/chicken β-actin promoter, was used for vector production. Chimeric EGFP.eTeNT.PEST was generated byfusing the human codon-optimized tetanus neurotoxin lightchain (eTeNT) with the EGFP derived from pEGFP-N1 vector(Clontech, Mountain View, CA, USA) and the PEST sequence ofornithine decarboxylase, as reported previously (Kinoshita et al.,2012). The transfer plasmid pLV-TRE-EGFP.eTeNT.PEST wasbased on pFUGW (a gift from D. Baltimore, California Instituteof Technology, Pasadena, CA, USA), and constructed by swapping the ubiquitin promoter-EGFP sequence with the TRE/CMV
promoter of the pTRE-Tight vector (Clontech, Mountain View,
CA, USA) and EGFP.eTeNT.PEST. The NeuRet vector encoding EGFP.eTeNT was placed downstream of the TRE promoter (termed NeuRet-TRE-EGFP.eTeNT) and was prepared as
described previously (Inoue et al., 2012).
AAV2-CMV-rtTAV16

The rtTA variant rtTAV16 was generated by introducing theV9I, G12S, F67S, F86Y, R171K, and A209T mutations into
rtTA2S-M2 of the pTet-ON advanced vector (Clontech, Mountain
View, CA, USA), as reported previously (Kinoshita et al., 2012).
Plasmid pAAV2-CMV-rtTAV16 (Figure 1A) is based on pAAVMCS (Agilent Technologies, Inc., Santa Clare, CA, USA), andwas constructed by inserting the CMV promoter sequence ofthe pTet-On advanced vector rtTAV16, the woodchuck hepatitis
virus posttranscriptional regulatory element WPRE sequence ofpFUGW, and the SV40 polyadenylation signal (SV40pA) of thepCMV-script vector (Agilent Technologies, Tokyo, Japan) into a
multiple cloning site. The AAV vector for in vivo injection was
produced as described previously (Kaneda et al., 2011).
INJECTIONS OF NeuRet-TRE-EGFP.eTeNT AND AAV2-CMV-rtTAV16

PREPARATION OF VIRAL VECTORS

Vector preparation was described by Kinoshita et al. (2012).
However, a major difference was that we used a neuron-specifichighly efficient retrograde gene transfer lentivirus (NeuRet) vector (Kato et al., 2011b) instead of the HiRet vector, because the
NeuRet vector is specifically taken up by axons at the injection
site.
NeuRet-TRE-EGFP.eTeNTThe NeuRet vector is a pseudotype of a human immunodeficiency
virus type 1-based lentiviral vector with fusion glycoprotein

Frontiers in Neural Circuits

We anesthetized 6–10-week-old male C57BL/6 mice with an
intraperitoneal injection of a mixture of ketamine (60 mg/kg
body weight) and xylazine (10 mg/kg body weight). In addition,
dexamethasone (5.5 mg/kg body weight) was injected intramuscularly as premedication. The head of the mouse was fixed to thestereotaxic apparatus (Narishige, Tokyo, Japan) and injections of
the vectors were made from the dorsal approach. NeuRet-TREEGFP.eTeNT (0.8–1.2 μL); titer, 3.3–12.1 × 1011 copies/mL) was
injected into the medial pontine reticular formation (MPRF)
on the left side (Figures 1B,C) using a thin glass micropipette(tip diameter, 50–70 μm) inclined by 45◦ caudally to the verticalwww.frontiersin.org

October 2013 | Volume 7 | Article 162 | 2

Sooksawate et al.

Selective blockade of orienting pathway

FIGURE 1 | Experimental protocols for the selective blockade of thecrossed tectoreticular pathway by double infection with the NeuRet
lentiviral vector (carrying EGFP.eTeNT) and the AAV2 vector (carrying the
Tet-ON sequence, CMV-rtTAV16). (A) The design of the two viral vectors.
(B) Photomicrographs of the superior colliculus (SC) and medial pontine

axis of the stereotaxic coordinates (Franklin and Paxinos, 2008),−8.3 mm from the bregma, 0.7 mm lateral to the midline, andat 3.6 and 4.4 mm from the presumed dorsal surface of the cerebellar cortex (0.4–0.6 μL/point of injection). 7–17 days after the
NeuRet-TRE-EGFP.eTeNT injection, AAV2-CMV-rtTAV16 (0.8–1.0 μL; titer, 1.96 × 1013 particles/mL) was injected into the SC
on the right side (Figures 1B,C). A small hole was made in theskull over the occipital cortex and a thin glass micropipette (tipdiameter; 50–70 μm) was inserted vertically into the right SC,−4.0 to −4.2 mm from the bregma, 1.1–1.2 mm lateral to themidline, and at 1.1–1.6 and 1.4–2.3 mm from the presumed dorsal surface of the cerebral cortex (0.4–0.5 μL/point of injection).
We used a syringe pump (ESP-32; Eicom, Kyoto, Japan) for the
injection; the injection rate was 0.1 μL/min. Before removing theglass micropipette from the injection site, we waited for 5 min.

Frontiers in Neural Circuits

reticular formation (MPRF) showing the viral vector injection targets. (C)Diagram for the double injection of the viral vectors into the MPRF and SC,
and the interaction of NeuRet-TRE-EGFP.eTeNT and AAV2-CMV-rtTAV16 in
the double infected crossed tectoreticular neurons (cTRNs). (D) The
experimental schedule of this study.

