LETTER

doi:10.1038/nature11206

Genetic dissection of the circuit for hand dexterity
in primates
Masaharu Kinoshita1, Ryosuke Matsui2, Shigeki Kato3, Taku Hasegawa2, Hironori Kasahara2, Kaoru Isa1, Akiya Watakabe4,5,
Tetsuo Yamamori4,5, Yukio Nishimura1,5,6, Bror Alstermark7, Dai Watanabe2, Kazuto Kobayashi3 & Tadashi Isa1,5gene transfer (HiRet) lentiviral vector11 carrying enhanced tetanus
neurotoxin light chain (eTeNT)12 (Supplementary Fig. 1) and theenhanced GFP (EGFP) downstream of the tetracycline-responsive
element (TRE)13; and second, the adeno-associated virus serotype 2(AAV2) vector carrying the Tet-on sequence, a variant of reverse
tetracycline transactivator (rtTAV16)14 (see Methods) under thecontrol of the cytomegalovirus (CMV) promoter.
a
HiRet-TRE-EGFP.eTeNTLTR psiRRE

HiRet-TRE-EGFP
LTR psiRRE

CMV

AAV2-CMV-rtTAV16

TRE

EGFP

TRE

EGFP

eTeNT

cPPT

PEST

cPPT

WPRE

L-ITR

WPRE

LTR

LTR

1 kb

rtTAV16
WPRE

SV40 R-ITR
pAb

Double infected PNs
in C3–C5

Cortex
AAV

CMV

Dox

rtTAV16

C3–C5

rtTAV16 Dox

PNs
TRE

eTeNT

Retrograde
transport

eTeNT

C6–T1

It is generally accepted that the direct connection from the motor
cortex to spinal motor neurons is responsible for dexterous hand
movements in primates1–3. However, the role of the ‘phylogeneticallyolder’ indirect pathways from the motor cortex to motor neurons,
mediated by spinal interneurons, remains elusive. Here we used anovel double-infection technique to interrupt the transmission
through the propriospinal neurons (PNs)4–6, which act as a relayof the indirect pathway in macaque monkeys (Macaca fuscata andMacaca mulatta). The PNs were double infected by injection of ahighly efficient retrograde gene-transfer vector into their target areaand subsequent injection of adeno-associated viral vector at thelocation of cell somata. This method enabled reversible expressionof green fluorescent protein (GFP)-tagged tetanus neurotoxin,
thereby permitting the selective and temporal blockade of the motor
cortex–PN–motor neuron pathway. This treatment impaired reachand grasp movements, revealing a critical role for the PN-mediatedpathway in the control of hand dexterity. Anti-GFP immunohistochemistry visualized the cell bodies and axonal trajectories of theblocked PNs, which confirmed their anatomical connection to
motor neurons. This pathway-selective and reversible techniquefor blocking neural transmission does not depend on cell-specificpromoters or transgenic techniques, and is a new and powerful tool
for functional dissection in system-level neuroscience studies.
Lesion studies at the level of the brainstem1 in macaque monkeys
showed that the performance of highly fractionated finger movements
primarily depends on the pyramidal tract. Later studies proposed thatthe direct corticomotoneuronal connection (the monosynaptic
connection from cortical neurons to motor neurons)2,3, which is
unique to higher primates, is the basis for dexterous hand movements.
In less dexterous animals such as cats, the corticospinal tract (CST) is
not directly connected to hand–arm motor neurons. In these animals,
the shortest pathway from the cortex to motor neurons is disynaptic,
mainly involving spinal interneurons located either in the same
segments as motor neurons (caudal to C6; segmental interneurons)
or in more rostral segments (mainly in C3–C5; PNs)4. Evidence thatthe non-monosynaptic pathway is conserved in monkeys was provided recently5,6. Contribution of this phylogenetically older PNmediated pathway has been assessed in behavioural experiments that
determined the effects of specific lesions of the CST at the C5 and C2
segments, thereby leaving the PN axons intact7–10. The results indicated that the PN-mediated pathway could control independent finger
movements. However, because the lesions were irreversible, it ispossible to consider that the indirect pathway was responsible forthe recovery of function, rather than contributing to hand dexterityin the intact state3. This problem could be solved if it is possible toblock the PNs specifically and temporarily. For this purpose, we combined two viral vectors for the selective and reversible blockade of the
PN-mediated pathway (Fig. 1); first, the highly efficient retrograde

MNs
eTeNT

HiRet

VAMP2

Musclec

MNs in C6–T1

HiRet injection
AAV injection
Dox administration
Dox1
7–10 1–2
Days Month

6–9
Days

Terminal experiment

Dox2
23–33
Days

Dox3

Behavioural analysis

Figure 1 | The pathway-specific and reversible blockade of synaptictransmission. a, The design of viral vectors. b, Schematic diagrams of vector
injections and how HiRet-TRE-EGFP.eTeNT and AAV2-CMV-rtTAV16interact in the double-infected cells. c, The experimental schedule. cPPT,
central polypurine tract; Dox, doxycycline; L- and R-ITR, left and right inverted
terminal repeat; LTR, long terminal repeat; MNs, motor neurons; RRE, Rev
responsive element; PEST, PEST sequence; psi, packaging signal; VAMP2,
vesicle-associated membrane protein 2 sequence; WPRE, Woodchuck hepatitis
virus post-transcriptional regulatory element.

