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. Author manuscript; available in PMC: 2009 Oct 30.
Published in final edited form as: J Neurosci Methods. 2008 Aug 19;175(1):125–132. doi: 10.1016/j.jneumeth.2008.08.014

Transient silencing of synaptic transmitter release from specific neuronal types by recombinant tetanus toxin light chain fused to antibody variable region

Tomoko Kobayashi a,b, Nobuyuki Kai a, Kenta Kobayashi a, Tomonori Fujiwara c, Kimio Akagawa c, Masanori Onda d, Kazuto Kobayashi a,b,*
PMCID: PMC2591090  NIHMSID: NIHMS70051  PMID: 18775748

Abstract

We developed a novel strategy for conditional silencing of synaptic transmission in specific neuronal types in transgenic animals. We generated a recombinant protein termed immuno-tetanus toxin (ITet), which contains a monoclonal antibody variable region for human interleukin-2 receptor α-subunit (IL-2Rα) fused to tetanus toxin light chain. ITet was designed to transiently suppress transmitter release from target neurons genetically engineered to express human IL-2Rα via proteolytic cleavage of vesicle-associated membrane protein-2 (VAMP-2). The in vivo actions of ITet were investigated by using mutant mice that express IL-2Rα in striatal neurons under the control of the gene encoding dopamine D2 receptor. Unilateral ITet injection into the striatum induced rotational behavior in the mutant mice and the rotations gradually reversed to the normal level. The behavioral alteration was accompanied by a transient decrease in the striatal VAMP-2 level and depolarization-evoked transmitter release in synaptic target region. However, ITet injection caused no structural change in striatal cells and nerve terminals in the mutants. These data indicate that ITet acts on striatal neurons bearing human IL-2Rα and temporally reduces their VAMP-2 content, thereby causing the blockade of transmitter release. Our ITet technology provides a useful approach for inducible and reversible control of synaptic transmission in specific neuronal types in the brain.

Keywords: cell targeting, monoclonal antibody, tetanus toxin light chain, transmitter release; striatum; motor control

1. Introduction

An understanding of the neural circuit mechanisms that mediate brain functions has progressed with the development of methods that manipulate the functions of different types of neurons. One approach for this purpose is the use of immunotoxin-mediated cell targeting (IMCT), which is a transgenic technology for conditional cell ablation based on the specificity of a recombinant immunotoxin (Kobayashi et al., 1995; see Kobayashi, 2007 for a review). IMCT permits the elimination of neurons that are genetically engineered to express human interleukin-2 receptor α-subunit (IL-2Rα), a target molecule of the immunotoxin. The immunotoxin injection induces neuronal elimination in a specified brain region at a desired time. This approach has been used to study behavioral and physiological roles of a variety of neuronal types in the central nervous system (Watanabe et al., 1998; Kaneko, et al. 2000; Hikida et al., 2003; Sano et al., 2003; Yasoshima et al., 2005).

In addition, systems that genetically modulate firing activity or synaptic transmission of specific neurons provide a useful approach for functional dissection of the neural circuit (Wulff and Wisden, 2005). For instance, the use of insect G-protein-coupled receptor for allatostatin enables rapid and reversible inactivation of cortical neurons that express the receptor in response to peptide application (Lechner et al., 2002; Tan et al., 2006). However, this system requires the expression of other components (G-protein-coupled inwardly rectifying K+ channel subunits) to inactivate the target neurons. Recent advances in the technology of light–gated cation channels permit an excellent system for manipulation of neuronal activity dependent on light stimulation (Nagel et al., 2003; Zemelman et al., 2003; Arenkiel et al., 2007), but the use of this technology is limited to target neurons localized close to the brain surface.

Another possible approach for dissecting the neural circuit is the expression of tetanus toxin light chain (TeTx-L), which blocks synaptic vesicular release of neurotransmitters by causing proteolytic cleavage of vesicle-associated membrane protein-2 (VAMP-2) (Yamamoto et al., 2003; Yu et al., 2004). The tetracycline-controlled system is used to express TeTx-L, which confers the reversibility of suppression of synaptic transmission in target cells (Yamamoto et al., 2003). However, the tetracycline-controlled system displays a slow response in the activation/deactivation of gene expression because of the pharmacological property of tetracycline derivatives.