The transfer plasmid pLV-TRE-EGFP.eTeNT.PEST is switchedon to produce the tetanus neurotoxin only when the rtTAV16
sequence provided by the AAV2-CMV-rtTAV16 vector isexpressed in the same neuron and activated by Dox (Figure 1C).
The tetanus neurotoxin blocks the transmitter release by cleaving VAMP-2 at the nerve terminals, but does not kill the cell (forreview see Montecucco and Schiavo, 1994).
DOX ADMINISTRATION

5–9 weeks after the injection of AAV2-CMV-rtTAV16, the dailyadministration of Dox was initiated by a single intraperitoneal injection (10 μg/g body weight) of Dox in a 0.9% NaClsolution, followed by the continuous oral administration ofDox in the drinking water (3 mg/mL in a 5% sucrose solution) for 7 days (Figure 1D). In some animals, the second

www.frontiersin.org

October 2013 | Volume 7 | Article 162 | 3

Sooksawate et al.

Selective blockade of orienting pathway

period of Dox administration (2nd Dox administration) was
conducted from 28 days after the offset of the first periodof Dox administration (1st Dox administration). One groupof mice received Dox continually for 21 days for histologicalanalysis.
BEHAVIORAL TESTS FOR VISUAL ORIENTING AND TURNING BEHAVIOR

We utilized three tests to assess visual orienting and turning
behaviors: (1) the visual placing response, (2) the visual orienting
response, and (3) a turning behavior test (Figure 1D). The behavioral tests began on the day before Dox administration, continued
during Dox administration for 7 days, and after Dox administration for 7 days. All tests were performed between 6.00 AM and3.00 PM.
Visual placing response

A modified visual placing response test was used to evaluate visualorienting behavior toward the affected and unaffected sides of
the tested mouse. This test was modified from Metz and Schwab(2004) and Pinto and Enroth-Cugell (2000). In this test, themouse was suspended by holding its tail and then lowered towarda plastic plate either on the left or right side of the head without any contact to the vibrissae. Normally, when the head ofa mouse was lowered to near the edge of the plastic plate, itturned its head and trunk, and extended its forelimbs to placethem on the plate (see Figure 2A). The procedure was conductedbilaterally (10 trials per side each day). The number of times
the mouse successfully placed its forelimbs on the plate wascounted.
Visual orienting response

A visual orienting response test was used to evaluate visual orienting behavior toward a visual target presented either on theaffected or unaffected side of the tested mouse. The mouse wasplaced inside a small glass cylinder (15 cm height, 14 cm innerdiameter), and a stimulus (a small long-handle stainless steel
chemical spoon) was quickly moved into the visual field of themouse from behind, in the same horizontal plane as its eyes, until
the stimulus was located between its eyes (Smith et al., 1998).
Normally, when the stimulus entered its visual field, the mouseturned its head toward the stimulus (Figure 3A). The procedure
was conducted bilaterally in a pseudorandom sequence (10 trials

FIGURE 2 | (A) Series of photographs showing the effect of the
Tet-induced tetanus neurotoxin-expressing system on the visual placing
response toward the affected side (Dox-on day 3) and disappearance of theeffect (Dox-on day 6), compared with the unaffected side. (B) Effect of the

Tet-induced tetanus neurotoxin-expressing system on the visual placing
response toward the affected side after the 1st Dox administration for 7days, termination of Dox administration for 28 days, and the 2nd Dox
administration for 7 days.

FIGURE 3 | (A) A photograph showing the visual orienting response ofa mouse toward the unaffected side. (B) Effect of the Tet-induced
tetanus neurotoxin-expressing system on the visual orienting responseof the affected side after the 1st Dox administration for 7 days,
termination of Dox administration for 28 days, and the 2nd Dox
administration for 7 days.

Frontiers in Neural Circuits

www.frontiersin.org

October 2013 | Volume 7 | Article 162 | 4

Sooksawate et al.

Selective blockade of orienting pathway

per side each day). The number of times the mouse successfullyoriented itself to the stimulus was counted.
Turning behavior

A turning behavior test was used to evaluate the effect of the Tetinduced tetanus neurotoxin-expressing system on the preferredside of turning of the tested mouse after Dox administration. The
mouse was placed into a small glass cylinder (15 cm height, 14 cm
inner diameter), which forced it to turn left or right inside thecylinder (Figure 4A). The number of complete turns (360◦ ) toeach side was recorded for 10 min each day.
IMMUNOHISTOCHEMICAL ASSESSMENTS

At the end of the experiments, the mice were deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (80 mg/kg body weight) (Hospira Inc., Lake Forest, IL, USA)and transcardially perfused with 0.05 M phosphate-bufferedsaline and then 4% paraformaldehyde in a 0.1 M phosphate buffer(pH 7.4). The brainstem and spinal cord were cryoprotected andsectioned at a thickness of 40 μm using a sliding microtome (HM450; Microm, Walldorf, Germany). To obtain a clearer image ofthe cell bodies, dendrites and axons of the double infected neurons, we adopted permanent visualization of EGFP-positive cellswith anti-GFP immunohistochemistry. In particular, the permanent visualization technique made the axon tracing much easier.
Moreover, the distribution of neurons infected by AAV2-CMVrtTAV16 was assessed with in situ hybridization to detect thertTA sequence (the antisense probe for the tetracycline transactivator, tTA, was a gift from T. Yamamori, National Institute for
Basic Biology, Okazaki, Japan). The technical details of the antiGFP immunohistochemistry and in situ hybridization against thertTAV16 sequence were described previously (Kinoshita et al.,2012) and are available on the web (http://www.nibb.ac.jp/brish/
indexE.html), respectively. The number of GFP-positive neurons and tracings of the axons originating from their cell bodies
to their target areas were counted and drawn using a camera
lucida attached to a light microscope (BX51; Olympus, Tokyo,
Japan). Photomicrographs of the histological slices were takenusing light microscopes (Axioplan2; Zeiss, Göttingen, Germany
and BZ-9000; Keyence, Elmwood Park, NJ, USA).