1

Department of Developmental Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan. 2Department of Molecular and System Biology, Graduate School of Biostudies,
Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. 3Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima 960-1295, Japan.
4
Division of Brain Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan. 5The Graduate University for Advanced Studies (Sokendai), Hayama, Kanagawa 240-0193, Japan. 6Precursory
Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Chiyoda, Tokyo 102-0076, Japan. 7Department of Integrative Medical Biology, section of
Physiology, Umeå University, S-901 87 Umeå, Sweden.
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RESEARCH LETTER

0

0.13

0.53

e

0.27

Monkey H

00.83

Monkey K

Monkey Ha

Dox1 (Day +2) Pre (Day 0)

HiRet-TRE-EGFP.eTeNT was injected into the region containing
motor neurons targeted by the PNs (that is, the ventral horn of the C6–
T1 segments (T1 is the first thoracic segment)), and AAV2-CMVrtTAV16 was injected 7 to 10 days later, into the intermediate zoneof the caudal C2 to rostral C5 segments where their cell bodies arelocated (Supplementary Table 1). Thus, PNs with cell bodies in the
mid-cervical segments and with axons that contact the hand–arm
motor neurons would be double infected. During administration of
doxycycline, expression of eTeNT depresses synaptic transmission in
the PNs selectively and spares other inputs to the motor neurons.
We tested four monkeys (H, K, B and N) with double injection of
HiRet-TRE-EGFP.eTeNT and AAV2-CMV-rtTAV16, and one additional monkey (S) was injected with HiRet-TRE-EGFP, which lacks theeTeNT of HiRet-TRE-EGFP.eTeNT (Fig. 1a), and used as a control.
One to two months after the injections, the oral administration of
doxycycline was initiated to induce the expression of eTeNT. Two to
five days after beginning administration, all four monkeys transfectedwith the eTeNT gene had profound deficits in reach and grasp movements. Typically, their movements were slowed with increased errors
in precision grip and/or reach (Fig. 2e). Monkeys H and N showed
deficits mainly in precision grip, in which thumb extension was particularly problematic (Fig. 2a, b and Supplementary Movies 1 and 4).
Conversely, monkeys B and K had deficits in reach; their hands often
hit the edge of the apparatus before reaching the food piece (Fig. 2c, d,and Supplementary Movies 2, 3 and 5). Hand movements in thecontrol animal, monkey S, were not affected.
Despite the continued administration of doxycycline, impairmentof the reach and grasp movements resolved over a period of 2 to 3 days.
On day 6 to 8 of doxycycline administration, when the behaviouralb
Monkey H
Day 0

deficits disappeared, the blood concentration of doxycycline remained
high (Supplementary Fig. 2) and transmission through the PNs was
still largely blocked as revealed by electrophysiological experiments(see below; Fig. 3). These results indicated that the expression ofeTeNT did not abate during doxycycline administration. Taken
together, the results show that intact neural systems, including thedirect corticomotoneuronal pathway or other indirect pathways(see the circuit diagram in Fig. 3a), would have compensated for theloss of control by the blocked PNs. When doxycycline administration
was terminated and re-started 3 to 4 weeks later, all animals again
showed deficits in reach and grasp movements. This indicates thatthe compensation by other pathways was only temporary and that
the PNs recovered their control during the doxycycline-off period.
After behavioural testing was completed, electrophysiologicalexperiments were performed to confirm the blockade of synaptictransmission through PNs in monkeys B, N and S. Stimulation of
the contralateral medullary pyramid at 200 mA induced monosynaptic
excitatory postsynaptic potentials (EPSPs) in motor neurons on bothsides of the spinal cord (Supplementary Fig. 3b). The amplitudes of theevoked monosynaptic field potentials in the deep radial motor nucleus
were not significantly different between the two sides (Fig. 3b, onetailed t-test; P 5 0.34 and 0.71, in monkeys B and N, respectively).
These data indicate that the vectors did not adversely affect the direct
corticomotoneuronal pathway. Indirect input to motor neurons
mediated by the PNs was assessed by recording the disynaptic negative
field potentials in the deep radial motor nucleus, which had segmental
latencies in the range of 0.9–1.9 ms. This component remained after
CST lesioning at the C5 level, but was almost completely abolishedafter C2 lesioning (Fig. 3c, d and Supplementary Fig. 4). This indicates

Dox1

(%)
80

Dox2
(s)
1

40

0.6
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–2 0

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Monkey B

1 cm

Monkey N

Monkey K

Dox3 (Day +3) Pre (Day –2)

c

0.30

d
Monkey S(control)Monkey K
Day –2
Monkey K
Day +2,+3
1 cm

0.2
80
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40

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Monkey H
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–2 0