In the present study, we develop a novel system for conditional suppression of synaptic transmitter release from specific neuronal types in transgenic animals. We produce a recombinant protein termed immuno-tetanus toxin (ITet) that is composed of a monoclonal antibody variable region against human IL-2Rα fused to TeTx-L. In this system, ITet acts on target neurons genetically engineered to express IL-2Rα and inhibits transmitter release through proteolytic cleavage of VAMP-2. Our results show that ITet technology enables inducible and reversible silencing of transmitter release from target neurons in specified brain regions of transgenic animals.

2. Materials and methods

2.1. Plasmid construction

The expression vector for the recombinant immunotoxin anti-Tac(Fv)-PE38, which consists of the variable region of the monoclonal antibody recognizing human IL-2Rα [anti-Tac(Fv)] and the translocation/catalytic domains of truncated Pseudomans exotoxin A (PE38) was described previously (Chaudhary et al., 1989; Batra et al., 1990; Kreitman et al., 1994). The plasmid containing cDNA for Chlostridium tetani TeTx-L (Eisel et al., 1993) was kindly provided by Dr. Joseph Gogos at Columbia University. The C-terminal 30 amino acids of TeTx-L were deleted to increase the production efficiency of the recombinant protein in the bacterial expression system. To construct the expression vector for ITet (pEX-ITet), we substituted a region corresponding to the PE38 catalytic domain in the anti-Tac(Fv)-PE38 expression vector with the cDNA part encoding the truncated TeTx-L form. For purification of the recombinant protein by affinity chromatography, a FLAG peptide sequence was introduced into the C-terminal region of the pEX-ITet vector.

2.2. Protein purification

E. coli, the BL21 (DE3) strain (Promega, Madison, WI) carrying the pEX-ITet vector was grown at 37°C for 2−3 hr, and the proteins were then induced by 1 mM isopropylthio-β-d-galactoside (IPTG) during an incubation for 7−8 hr at 30°C. The cells were then harvested, suspended in 50 mM Tris-HCl buffer (pH8.0) containing 100 mM NaCl and 20 mM EDTA, sonicated for 20 sec, and centrifuged at 13,000 rpm for 50 min at 4°C. The supernatant was dialyzed against 20 mM Tris-HCl buffer (pH7.4) and applied to a HiTrap™ DEAE FF ion-exchange column (GE Healthcare, Buckinghamshire, UK) in the same buffer, and the proteins were eluted with a linear 0−0.5 M NaCl gradient. The peak fractions were applied to an anti-FLAG® M2 affinity gel (Sigma, St. Louis, MO) in 50 mM Tris-HCl (pH7.4) containing 150 mM NaCl, and eluted with 100 mM glycine-HCl (pH3.5). The eluted fractions were immediately neutralized with 1 M Tris-HCl (pH8.0). The peak fractions were further separated by using a Superdex 200 gel filtration column (GE Healthcare) with phosphate-buffered saline (PBS) as the eluent. Protein concentrations were determined by Bradford protein assay (Bio-Rad, Burlington, MA) with bovine serum albumin (BSA) as a standard.

2.3. Protein analysis

Binding activity to human IL-2Rα was measured by using ELISA (Onda et al., 2005; 2006). Microtiter plates (96 well) were coated with extracellular domain containing human IL-2Rα fused to Fc fragment (2.0 μg/ml) in PBS, blocked with 1% BSA in PBS, and washed with PBS containing 0.05% Tween 20. The plates were incubated with serial dilutions of purified ITet, mouse anti-PE38 monoclonal antibody, and anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (HRP). Assays were developed with 3,3’,5,5’-tetramethyl benzine/H2O2 substrate, and the absorbance was detected at 450 nm.