DATA ANALYSIS

Data are expressed as the mean ± standard error of the mean.
Significance was tested by Student’s t-test, and a P value of lessthan 0.05 was considered to be significant.

RESULTSA total of 61 C57BL/6 mice were used in this study; the twoviral vectors were injected into 56 mice. As controls, somemice received an injection of NeuRet-TRE-EGFP.eTeNT without Dox administration (n = 2), NeuRet-TRE-EGFP.eTeNT and
AAV2-CMV-rtTAV16 without Dox administration (n = 2), or
NeuRet-TRE-EGFP.eTeNT with Dox administration (n = 3), andone group of mice was not injected (n = 5). No clear behavioral effects could be observed in any of these control groups.
Specifically, the % success rate for the visual placing responseon the side contralateral to the collicular injection ranged from95.00 ± 5.00 to 100.0 ± 0.00%, which was not significantly different from the side ipsilateral to the collicular injection (100.0 ±0.00%). The % success rate for the visual orienting responses
on the side contralateral to the collicular injection ranged from95.00 ± 5.00 to 100.0 ± 0.00%, which was also not significantly
different from the side ipsilateral to the collicular injection that
ranged from 95.00 ± 5.00 to 100.0 ± 0.00%. Moreover, the %turning rate to the side contralateral to the collicular injection was
not significantly different from the side ipsilateral to the collicular injection on any of the testing days (Dox-on day 0 to Dox-on
day 7). It ranged from 44.60 ± 5.63 to 50.40 ± 3.15%.
BEHAVIORAL OBSERVATIONSVisual placing response

After Dox administration, the visual placing response toward theaffected side (left side) of the double infected mice was impaired
from Dox-on day 1 to day 5, with the maximal impairment on
Dox-on day 3 compared with the unaffected side (right side).
The mice failed to turn their head and trunk and place theirforelimbs onto the plastic plate when they were lowered withthe plastic plate on the affected side (Figure 2A). The % successrate for placing toward the affected side was reduced to 58.95 ±
5.41%, compared with 99.47 ± 0.3671% for the unaffected side

FIGURE 4 | (A) A photograph showing the turning behavior of a mouse to the unaffected side (right). (B) Effect of the Tet-induced tetanus neurotoxin-expressing
system on turning behavior after the 1st Dox administration for 7 days, termination of Dox administration for 28 days, and the 2nd Dox administration for 7 days.

Frontiers in Neural Circuits

www.frontiersin.org

October 2013 | Volume 7 | Article 162 | 5

Sooksawate et al.

Selective blockade of orienting pathway(P < 0.0001, n = 38, unpaired t-test; Figure 2B). However, thisbehavioral effect was gradually reduced from Dox-on day 4 andcompletely disappeared on Dox-on days 6–7. After the termination of Dox administration, no behavioral effect could beseen on the affected and unaffected sides for 7 days (Dox-offdays −1 to −7).
Visual orienting response

Impairment of the orienting response could also be found in
the visual orienting response test. After Dox administration, thevisual orienting response toward the affected side (left side) was
impaired from days 1–6, while it was not impaired toward theunaffected side (right side) (Figure 3A). The % success rate of
the affected side was reduced to 64.21 ± 4.473%, compared tothe 96.32 ± 0.793% of the unaffected side (P < 0.001, n = 38,
unpaired t-test; Figure 3B). The behavioral effect was gradually reduced from Dox-on day 4, and completely disappeared by
Dox-on day 7. After the termination of Dox administration, no
behavioral effect could be seen on the affected and unaffectedsides for 7 days (Dox-off days −1 to −7).
Turning behavior

Before Dox administration and in the control groups, the micealmost equally preferred to turn toward the left and right.
However, after Dox administration, the mice preferred to turnto the unaffected side (right) (Figure 4A). The % turning to theaffected side (left side) was reduced from Dox-on days 1 to 4,
with the maximal reduction on day 3 (Figure 4B). The % turning to the affected side was reduced to 23.92 ± 2.721% from the
42.77 ± 1.824% of the controls on day 0 (P < 0.001, n = 38,
unpaired t-test). Then, the % turning to the affected side gradually returned to the same level as before Dox administration
by Dox-on day 7. It is noteworthy that after the termination ofDox administration, % turning to the affected side (left side)increased to its highest level on Dox-off day −2 (P < 0.05, n =15, unpaired t-test), before returning to the same level as before
Dox administration on Dox-off days −5 to −7.
REVERSIBILITY OF THE DOX-ON EFFECTS

28 days after the termination of the 1st Dox administration (for7 days), the 2nd Dox administration was started for another 7
days. All of the behavioral tests for visual orienting were foundto be impaired after the 2nd Dox administration. The pattern
and time course of the impairments were almost the same as
for the 1st Dox administration. The % success rates for thevisual placing response (Figure 2B) and visual orienting response(Figure 3B) and % turning (Figure 4B) toward the affected side
were reduced on Dox-on days 1–2 to days 4–5 and, then, gradually increased to the same levels as before Dox administration
on days 6–7. Although, the maximal effects of the 2nd Dox
administration on all behavioral tests appeared to be greater,
they were not significantly different from the 1st Dox administration (visual placing response, P = 0.0531; visual orienting
response, P = 0.2123; turning behavior, P = 0.1544, unpaired
t-test). These results indicate that the selective blockade of thecrossed tectoreticular pathway by double infection with NeuRetTRE-EGFP.eTeNT and AAV2-CMV-rtTAV16 can be performed
repeatedly with an interval of 28 days.