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–4 –2 0

2

4

Figure 2 | Reversible impairment of reach and grasp movements during
doxycycline administration. a–d, An example of reach and grasp movements
before (Pre) and 2 days after doxycycline administration in monkey H (a). The
monkey showed ‘precision grip error’ (Supplementary Movie 1). The first
frame shows the initial contact of the index finger with the food piece and thesecond frame shows the first contact of the thumb with the food. The time fromthe first frame (in s) is indicated in each panel. Stick diagrams of reach and grasp
movements of monkey H are shown in b. The index finger and wrist joint are
shown in blue and the thumb in red, and five trials are superimposed. Reach

6

8

0.2

0.2
1

40

0.6
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0.2

0.6
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40

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–4 –2 0

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0.2

Error rate (%)> mean + 2.58 s.d. (99%)> mean + 1.96 s.d. (95%)
Movement
time (s)P < 0.001
P < 0.01
Days from startof Dox administration
Day of Dox administrationand grasp movement before and 3 days after the doxycycline administration inmonkey K, ‘slit-hit error’ (c) (Supplementary Movie 2). Stick diagrams of reachand grasp movements of monkey H are shown in d. Five trials aresuperimposed, and colours are as in b. The left panels of b and d correspond to
the timing of the first frame in a and c, respectively. The right panels correspondto the second frame in a and c, respectively. e, Movement time (blue) and error
rate (red) before and during doxycycline administration in all monkeys. The
means (thick lines) and s.d. (thin vertical lines) of 30–100 trials are shown.

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LETTER RESEARCH

Intact

1 ms
–

CDP

RSNs
C2 lesion

MNs+

Monkey N

Affected

IntactIntact1 ms

Monkey S(control)Affected

1.5

1.5

**
**

First stim.
Third stim.

1 ms–

Monkey B
Intact1.0

AffectedsINs
Field rec.

1 mV

Affected
CDP
Field

C5 lesion

(mV)
1.5

3 ms

+

PNs

d

Pyr 200 μA × 3
C5DLF lesion

0.2 mV

Intact

1 mV

Field

Pyr
stim.

c

Pyr 200 μA × 1
No lesion

0.2 mVbCortexAffected

a

1.0

0.5

1.0
**

**
0.5

0
Lesion No C5 C2 No C5(n) (4) (10) (4) (3) (14)

0.5

0

0
No C5 C2 No C5

No C5 C2(6) (9) (7) (6) (6)(6) (3) (5)Figure 3 | Blockade of synaptic transmission through the PNs. a, The
experimental arrangement. In addition to the direct corticomotoneuronalconnection, there could be three possible routes of indirect pathways; throughreticulospinal neurons (RSNs), PNs and segmental interneurons (sINs).
During the experiments, the dorsolateral funiculus (DLF) was transectedsuccessively at the C5 and C2 levels (Supplementary Methods). Dashed lines,
transected axons. Pyr, contralateral medullary pyramid. Arrowheads,
presynaptic terminals. b, Monosynaptic field potentials (grey arrowheads) inthe deep radial motor nucleus (top panel), and cord dorsum potential (CDP;

bottom panel) on the intact and affected sides. c, Disynaptic field potentials
evoked by triple stimulation of the contralateral medullary pyramid at 200 mA.
Full records (top panel) and superimposed records of the expanded view of the
responses following the first and third stimuli (bottom panel). d, Quantitative
analysis of the disynaptic field potentials with no lesion, after CST lesioning at
C5 and C2 in monkeys B, N and S. Means and s.d. values are also shown. P
values showing a statistical difference are marked with asterisks (**, P , 0.01).
The number of records are shown below.

that the field potential observed after C5 lesioning was mostly
mediated by the PNs. On the affected sides of monkeys B and N, thedisynaptic field potential became much smaller after the C5 lesioning(13% in monkey B and 6.8% in monkey N) compared to the intact side
(Fig. 3c, d). In contrast, the disynaptic field potential on the injected sideof the sham control (monkey S) showed similar amplitudes to the intact
side of the other animals (Fig. 3d). These results confirmed that neuraltransmission by the majority of PNs (90%) had been blocked on theaffected side. Corroborating results were observed from intracellular
recordings in motor neurons (Supplementary Fig. 3). Finally, stimulation of the lateral reticular nucleus (LRN) in the brainstem induces

the monosynaptic excitation of motor neurons that is mediated byaxon reflex of the PNs6,15 (Supplementary Fig. 5a). The results of LRN
stimulation also indicated that the PN-mediated transmission was
severely affected (Supplementary Figs 5b, c and 6).
Because EGFP was tagged to eTeNT, the additional expression of
EGFP provided neuroanatomical confirmation that the neurons affected by eTeNT were PNs (Fig. 4a). Neurons marked with anti-GFP
immunohistochemistry were concentrated in the lateral part oflamina VII and partly in laminae V, VI and VIII (Fig. 4b, c, e andSupplementary Fig. 7). This fits well with the location of previously
reported PNs in cats16,17 and monkeys6. In situ hybridization for thetTA sequence revealed that the AAV vector had infected many neurons
in a wide area of the grey matter on the injected side (Fig. 4d, f).
The GFP-positive cells comprised 1–2% of AAV2-CMV-rtTAV16infected cells (Supplementary Table 2). They were most abundant in
the C4 to rostral C5 segments and decreased in number at the C3
segment (Fig. 4f). Again, this distribution fits well with the distributionof the PNs reported in cats17. The total number of GFP-positive cells inmonkeys K, B, N and S was in the range of 865–2,760. This number
was smaller (n 5 294) in monkey H, presumably reflecting its relatively short survival time (Supplementary Table 2). Interestingly, antiGFP immunohistochemistry labelled not only the PN cell bodies but
also their axons, particularly in monkeys N and S. In these animals,
labelled axons were traced to the motor nuclei in the C6–T1 segments