For determination of proteolytic activity toward VAMP-2 (Schiavo et al., 1994), the synaptosomal fraction isolated from rat brain cortex (12.5 μg) was incubated for 3 hr at 37°C with different doses of purified ITet in the reaction mixture (30 μl) of 20 mM Na-HEPES buffer (pH 7.4) containing 140 mM NaCl, 5 mM KCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM Na2HPO4, 10 mM glucose, 0.8% 1-O-n-octyl-β-D-glucopyranoside. Proteins (2 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blotting. The proteins were transferred onto a nitrocellulose membrane. The membrane was incubated with rabbit anti-VAMP-2 polyclonal antibody (1:1000 dilution, WAKO Inc., Osaka, Japan) and then incubated with anti-rabbit secondary antibody conjugated with HRP. The same membrane, after having been washed, was serially incubated with mouse anti-syntaxin-1A monoclonal antibody (1:10000 dilution, WAKO Inc.) and anti-mouse secondary antibody conjugated with HRP. The labeled signals were visualized with enhanced chemiluminescence (ECL) reagents (GE Healthcare). The intensity of immunoreactive bands was quantified by using the computer program with NIH Image J.

2.4. Stereotaxic surgery

Mutant mice in which the human IL-2Rα gene cassette was introduced into the mouse dopamine D2 receptor (D2R) locus by gene targeting (Sano et al., 2003) were used. Genotyping was carried out by PCR amplification of genomic DNA prepared from tail tissue. The wild type and heterozygous mutant littermates at 10−12 weeks old were used for stereotaxic surgery. Animal care and handling procedures were carried out according to the guidelines established by the Animal Research Committee of Fukushima Medical University.

Mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and subjected to a unilateral intrastriatal injection of ITet. The protein was diluted to a final concentration of 50 μg/ml in PBS containing 0.1% mouse serum albumin. Solution (0.5 μl/site) was injected into four sites on one side of the dorsal striatum through a glass micropipette, which was stereotaxically introduced by using the coordinates according to an atlas of the mouse brain (Paxinos and Franklin, 2001). The anteroposterior, mediolateral, and dorsoventral coordinates (mm) from bregma and dura were 1.20/1.25/2.00 (site 1), 1.20/2.00/2.00 (site 2), 0.50/1.50/2.00 (site 3), and 0.50/2.25/2.50 (site 4). Injection was carried out at a constant flow rate of 0.25 μl/min with a microinfusion pump connected to the glass micropipette. To estimate the diffusion range of the recombinant protein, mice were transcardially perfused and brains were dissected for cryostat sectioning after the intrastriatal injection. Sections through the striatum were immunostained with anti-FLAG monoclonal antibody. The recombinant protein diffused to a large area in the dorsal region of the striatum (see Supplementary material 1).

2.5. Behavioral test

Mice were placed in a spherical bowl (25 cm diameter), and their behavior was monitored with a digital video camera. One rotation was defined as a complete 180° turn. The number of spontaneous rotations in a 30-min test period was counted.

2.6. Neurochemical analysis

For measurement of the striatal VAMP-2 level, the striatum was dissected and homogenized in nine volumes of 100 mM Tris-HCl buffer (pH 7.4) containing 320 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors (Roche, Basel, Switzerland). Striatal homogenates (150 μg of proteins) were separated by SDS-PAGE and analyzed by Western blotting. Proteins were labeled with rabbit anti-VAMP-2 polyclonal antibody or mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (1:5000 dilution, Chemicon, Temecula, CA) and the corresponding HRP-conjugated secondary antibodies. The labeled signals were visualized with ECL reagents.

The extracellular GABA level was determined by using the microdialysis technique (Kobayashi et al., 2000) with some modifications. Mice were anesthetized with sodium pentobarbital and surgically implanted with a 33-gauge guide cannula into the globus pallidus (GP) by using the following coordinates (mm): anteroposterior −0.45, mediolateral 1.8, and dorsoventral 2.8 from bregma and dura. A microdialysis probe (EICOM, Kyoto, Japan) was constructed with 1.0 mm of active dialysis membrane extending beyond the tip of the guide cannula. On the day of sampling, the probe was inserted into the cannula and artificial cerebrospinal fluid (ACSF) was perfused through the probe with a microinfusion pump at 2 μl/min for more than 5 hr. Dialysis samples were fractionated at 20-min intervals for 1 hr, and ACSF containing 100 mM KCl was then perfused for 40 min. The dialysis buffer was then reversed to ACSF and washing was carried out for 1 hr. The concentration of GABA in each fraction was measured by using a high performance liquid chromatography (HPLC)-electrochemical detection system including precolumn derivation of amino acids with o-phtalaldehyde and a reversed-phase column (Eicompak SC-5ODS, EICOM)). The mobile phase was 50 mM sodium phosphate buffer (pH 6.0) containing 0.1 mM EDTA and 50% methanol. The detector potential was set at 0.6 V versus the Ag/AgCl electrode. After the microdialysis experiments, the placement site of the probe was confirmed by histological examination (see Supplementary material 2).