Frontiers in Neural Circuits

HISTOLOGICAL ANALYSISThe number of GFP-positive crossed tectoreticular neurons(cTRNs) in the intermediate and deep layers of the SC of the
double infected mice increased sharply after the start of Dox
administration for 1 day and reached its maximum on Dox-on
day 3 (Figures 5A,C). The maximum number of GFP-positivecTRNs was 458.8 ± 141.4 neurons (n = 6). After Dox administration was terminated, the number of GFP-positive cTRNs was
gradually reduced on Dox-off days −3 to −14, and had returnedto the baseline level by Dox-off day −28. To confirm the injection site of AAV2 in the right SC, in situ hybridization of rtTAV16
was performed. The injection sites were found to be located in
the intermediate to deep layers of the right SC (Figure 5B). To
demonstrate the reversibility of the Dox-on effects in these doubled infected mice, the 2nd Dox administration was started aftera 28-day Dox-off period. The number of GFP-positive cTRNs in
the intermediate and deep layers of the right SC increased again
to 400.0 ± 218.3 (n = 3; Figure 5C). The histological results paralleled the impairment of visual orienting behavior after the 2nd
Dox treatment in this group (Figures 2B, 3B, 4B).
In another group of mice (n = 3), the period of Dox administration was extended to 21 days, which resulted in strong stainingof the cTRNs with anti-GFP immunohistochemistry that filled
their distal dendrites. The axons from their cell bodies in theintermediate and deep layers of the right SC (Figure 6A) werelabeled all the way to their target areas. Such tracing was difficult to perform in the mice with Dox administration for 7 days.
The axons and terminals (Figures 6B,C) of these GFP-positivecTRNs could be found in the right mesodiencephalic junction[e.g., fields of Forel (FF), zona incerta (ZID, ZIV)] (Figure 6Ca),
mesencephalic reticular formation (mRt) (Figure 6Cb), nucleus
reticularis tegmenti pontis (RtTg) (Figures 6Cc,d), pontine reticular formation (PRF; including the injection site of NeuRet-TREEGFP.eTeNT) (Figures 6Cc,d,e), gigantocellular reticular nucleus(Gi) (Figure 6Ce), inferior olivary nuclei (IO) (Figure 6Cf), etc.
Thus, in addition to the injection site of the NeuRet vector, positive axons and terminals were found in many other target areas ofcollaterals that originated from the cTRNs (see Discussion).
TIME COURSE OF THE DOX-ON AND DOX-OFF PERIODS

To compare the time course of the behavioral effects and theappearance of anti-GFP immunoreactivity in cTRNs for the
Dox-on and Dox-off periods, the results of the three behavioral tests were normalized to the maximal impairment andaveraged. Then, the normalized values were compared to thenormalized number of GFP-positive cTRNs during the Dox-on
and Dox-off periods (Figure 7). The number of GFP-positivecTRNs increased sharply after the administration of Dox for
1 day, reached its maximum on Dox-on day 3, and wasmaintained until the end of Dox administration on day 7.
Although behavioral impairment was also found from Doxon day 1, it increased slower than the appearance of GFPpositive cTRNs. In contrast to the number of GFP-positivecTRNs, the behavioral impairment, after reaching its maximal effect on Dox-on day 3, was reduced to near the baseline
level on Dox-on days 6–7. After Dox administration was terminated on day 7, the number of GFP-positive cTRNs waswww.frontiersin.org

October 2013 | Volume 7 | Article 162 | 6

Sooksawate et al.

Selective blockade of orienting pathway

FIGURE 5 | Photomicrographs of GFP-positive cTRNs and in situ
hybridization to visualize rtTAV16 in the superior colliculus (SC) of
double infected mice. (A) Schematic diagram of the laminar structure of themouse SC (frontal plane) and photomicrographs of the SC slices processedwith anti-GFP immunohistochemistry (frontal section, inset area in the SC
diagram), before Dox administration (Dox-on day 0), Dox administration for 1
day (Dox-on day 1), 3 days (Dox-on day 3), 7 days (Dox-on day 7), and after thetermination of Dox administration for 3 days (Dox-off day −3), 7 days

(Dox-off day −7), 14 days (Dox-off day −14), and 28 days (Dox-off day −28).
(B) In situ hybridization against the tTA sequence in an SC slice indicating thecells infected by AAV2. (C) Average number of GFP-positive neurons in the
SC after the 1st Dox administration for 7 days, termination of Dox
administration for 28 days, and after the 2nd Dox treatment for 7 days (n = 3).
Abbreviations: SGS, superficial gray layer; SO, optic layer; SGI, intermediategray layer; SAI, intermediate white layer; SGP, deep gray layer; SAP, deep
white layer; PAG, periaqueductal gray.

gradually reduced and reached the baseline level on Dox-offday −28.

1988) impairs orienting responses; (3) single unit recordings
either from the SC (Sparks, 1975, 1978) or the medial pontomedullary reticular formation (Grantyn and Berthoz, 1987;
Isa and Naito, 1995; Isa and Sasaki, 2002) reveal the existenceof neurons that show increased firing preceding the orienting
response; and (4) anatomically, the medial pontomedullary reticular formation receives massive inputs from the intermediate anddeep layers of the contralateral SC (Kawamura and Brodal, 1973;
Huerta and Harting, 1982). Thus, there is overwhelming evidence
that the tectoreticular pathway plays a major role in the control of orienting responses (Sparks, 1986; Isa and Sasaki, 2002).
However, the specific role of cTRNs in gaze has been difficult
to resolve. In fact, this difficulty in precisely attributing function

DISCUSSION
The crossed tectoreticular pathway has been suggested to be
involved in the control of orienting responses based on the following evidence: (1) repetitive electrical stimulation of the SC
induces contraversive orienting responses (Cowie and Robinson,
1994; Corneil et al., 2002) and that of the medial pontomedullary reticular formation induces ipsiversive orienting
responses (Cowie and Robinson, 1994); (2) lesion or reversible
inactivation of the SC (Rosenquist et al., 1996; Quaia et al., 1998)
or medial pontomedullary reticular formation (Isa and Sasaki,

Frontiers in Neural Circuits

www.frontiersin.org

October 2013 | Volume 7 | Article 162 | 7

Sooksawate et al.