a

d

rtTA

e

b

GFP

I–III
IV

V
VI
VII

X

VIII

10,0005,000

200Lamina

g

T1

C3

h

C4

C5

5 mm

rtTA+ cells per 400 μm

I–III IV V VI VII VIII IX X

0

0

100

50

f

0

GFP+ cells (%)

Monkey K

GFP+ cells per 400 μm

IX

C4

c 100

Figure 4 | Visualization of the blocked PNs. a, An example of GFP-positive
cells. Scale bar, 100 mm. b, c, Distribution of GFP-positive cells in the C4
segment of monkey K (b) and their numbers in individual layers of Rexed(c). I–X, laminae. d, e, In situ hybridization and anti-GFP
immunohistochemistry of the C4 segment of monkey K, which shows the
AAV2-CMV-rtTAV16-infected neurons in dark blue (d) and GFP-positive
cells shown in black by anti-GFP immunohistochemistry in an adjacent section
counterstained with Neutral Red (e). Arrows, centre of the injection site. Leftpanel, scale bars are 1 mm. Right panel, scale bars are 500 mm. f, Longitudinal
distribution of the GFP-positive cells (green columns). Blue lines show the
distribution of the rtTA-positive cells. Black arrowheads, the rostral and caudal
ends of the AAV2-CMV-rtTAV16 injections. g, Camera lucida drawings of
GFP-positive axons and terminals at the level of the motor nucleus at the T1
segment in monkey N. Scale bar, 1 mm. h, Photomicrographs of axons andterminals (black) in the motor nucleus. The sections were counterstained with
Neutral Red. Scale bar, 50 mm.
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RESEARCH LETTER(Fig. 4g, h) and in the LRN (Supplementary Fig. 8b, c) showing that the
injected vectors were effectively directed to the PNs.
Thus, we have succeeded in the pathway-selective and reversible
blockade of synaptic transmission in primates by using double infectionof viral vectors. Blockade of synaptic transmission through the PNs
caused substantial, albeit temporary disruption of hand reach and ofgrasp dexterity. It has been proposed previously that a major advantageof the direct corticomotoneuronal connection for the control of independent finger movements is that the direct corticomotoneuronal connection bypasses these spinal interneurons, which were considered tohave too widespread connections with motor neurons to control thefractionated finger movements2. However, our results indicate strongly
that the PN-mediated indirect pathway is required for the control ofhand dexterity in the intact state. The current technique is a notable
extension of the permanent pathway-selective lesioning of thethalamostriatal pathway in mice reported recently18. This method
can be used as a powerful tool for functional dissection of specificcentral pathways whose cell-specific promoters12,19 are not known.
This is particularly important in species such as primates, for whichmaking transgenic animals is more difficult (compared with mice, forexample) and for which no successful example of behaviouralmodification with optogenetics has been reported20–22.

METHODS SUMMARY
Five macaque monkeys were used in the present study. The experimental protocols
followed the principles of the National Institutes of Health, and the Ministry of
Education, Culture, Sports, Science and Technology of Japan, and were approvedby the Institutional Animal Care and Use Committee of the National Institutes of
Natural Sciences, Japan. The monkeys were trained to perform reach and grasp
movements in their home cage. Under anaesthesia, HiRet-TRE-EGFP.eTeNT was
injected into the ventral horn of the C6–T1 spinal segments (16–19 tracks at 1-mm
intervals, 0.5 ml vector solution in each track) in four monkeys (H, K, B and N). In
one monkey (S), HiRet-TRE-EGFP was injected as a control. Seven to ten days
later, the AAV2 vector carrying the rtTAV16 sequence was injected into theintermediate zone of the C2–C5 segments (8–13 tracks at 1-mm intervals, 0.5 ml
vector solution in each track). One to two months later, the oral administration of
doxycycline was initiated and continued for 6 to 9 days. Doxycycline administration was repeated for up to 3 times at 3- to 4-week intervals. Reach and grasp
movements were tested before, during and after each administration period. On
the last day of administration, electrophysiological experiments to evaluate transmission through the PNs were performed under anaesthesia and artificial ventilation. Under deep anaesthesia with sodium pentobarbital (50–100 mg kg21), all themonkeys were transcardially perfused and histological examinations were performed to visualize the blocked PNs using anti-GFP immunohistochemistry,
and the AAV-infected cells were assessed using in situ hybridization for rtTA.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 14 December 2011; accepted 1 May 2012.
Published online 17 June 2012.
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Alstermark, B., Isa, T., Ohki, Y. & Saito, Y. Disynaptic pyramidal excitation in forelimb
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C3–C4 segments mediating disynaptic pyramidal excitation to forelimb
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dexterity after spinal cord injury. Science 318, 1150–1155 (2007).
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12. Yamamoto, M. et al. Reversible suppression of glutamatergic neurotransmission of
cerebellar granule cells in vivo by genetically manipulated expression of tetanus
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system for regulated gene expression through viral evolution. Gene Ther. 13,
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15. Alstermark, B., Lindström, S., Lundberg, A. & Sybirska, E. Integration in descending
motor pathways controlling the forelimb in the cat. 8. Ascending projection to thelateral reticular nucleus from C3–C4 propriospinal also projecting to forelimb
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank S. Nakanishi, H. Jingami and C. Akazawa for continuous
encouragement. We thank P. Redgrave for comments on the earlier version of themanuscript. This study was supported by the Strategic Research Program for Brain
Sciences by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)
of Japan. B.A. was supported by the Swedish Research Council. We thank T. Oishi forproviding macaque brain sample tissue. We thank P. Phongphanphanee, M. Togawa,Y. Yamanishi, T. Katoh, K. Shimizu, N. Takahashi and K. Takada for technical support.
Author Contributions M.K., D.W., K.K., B.A. and T.I. designed the experiments and wrote
the paper. M.K., B.A., K.I. and T.I. conducted the surgery, behavioural,electrophysiological and histological experiments and analysed the data. R.M., T.H.,
S.K., H.K., D.W. and K.K. made the viral vectors. A.W. and T.Y. contributed to thehistological processing. Y.N. contributed to behavioural analysis andelectrophysiological experiments.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should be
addressed to T.I. (tisa@nips.ac.jp).

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LETTER RESEARCHMETHODS
Five macaque monkeys (three Macaca fuscata and two Macaca mulatta, bodyweight 3.1–6.0 kg) were used in the present study. The experimental protocols
followed the guideline of the National Institutes of Health, and the Ministry of
Education, Culture, Sports, Science and Technology (MEXT) of Japan, and wereapproved by the Institutional Animal Care and Use Committee of National
Institutes of Natural Sciences. The time course of the experiments on individual
animals is shown in Fig. 1c and Supplementary Table 1.
Immunoblot analysis. The soluble NSF attachment protein receptor (SNARE)
cDNAs (VAMP2, SNAP25 and SNAP23) were cloned from a macaque brain(Macaca fuscata) and subcloned into pCI (Promega) with an amino-terminal
Flag-tag sequence (to generate pVAMP2, pSNAP25 and pSNAP23, respectively).
Expression plasmids of wild-type TeNT and codon-optimized modified TeNTs(eTeNT and EGFP.eTeNT.PEST) contained the CMV. For Tet-on inducible
expression of eTeNT, an rtTA2 expression plasmid containing the CMV(prtTA2) and pLV-TRE-EGFP.eTeNT.PEST was used. Forty-eight hours after
transfection, the cells were collected. Immunoblots were performed using a mouseanti-Flag M2 monoclonal antibody (F1804, Sigma-Aldrich) and visualized with ahorseradish peroxidase-conjugated sheep anti-mouse immunoglobulin-G (IgG)
antibody (NA931V, GE Healthcare) (Supplementary Fig. 1).
Vector preparation. The highly efficient retrograde gene transfer (HiRet) vector
is a pseudotype of a human immunodeficiency-virus-type-1-based lentiviral
vector with fusion glycoprotein B type, which is composed of the extracellular
and transmembrane domains of rabies virus glycoprotein and the cytoplasmic
domain of vesicular stomatitis virus glycoprotein11,18. In the present study, theenvelope plasmid encoding fusion glycoprotein B2 type under the control of thecytomegalovirus enhancer and chicken b-actin promoter18 was used for vector
production. Chimaeric EGFP.eTeNT.PEST was generated by fusing the humancodon-optimized eTeNT with the EGFP cDNA of the pEGFP-N1 vector(Clontech) and the PEST sequence of ornithine decarboxylase, as reported previously12. The transfer plasmid pLV-TRE-EGFP.eTeNT.PEST was based thepFUGW23 (a gift from D. Baltimore) and constructed by swapping the ubiquitin
promoter-EGFP sequence with the tetracycline responsive element (TRE) of thepTRE-Tight vector (Clontech) and EGFP.eTeNT.PEST. For the control, thetransfer plasmid pLV-TRE-EGFP.PEST was constructed by removing theeTeNT sequence from pLV-TRE-EGFP.eTeNT.PEST. The HiRet vector encoding
EGFP.eTeNT or EGFP downstream of the TRE promoter (termed HiRet-TREEGFP.eTeNT or HiRet-TRE-EGFP) was prepared as described previously18.
HEK293T cells were transfected with transfer, envelope and packaging plasmids
by the calcium phosphate precipitation method. Viral vector particles were pelleted
by centrifugation at 6,000g for 16–18 h and resuspended in phosphate-bufferedsaline (PBS). The particles were then applied to a Sepharose Q FF ion-exchange
column (GE Healthcare) in PBS and eluted with a linear 0.0–1.5 M NaCl gradient.
The fractions were monitored at absorbance of 260 or 280 nm. The peak fractions
containing the particles were collected and concentrated by centrifugation througha Vivaspin filter (Vivascience). Functional titre (transduction unit) was measured
by flow cytometry (FACSCalibur, Becton Dickinson). To determine the RNA titre,viral RNA in vector preparations was isolated with a NucleoSpin RNA Virus Kit(Clontech), and the copy number of the RNA genome was determined by using a
Lenti-X qRT–PCR Titration Kit (Clontech). Polymerase chain reaction (PCR)
amplification was performed on duplicate samples by using a StepOne Real-time
PCR System (Life Technologies Corporation) under the following conditions: one
cycle of 95 uC for 3 min; and 40 cycles of 95 uC for 15 s and 54 uC for 1 min. Theselentiviral vectors were used within 2 weeks of their preparation.
The rtTA variant rtTAV16 was generated by introducing the V9I, G12S, F67S,
F86Y, R171K and A209T mutations into rtTA2S-M2 of the pTet-On advanced
vector (Clontech), as reported previously14. Plasmid pAAV2-CMV-rtTAV16 isbased on pAAV-MCS (Agilent Technologies), constructed by inserting the CMV
sequence of the pTet-On advanced vector, rtTAV16, the woodchuck hepatitis
virus post-transcriptional regulatory element (WPRE) sequence of pFUGW,
and the SV40 polyadenylation signal (SV40pA) of the pCMV-script vector(Agilent Technologies) into the MCS. The AAV vector for in vivo injection was
produced as described previously24.
To prevent adhesion of the AAV vector to the glass micropipettes, 0.001%
Pluronic-F68 Solution (Sigma-Aldrich) was added to the vector solution.
Because it was reported that the titre deteriorated by about 10% in 1 month in
this stock condition25, the vector solution was divided into small aliquots (eachcontained 10 ml of the vector solution) before being stored at 280 uC to reduce thenumber of freeze–thaw cycles. The vector solutions were used within 5 months of
their preparation.
The development of the highly efficient Tet-on transactivator, rtTAV16,
enabled the sharp onset of the effect of eTeNT transcription within 2 days, as