Selective blockade of orienting pathway

FIGURE 6 | Tracings of the axonal trajectories of the GFP-positivecTRNs in the superior colliculus (SC) in the mice with Dox treatment
for 21 days. (A) GFP-positive cTRNs in the right SC. (B) Axon terminals(arrows) in the left pontine reticular formation. (C) Tracings of the axons
and their terminals in the brainstem: a. Pretectum; b. Midbrain (SC level);c. Midbrain (inferior colliculus level); d. Pons (rostral part); e. Pons (caudalpart); f. Medulla. GFP-positive cell bodies are indicated as blue dots in Cb.
Abbreviations: 5N, trigeminal nucleus; 7N, facial nucleus; 7n, facial nerve;
12N, hypoglossal nucleus; CnF, cuneiform nucleus; cp, cerebral peduncle;
FF, fields of Forel; fr, fasciculus retroflexus; g7, genu of the facial nerve; Gi,

gigantocellular reticular nucleus; IC, inferior colliculus; IO, inferior olive; mlf,
medial longitudinal fasciculus; mRt, mesencephalic reticular formation;
PAG, periaqueductal gray; pc, posterior commissure; PRF, pontine reticularformation; py, pyramidal tract; RMg, raphe magnus nucleus; RN, red
nucleus; rs, rubrospinal tract; RtTg, nucleus reticularis tegmenti pontis;
SAI, intermediate white layer; SAP, deep white layer; scp, superior
cerebellar peduncle; SGI, intermediate gray layer; SGP, deep gray layer;
SGS, superficial gray layer; SO, optic layer; SNr, substantia nigra pars
reticulata; sp5, spinal trigeminal tract; ZID, zona incerta, dorsal part; ZIV,
zona incerta, ventral part.

to a specific cell population exists for many of the neural systems in the brain. In this regard, the present study, for the firsttime, showed that pathway-specific and reversible blockade ofsynaptic transmission worked as efficiently to cause behavioral
deficits in mice, as it has in non-human primates (Kinoshita et al.,2012). Moreover, it specifically provides clear evidence that thedirect crossed tectoreticular pathway is essential for the execution of orienting responses. Previous pharmacological techniques
were not able to dissect this pathway from other tectal output
pathways, such as the thalamic projection from the superficial
layer, which might be involved in the higher visual processing(May, 2006), and from the uncrossed tecto-reticular pathway,
which has been reported to control the avoidance behaviors(Sahibzada et al., 1986; Dean et al., 1989). However, the present
method will enable us to pin down the functions of these individual components of the output pathways from the SC in near
future.

Finally, we analyzed the methodological details of thistechnique, especially the time course of the effects of Dox-on
and Dox-off with this relatively simple behavioral system. Theseexperiments made use of a fair number of mice, which would be
difficult to perform in non-human primates.

Frontiers in Neural Circuits

TIME COURSE AFTER DOX-ON

In this study, the behavioral effects of Dox administration could
be partially observed as early as 24 h after initiation. This isearlier than observed in our previous study on macaque monkeys, in which the effects appeared on the 2nd day. This may bepartly because we used an intraperitoneal injection on the firstday to facilitate the quick onset of the effects (Perl et al., 2002).
However, a species-related difference cannot be excluded. The
effects became maximal on the 2nd and 3rd days after initiation,
but gradually became smaller and had mostly disappeared by the
6th day. It is clear that this is not due to the disappearance ofwww.frontiersin.org

October 2013 | Volume 7 | Article 162 | 8

Sooksawate et al.

Selective blockade of orienting pathway

FIGURE 7 | Schematic drawing of the time course for the emergenceand disappearance of blocking effects as measured by the behavioral
effects and detectability of anti-GFP immunoreactivity after the onset
and offset of Dox administration.

the Dox effects on the tetanus toxin production in the doubly
infected neurons because the number of GFP-positive cells did
not parallel the disappearance of the behavioral effects rather it
persisted, as long as Dox was administered continuously. Such adisappearance of the behavioral effects must instead be caused
by a compensatory mechanism in the residual pathways. This
assumption is partly supported by the observation of a contralateral postural deviation in the turning response at 2 days afterDox-off (Figure 4). It is not clear which neural systems were
responsible for such compensation, but it is likely due either tocTRNs, which were spared from double infection by vectors, orto direct corticoreticular pathways or to other unknown pathways
involved in the control of visual orienting responses. A combination of these must have taken over the function of the blockedneurons.
TIME COURSE AFTER DOX-OFF

In this study, the behavioral changes were mostly not observed
after Dox-offset following 7 days of Dox administration. We
noticed a postural deviation to the right side at 2 days after Doxoff, which might be due to a compensatory mechanism established during the Dox-on period. Histological analysis showedthat the number of GFP-positive cells gradually decreased afterDox-off, and returned to the baseline level on the 28th day afterDox-off. Furthermore, the 2nd Dox application on the 28th day
after Dox-off caused as potent behavioral effects as during the
1st Dox administration. At least a one-month interval was necessary to observe the reversibility of the Dox effect. Since thefunctional compensation for the loss of transmission through a
population of cTRNs may be caused by other compensatory neural circuits, this reversibility suggests that the mouse brain reused
the cTRNs that had been blocked, once their transmission was
restarted.
BLOCKADE OF TRANSMISSION THROUGH OTHER COLLATERALS OF
THE cTRNs

Our previous study in macaque monkeys (Kinoshita et al., 2012)
showed that anti-GFP immunohistochemistry clarified not only
the location of the cell bodies of the PNs that expressed EGFP,

Frontiers in Neural Circuits

but also their axonal trajectories. Similarly, a close examinationof histological samples after anti-GFP immunohistochemistry
revealed that although the number was small, the axons of thecTRNs could be traced from the cell bodies in the intermediate/deep layers of the right SC to their target areas. Axonsand terminals could be observed not only in the left medial
pontine reticular formation, the injection site of the NeuRet-TREEGFP.eTeNT, but also in several nuclei in the mesodiencephalicjunction, pons, and medulla, which are targets of the ascending and descending collaterals of tectoreticular neurons (Grantyn
and Grantyn, 1982; Huerta and Harting, 1982) (Figure 6). The
possibility cannot be excluded that NeuRet-TRE-EGFP.eTeNT
had diffused to the right side of the pontine reticular formationfrom the injection site, and some of these axons and terminals belonged to ipsilaterally projecting tectoreticular neurons.
However, this should be a minor occurrence. In fact, the GFPpositive neurons were found exclusively in the caudal and lateralSC, where the cTRNs are located (Figure 5A), despite the factthat injection of AAV2-CMV-rtTAV16 included more medial
parts of the SC (Figure 5B). If NeuRet-TRE-EGFP.eTeNT diffused to the right side, the GFP-positive cells should have beenfound also in more medial and rostral part of the SC where theuncrossed tectoreticular neurons are primarily located (Redgrave
et al., 1990), Furthermore, the behavioral effects were clearly
those expected from the impairment of cTRNs. In the previous
reports (Grantyn and Grantyn, 1982; Isa and Sasaki, 2002), thepontine projection of the cTRNs was described as almost exclusively contralateral. The present report suggests there may be
species differences in this regard between rodents and carnivores.
As eTeNT was fused with EGFP, it should be transported to theaxon terminals in these areas. These results indicate that synaptic transmission to these targets was also blocked, and that the
behavioral effects cannot always be ascribed only to the blockade of transmission from the SC to the medial pontine reticularformation, but also to the blockade of signal transmission to allof the target nuclei. To conclude which connection was critical
for the observed behavioral effects, another technique, such as
optogenetic blockade of synaptic transmission at the nerve terminal (Kaneda et al., 2011), will need to be combined with ourapproach.
OTHER POSSIBLE USES OF THIS TECHNIQUE

The present results showed that the successful blockade of aparticular central pathway with the double viral vector technique could result in behavioral effects in mice. Using a similar highly efficient retrograde gene transfer vector, some of
the authors of this article have produced the permanent ablation of a central pathway by using it in combination with
an immunotoxin (Inoue et al., 2012). This may represent a
good approach for creating animal disease models. However,
one of the major advantages of the present technique is its
reversibility. Basically, this technique could be applicable toany pathway in the brain. However, when we apply the current technique to other pathways in the brain, it might benecessary to use other serotypes of AAV, because efficiencyof transfection of each serotype could vary depending on thetarget cell types (Blits et al., 2010; Jakovcevski et al., 2010;www.frontiersin.org

October 2013 | Volume 7 | Article 162 | 9

Sooksawate et al.

Selective blockade of orienting pathway

Markakis et al., 2010). The success of the current method was primarily based on the following factors: (1) development of highly
efficient retrograde gene transfer using NeuRet; (2) very efficient amplification of gene expression by the recently developed
Tet-ON sequence rtTAV16; and (3) very effective expression of
the humanized tetanus neurotoxin eTeNT. Replacing eTeNT withother functional proteins, such as light-sensitive opsins for optogenetic control of neural activity (for reviews see Yizhar et al.,2011; Tye and Disseroth, 2012), may open up a novel direction
for neural circuit analysis.

ACKNOWLEDGMENTS

REFERENCES

Kato, S., Inoue, K., Kobayashi, K.,
Yasoshima, Y., Miyachi, S., Inoue,S., et al. (2007). Efficient genetransfer via retrograde transport inrodent and primate brains using
a human immunodeficiency virus
type 1-based vector pseudotyped
with rabies virus glycoprotein.
Hum. Gene. Ther. 18, 1141–1151.
doi: 10.1089/hum.2007.082
Kato, S., Kobayashi, K., Inoue, K.,
Kuramochi, M., Okada, T.,
Yaginuma, H., et al. (2011a).
A lentiviral strategy for highly
efficient retrograde gene transfer by pseudotyping with fusionenvelope
glycoprotein.
Hum.
Gene Ther. 22, 197–206. doi:
10.1089/hum.2009.179
Kato, S., Kuramochi, M., Takasumi, K.,
Kobayashi, K., Inoue, K., Takahara,D., et al. (2011b). Neuron-specificgene transfer through retrograde
transport of lentiviral vector pseudotyped with a novel type of
fusion envelope glycoprotein. Hum.
Gene Ther. 22, 1511–1523. doi:
10.1089/hum.2011.111
Kawamura, K., and Brodal, A. (1973).
The tectopontine projection in thecat: an experimental anatomical
study with comments on pathways for teleceptive impulses tothe cerebellum. J. Comp. Neurol.
149, 371–390. doi: 10.1002/cne.9014
90306
Kinoshita, M., Matsui, R., Kato, S.,
Hasegawa, T., Kasahara, H., Isa, K.,
et al. (2012). Genetic dissection of
the circuit for hand dexterity inprimate. Nature 487, 235–238. doi:
10.1038/nature11206
Kobayashi, K., Morita, S., Sawada, H.,
Mizuguchi, T., Yamada, K., Nagatsu,
I., et al. (1995). Immunotoxinmediated conditional disruptionof specific neurons in transgenic mice. Proc. Natl. Acad.
Sci. U.S.A. 92, 1132–1136. doi:
10.1073/pnas.92.4.1132
Markakis, E. A., Vives, K. P., Bober, J.,
Leichtle, S., Leranth, C., Beecham,J., et al. (2010). Comparative transduction efficiency of AAV vector serotypes 1-6 in the substantia