revealed in this study. The propriospinal (PN) blockade seemed to have occurredearlier than the compensatory process, which enabled us to observe the behavioural effects. The Tet-off system, which is commonly used as a highly sensitive
inducible system, requires 2 to 3 weeks to be effective26. Therefore, if we used the
Tet-off system, the compensatory process might have occurred in parallel to thegradual initiation of the transmission block and we would not have been able toobserve the behavioural effect.
Injection of vectors. Anaesthesia was induced by intramuscular injections ofketamine (1 mg kg21) (Daiichi Sankyo) and xylazine (1 mg kg21) (Bayer HealthCare), then by pre-medication with dexamethasone (1 mg) (Merck Sharp andDohme), atropine (0.5 mg) (Mitsubishi Tanabe Pharma) and gentamicin(10 mg) (Merck Sharp and Dohme). Under deep anaesthesia with isoflurane(1–2%) inhalation (Abbott Laboratories), the head of the monkey was fixed tothe stereotaxic apparatus and surgery was carried out on the cervical spine from thedorsal approach. During surgery, mannitol (20%, w/v) was supplemented up to a
total volume of 40 ml. Heart rate, O2 saturation, partial pressure of CO2 and bodytemperature were always monitored. In the case of the injections of HiRet-TREEGFP.eTeNT (titre, 3.9–7.5 3 1011 copies per ml) (or HiRet-TRE-EGFP; titre,
5.0 3 1011 copies per ml), laminectomy was performed on the C4–C7 vertebrae toexpose the C6–T1 segments on the left side (right side only in the case of monkey
S). A glass micropipette (with a tip diameter of 100 mm) was filled with the vector
solution. The injections were intended to be made at the point in the dorsal column
at 0.5–1.0 mm medial to the dorsal root entry zone to avoid the vessels on thesurface of the spinal cord. The pipette was inclined by 10 degrees towards the lateralside. A small hole was made on the dura with a 20-gauge injection needle and thepipette approached the spinal cord through the hole. The tip of the pipette wasinitially set at 4.5 mm from the dorsal surface of the spinal cord and then pulled up
to a depth of 4 mm and fixed. Then 0.5 ml vector solution was injected for 5 minunder the control of a microinfusion pump (Eicom) and after termination of theinjection, the pipette was fixed at the same position for another 5 min and thenpulled out. In the case of the HiRet-TRE-EGFP.eTeNT (or HiRet-TRE-EGFP)
injections, 16–19 injections were made into the ventral horn at 1-mm intervals
throughout the C6–T1 segments in four monkeys (H, K, B and N). In one monkey(S), HiRet-TRE-EGFP was injected in the same way, as a control. After injection,
the dura was covered with Sponzel (Astellas Pharma), and muscles and skins wereclosed by suturing. After the surgery, diclofenac sodium (Voltaren, Novartis) was
routinely applied to the anus for analgesia.
Seven to ten days after the HiRet-TRE-EGFP.eTeNT or HiRet-TRE-EGFP injection, AAV2-CMV-rtTAV16 (titre, 2.0 3 1013 particles per ml) was injected into theintermediate zone of the caudal C2 to rostral C5 segments. The surgical procedures
were similar to the previous surgery, except for the level of laminectomy (caudal
part of the C1 vertebra, and C2 and C3 vertebrae). The glass micropipette containing AAV2-CMV-rtTAV16 was inserted into the spinal cord perpendicularly withthe entrance at 500 mm medial to the dorsal root entry zone. The tip of the pipette
was initially inserted to a depth of 4 mm from the dorsal surface of the spinal cord
and then lifted to a depth of 3.5 mm before the AAV2-CMV-rtTAV16 was injected(0.5 ml for each site, 5 min for the injection and another 5 min leaving the pipette inposition). Injections were made at 1 mm intervals throughout the C2–C5 segments.
A total of 8–13 injections were made.
Administration of doxycycline. Doxycycline administration was initiated at 1 to
2 months after the vector injections (Fig. 1c and Supplementary Table 1).
Doxycycline was dissolved in 3% sucrose water and administered at a dose of
5–15 mg kg21 per day orally to the monkey every morning. Each period ofadministration lasted for 6 to 9 days. For measuring the blood doxycycline concentration, a blood sample (2 ml) was taken in the evening immediately before,
during and after doxycycline administration. Doxycycline concentration wasmeasured with high-performance liquid chromatography (LC-MS/MS system
6400, Agilent Technologies; an analysis service was provided by Towa
Environmental Science) as described previously27.
Behavioural tests. The five monkeys were trained to sit in their home cage andreach for pieces of sweet potato or carrot (7 3 7 3 7 mm3) through the window on