Blits, B., Derks, S., Twisk, J., Ehlert,
E., Prins, J., and Verhaagen, J.
(2010). Adeno-associated viral vector (AAV)-mediated gene transfer in the red nucleus of theadult rat brain: comparative analysis of the transduction propertiesof seven AAV serotypes and lentiviral vectors. J. Neurosci. Methods 185,
257–263. doi: 10.1016/j.jneumeth.
2009.10.009
Corneil, B. D., Olivier, E., and Munoz,
D. P. (2002). Neck muscle responses
to stimulation of monkey superior colliculus. I. Topography
and manipulation of stimulation
parameters. J. Neurophysiol. 88,
1980–1999.
Cowie, R. J., and Robinson, D. L.
(1994). Subcortical contributions
to head movements in macaques.
I. Contrasting effects of electrical stimulation of a medial pontomedullary region and the superior colliculus. J. Neurophysiol. 72,
2648–2664.
Dean, P., Redgrave, P., and Westby, G.
W. (1989). Event or emergency? Two
response systems in the mammaliansuperior colliculus. Trends Neurosci.
12, 137–147. doi: 10.1016/01662236(89)90052-0
Franklin, K. B. J., and Paxinos, G.
(2008). The Mouse Brain: in
Stereotaxic Coordinates. 3rd Edn.
New York, NY: Academic Press.
Grantyn, A., and Berthoz, A. (1987).
Reticulo-spinal neurons participating in the control of synergic eye andhead movements during orienting
in the cat. I. Behavioral properties.
Exp. Brain Res. 66, 339–354. doi:
10.1007/BF00243309
Grantyn, A., and Grantyn, R. (1982).
Axonal patterns and sites oftermination of cat superior colliculus neurons projecting in thetecto-bulbo-spinal
tract.
Exp.
Brain Res. 46, 243–256. doi:
10.1007/BF00237182
Huerta, M. F., and Harting, J. K.
(1982). Tectal control of spinal cord
activity: neuroanatomical demonstration of pathways connecting the

Frontiers in Neural Circuits

superior colliculus with the cervical spinal cord grey. Prog. Brain Res.
57, 293–328. doi: 10.1016/S00796123(08)64135-7
Inoue, K., Koketsu, D., Kato, S.,
Kobayashi, K., Nambu, A., andTakada, M. (2012). Immunotoxinmediated tract targeting in
the primate brain: selective
elimination of the corticosubthalamic“hyperdirect” pathway.
PLoS ONE 7:e39149. doi: 10.1371/
journal.pone.0039149
Isa, T., and Hall, W. C. (2009).
Exploring the superior colliculus in vitro. J. Neurophysiol. 102,2581–2593. doi: 10.1152/jn.00498.
2009
Isa, T., and Naito, K. (1995). Activityof neurons in the medial pontomedullary reticular formation
during orienting movements inalert head-free cats. J. Neurophysiol.
74, 73–95.
Isa, T., and Sasaki, S. (1988). Effectsof lesion of paramedian pontomedullary reticular formation
by kainic acid injection on thevisually triggered horizontal orienting movements in the cat.
Neurosci. Lett. 87, 233–239. doi:
10.1016/0304-3940(88)90454-5
Isa, T., and Sasaki, S. (2002). Brainstem
control of head movements during orienting; organization of the
premotor circuits. Prog. Neurobiol.
66, 205–241. doi: 10.1016/S03010082(02)00006-0
Jakovcevski, M., Guo, Y., Su, Q.,
Gao, G., and Akbarian, S. (2010).
rAAV9–a human-derived adenoassociated virus vector for efficienttransgene expression in mouse
cingulate cortex. Cold Spring
Harb. Protoc. 4, pdb.prot5417. doi:
10.1101/pdb.prot5417
Kaneda, K., Kasahara, H., Matsui,R., Katoh, T., Mizukami, H.,
Ozawa, K., et al. (2011). Selective
optical control of synaptic transmission in the subcortical visual
pathway by activation of viral
vector-expressed
halorhodopsin.
PLoSONE
6:e18452.
doi:
10.1371/journal.pone.0018452

This study is the result of “Highly Creative Animal Model
Development for Brain Sciences” carried out under the Strategic
Research Program for Brain Sciences (to Dai Watanabe, Kazuto
Kobayashi, and Tadashi Isa) and a Grant-in-Aid for Scientific
Research on Innovative Areas “Mesoscopic Neurocircuitry” (No.
25115731 to Tadashi Isa) by the Ministry of Education, Culture,
Sports, Science, and Technology of Japan. We thank Kotomi
Shimizu for assistance with immunohistochemistry and in situ
hybridization.

www.frontiersin.org

nigra and striatum of the primate
brain. Mol. Ther. 18, 588–593. doi:
10.1038/mt.2009.286
May, P. J. (2006). The mammaliansuperior
colliculus:
laminarstructure and connections. Prog.
Brain Res. 151, 321–378. doi:
10.1016/S0079-6123(05)51011-2
Metz, G. A., and Schwab, M. E. (2004).
Behavioral characterization in acomprehensive mouse test battery reveals motor and sensory
impairments in growth-associatedprotein-43 null mutant mice.
Neuroscience. 129, 563–574. doi:
10.1016/j.neuroscience.2004.07.053
Montecucco, C., and Schiavo, G.
(1994). Mechanism of action oftetanus and botulinum neurotoxins. Mol. Microbiol. 13, 1–8.
doi:
10.1111/j.1365-2958.1994.
tb00396.x
Perl, A.-K. T., Tichelaar, J. W., andWhitsett, J. A. (2002). Condition
gene expression in the respiratory
epithelium of mouse. Transgenic
Res. 11, 21–29. doi: 10.1023/A:101
3986627504
Pinto, L. H., and Enroth-Cugell,C. (2000). Tests of the mouse
visual system. Mamm. Genome.
11, 531–536. doi: 10.1007/s003350
010102
Quaia, C., Aizawa, H., Optican, L.
M., and Wurtz, R. H. (1998).
Reversible inactivation of monkey
superior colliculus. II. Maps of saccadic deficits. J. Neurophysiol. 79,
2097–2110.
Redgrave, P., Dean, P., and Westby,
G. W. (1990). Organization of thecrossed tecto-reticulo-spinal projection in rat - I. Anatomical evidence for separate output channels to the periabducens area andcaudal medulla. Neuroscience 37,
571–584. doi: 10.1016/0306-4522(90)90092-I
Rosenquist, A. C., Ciaramitaro, V. M.,
Durmer, J. S., Wallace, S. F., andTodd, W. E. (1996). Ibotenic acidlesions of the superior colliculus
produce longer lasting deficits invisual orienting behavior thanaspiration lesions in the cat. Prog.

October 2013 | Volume 7 | Article 162 | 10

Sooksawate et al.

Brain Res. 112, 117–130. doi:
10.1016/S0079-6123(08)63324-5
Sahibzada, N., Dean, P., and Redgrave,P. (1986). Movements resemblingorientation or avoidance elicited byelectrical stimulation of the superior colliculus in rats. J. Neurosci. 6,
723–733.
Smith, D. R., Striplin, C. D., Geller,
A. M., Mailman, R. B., Drago,
J., Lawler, C. P., et al. (1998).
Behaviouralassessment
of
mice lacking D1A dopamine
receptors.
Neuroscience86,
135–146. doi: 10.1016/S0306-4522(97)00608-8
Sooksawate, T., Isa, K., and Isa, T.
(2008). Cholinergic responses
in crossed tecto-reticular neurons of rat superior colliculus.
J. Neurophysiol. 100, 2702–2711.
doi: 10.1152/jn.90723.2008
Sooksawate, T., Saito, Y., and Isa,T. (2005). Electrophysiologicaland morphological properties ofidentified cross tecto-reticular neurons in the rat superior colliculus.

Frontiers in Neural Circuits

Selective blockade of orienting pathway

Neurosci. Res. 52, 174–184. doi:
10.1152/jn.90723.2008
Sparks, D. L. (1975). Response
properties of eye movementrelated neurons in the monkey
superior colliculus. Brain Res. 90,
147–152. doi: 10.1016/0006-8993(75)90690-3
Sparks, D. L. (1978). Functional properties of neurons in the monkey superior colliculus: couplingof neuronal activity and saccade
onset. Brain Res. 156, 1–16. doi:
10.1016/0006-8993(78)90075-6
Sparks, D. L. (1986). Translationof sensory signals into commands for control of saccadic
eye movements: role of primate
superior colliculus. Physiol. Rev. 66,
118–171.
Tye, K. M., and Disseroth, K. (2012).
Optogenetic investigation of neuralcircuits underlying brain disease inanimal models. Nat. Rev. Neurosci.
13, 251–266. doi: 10.1038/nrn3171
Watanabe, D., Inokawa, H., Hashimoto,K., Suzuki, N., Kano, M.,

Shigemoto, R., et al. (1998).
Ablation of cerebellar Golgi cells
disrupts
synaptic
integration
involving GABA inhibition and
NMDA receptor activation in
motor coordination. Cell 95,
17–27. doi: 10.1016/S0092-8674(00)81779-1
Wurtz, R. H., and Albano, J. E. (1980).
Visual-motor function of the primate superior colliculus. Annu. Rev.
Neurosci. 3, 189–226. doi: 10.1146/
annurev.ne.03.030180.001201
Yizhar, O., Fenno, L. E., Davidson, T.
J., Mogri, M., and Deisseroth,K. (2011). Optogenetics inneural systems. Neuron 71,
9–34.
doi:
10.1016/j.neuron.
2011.06.004

Conflict of Interest Statement: The
authors declare that the research
was conducted in the absence of any
commercial or financial relationshipsthat could be construed as a potential
conflict of interest.

www.frontiersin.org

Received: 01 July 2013; accepted: 21
September 2013; published online: 11
October 2013.
Citation: Sooksawate T, Isa K, Matsui
R, Kato S, Kinoshita M, Kobayashi K,
Watanabe D, Kobayashi K and Isa T(2013) Viral vector-mediated selective
and reversible blockade of the pathway for visual orienting in mice. Front.
Neural Circuits 7:162. doi: 10.3389/fncir.
2013.00162
This article was submitted to the journal
Frontiers in Neural Circuits.
Copyright © 2013 Sooksawate, Isa,
Matsui, Kato, Kinoshita, Kobayashi,
Watanabe, Kobayashi and Isa. This is
an open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in otherforums is permitted, provided the original author(s) or licensor are credited andthat the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which does
not comply with these terms.

October 2013 | Volume 7 | Article 162 | 11