the acrylic front panel of the cage. A vertical slit of 1 cm width was set in front of themonkey and the food piece was presented between the slit (Fig. 2a, c), so that themonkey could only use its index finger and thumb to pick up the food. The
distance between the food piece and entrance edge of the slit was kept constantfor each monkey throughout the experiments. Each session consisted of 30–100
reach and grasp movements and were performed once or twice a day. The hand
movements were video filmed from the side and the movement parameters wereanalysed off-line. We measured the number of error trials in each session. We
defined the three following types of errors. A ‘precision grip error’ was judged by
the same criteria as those used in a previous report8,9 (Fig. 2a, b and Supplementary
Movie 1). If the finger touched but did not pass the edge of slit and stayed there formore than 66 ms (2 video frames), we judged it to be a ‘slit-hit error’ (Fig. 2c, d and©2012 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER
Supplementary Movie 2). If both fingers touched the food but the monkey released
it and tried to pick it up again, we judged it as a ‘wandering error’ (Supplementary
Movie 3). The error rate was calculated as the number of error trials divided by the
total number of trials in the session. For the baseline, we calculated the average ands.d. of the error rates in the sessions performed between days 24 and 0 from thestart of doxycycline administration. Day 0 was included in the baseline period,
because doxycycline administration was performed just after the behavioural test
session in the morning. Filled red circles in Fig. 2e indicate that the error rate in thesession was higher than the average plus 2.58 s.d. (representing the 99% level ofthe normal distribution) of the baseline period. Movement time was defined as theinterval between the moment when any finger touched or passed the edge of theslit, and the moment when all the fingers exited from the edge of slit. Filled blue
circles in Fig. 2e indicate that the movement times in the session were significantly
longer than those during the baseline period (days 24 to 0) (two-tailed t-test,
P , 0.001). The trials in which the monkey dropped the food piece were excluded
from the analysis of the movement time.
Electrophysiological experiments. On the last day of the final period of
doxycycline administration, synaptic transmission through the PNs was investigated with electrophysiological experiments under anaesthesia. The basic experimental design has been described previously5–7,10.
The animals were first anaesthetized with ketamine (1 mg kg21) and xylazine(1 mg kg21) and after the tracheotomy, isoflurane (1–2%) inhalation was used foranaesthesia throughout surgery. After surgery, anaesthesia was changed toa-chloralose (75–150 mg kg21) (Sigma-Aldrich). Blood pressure was maintained
at ,100 mm Hg and with a partial pressure of CO2 at ,4%. The depth ofanaesthesia was continuously monitored by checking the stable blood pressure
and lack of a pupillary reflex. A drip of Ringer’s solution with glucose and doxycycline was given during the entire experiment and the urinary bladder was
emptied regularly. Dexamethasone (1 mg) and gentamicin (10 mg) were given
just after anaesthesia. Atropine was given at intervals of 4 to 5 h. The animals wereparalysed with pancuronium bromide (1 ml, 0.2 mg ml21) (Merck Sharp andDohme) given at 30-min intervals, and artificially ventilated with a pump. A
pneumothorax was made just before electrophysiological recordings.
A craniotomy was made to expose the posterior part of the cerebellum and thecaudal brainstem in order to place the pyramidal electrode. The electrode position
was calibrated at the obex (65u from the vertical line) and placed ,2.5 mm
rostrally, 1.25 mm laterally and at a depth of ,5.0 mm from the dorsal surfaceof the brainstem. The threshold for eliciting the descending pyramidal volley was,5 mA. Monopolar cathodal pulses (0.1 ms duration) were applied using tungsten
electrodes with an impedance of ,50–100 kV and a tip diameter of 10 mm. The
stimulating electrode in the lateral reticular nucleus (LRN) was set 2 mm caudal tothe obex, 3.5 mm lateral from the midline, and at 5.5 mm from the surface ofthe brainstem, which corresponded to the mediodorsal edge of the LRN6. A
laminectomy was made at the C2–T1 segments. The deep radial nerve was stimulated with needle electrodes inserted through the skin. Intracellular recordings
from antidromically identified forelimb motor neurons (and unidentified motor
neurons) in the lateral motor nuclei of the C6–T1 segments were performed as
described previously5–7,10, and field potential recordings from the deep radial
motor nuclei were made using glass microelectrodes filled with 2 M potassium
citrate (impedance 2–5 MV). The location of the deep radial motor nucleus was
identified by the criterion that the amplitude of antidromic field potentials fromthe deep radial nerve was larger than 0.2 mV. The depths of recordings from thedorsal surface of the spinal cord ranged from 2.8 to 4.1 mm. Individual recordingsites were separated more than 200 mm from each other and randomly sampled.
The cord dorsum potential (CDP) was recorded using a silver ball electrode put on
the dorsal surface of the spinal cord in proximity to the microelectrode penetrationsite to determine the timing of the arrival of the corticospinal tract (CST) volley.
Segmental latencies of synaptic responses, that is, the latencies following thepositive peak of the direct CST volley on the CDP, indicate the time spent in
the same segment after the arrival of the fastest conducting fibres. These times
were measured to identify of the synaptic linkage of the evoked responses.
Strychnine (0.1 mg kg21, given repeatedly to maintain the excitability level)(Sigma-Aldrich) was injected intravenously at the early stage of the experiments
to reduce glycinergic inhibition, by which the disynaptic excitation of motor
neurons from the CST5,6 was unmasked. During the experiment, after recording
the monosynaptic corticomotoneuronal effects, the CST was lesioned at the C5 or
C2 level by mechanical transection of the dorsolateral funiculus (DLF) with fineforceps to localize the interneurons mediating the disynaptic excitation from the
CST, either on the segmental interneurons (sINs), PNs or reticulospinal neurons(RSNs). Completeness of the lesion was assessed by measuring the CDP responses
to contralateral medullary pyramid stimulation recorded caudally to the lesion.
Histological assessment. At the end of the experiments, the monkeys were deeply
anaesthetized with intravenous injection of sodium pentobarbital (80–100 mg kg21)(Hospira) and transcardially perfused with 0.05 M PBS and then 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brainstem and spinal cord were
cryoprotected and sectioned at a thickness of 40 mm using a cryostat. Spinal cord
tissues were examined for the distribution of EGFP-positive neurons with anti-GFP
immunohistochemistry and the distribution of AAV2-CMV-rtTAV16-infected
neurons with in situ hybridization to detect the rtTA sequence.
Anti-GFP immunohistochemistry. After observation and photographing of GFP
fluorescence using fluorescent microscopes (Axioplan 2, Zeiss; and BIOREVO,
BZ-9000, Keyence), the sections were immunohistochemically stained with a GFP
antibody28. Every three to five sections were dipped in PBS with 0.3% Titon-X(PBST) containing 5% skimmed milk at room temperature and then with a rabbit
anti-GFP antibody (1:2,000; Life Technologies) in PBST for 16 h at 4 uC. The
sections were washed in PBST and incubated in biotinylated goat anti-rabbit IgG(1:200; Vector Laboratories) and then ABC-Elite (1:200; Vector laboratories) andvisualized with diaminobenzidine (1:10,000; Wako) containing 1% Nickel sodiumammonium and 0.0003% H2O2 in Tris-buffered saline. The sections were counterstained with Neutral Red.
The number of GFP-positive cells was counted in every fifth section (eachsection was 40 mm thick) using a BZ-51 microscope (Olympus). The number ofcells in two adjacent sections was averaged to estimate the number of PNs withinevery 400 mm of the rostrocaudal extent.
In situ hybridization to visualize rtTAV16. Infection of AAV2-CMV-rtTAV16
carrying rtTAV16 was examined by in situ hybridization. This method has been
described elsewhere29–31. Briefly, free-floating sections (40 mm) were treated withproteinase K (5 mg ml21) and hybridized with the rtTA antisense probe at 60 uC
overnight. After washing, the hybridized probe was detected using an alkalinephosphatase-conjugated anti-digoxigenin antibody followed by nitro blue
tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) colorization. The plasmid for the probe was constructed by cloning the EcoRI- andBamHI-digested fragment of the lentiviral plasmid, STB32 into pBlueScriptII.
This plasmid was linearized by EcoRI and used for digoxigenin labelling of theantisense RNA probe. This probe detected the expression of both Tet-off and Teton transactivators including rtTAV16. The detailed protocol for in situ hybridization is also available on the web (http://www.nibb.ac.jp/brish/indexE.html).
The number of rtTA-positive cells was counted in every 15th section throughout
the AAV2 vector injected sites.
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©2012 Macmillan Publishers Limited. All rights reserved