2.7. Histology

Mice were anesthetized with sodium pentobarbital and perfused transcardially with 4% paraformaldehyde in 100 mM phosphate buffer (PB) (pH 7.4). Fixed brains were cut into sections (30 μm thick) through the coronal plane with a cryostat. Free-floating sections were stained with neutral red. For immunohistochemistry (Sano et al., 2003), sections were incubated with primary antibody against tyrosine hydroxylase (TH) (0.4 μg/ml, Roche) and biotinylated secondary antibody at a 1:1000 dilution and visualized with a Vectastain Elite ABC kit (Vector Laboratories, Burmingame, CA). For in situ hybridization (Sano et al., 2003), fresh-frozen sections (10 μm thick) were fixed in a solution of 4% paraformaldehyde in PB and treated with 0.1 M triethanolamine (pH 8.0) containing 0.25% acetic anhydride. The sections were hybridized with antisense RNA probes for mouse D2R or dopamine D1 receptor (D1R) labeled by using in vitro transcription with digoxigenin-11-UTP (Roche). The hybridized signals were visualized with a nonradioactive detection system using anti-digoxigenin Fab fragments conjugated to alkaline phosphatase.

2.8. Statistical analysis

ANOVA and post-hoc Tukey-Kramer test were used for statistical comparisons. Values were expressed as the mean ± SEM of the data.

3. Results

3.1. Strategy for conditional silencing of synaptic transmitter release

To generate the recombinant protein ITet, we revised the E. coli expression vector of the recombinant immunotoxin anti-Tac(Fv)-PE38, which contains the anti-Tac(Fv) monoclonal antibody variable region for human IL-2Rα and the translocation/catalytic domains of PE38 (Chaudhary et al., 1989; Batra et al., 1990; Kreitman et al., 1994). A region corresponding to the PE38 catalytic domain in this vector was exchanged for the cDNA encoding the truncated form of TeTx-L (C-terminal 30 amino acids deleted), resulting in the expression vector for ITet (termed pEX-ITet). The pEX-ITet vector contained a T7 promoter and a coding region for ITet consisting of anti-Tac(Fv), PE38 translocation domain, and truncated TeTx-L form (see Fig. 1A).

Fig. 1.

Fig. 1

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Experimental design for conditional silencing of neurotransmitter release. (A) Structure of ITet expression vector. The pEX-ITet vector contains a coding region of ITet consisting of anti-Tac(Fv), PE38 translocation domain (PE38-DII), mutated TeTx-L (mTeTx-L), FLAG peptide, and KDEL sequence, under the control of the T7 promoter (PT7). A, ApaI; H, HindIII; N, NdeI; S, SacI. Ampr, β-lactamase gene. (B) Schematic illustration of ITet reaction. ITet binds to cells expressing human IL-2Rα and is incorporated into the cells through endocytosis. It is then processed and the C-terminal TeTx-L fragment is translocated into the cytosol. The fragment cleaves VAMP-2, and thereby blocks transmitter release from target cells.

The possible reaction mechanism of ITet is shown in Fig. 1B. Transgenic mice that express human IL-2Rα in selective neuronal types under the control of a cell type-specific gene promoter are treated with ITet by an appropriate injection procedure. ITet selectively binds to cells bearing human IL-2Rα and is incorporated into the cells through endocytosis. It is then processed and the C-terminal fragment containing TeTx-L is transported into the cytosol. This processing requires the domain II in PE38. The fragment degrades VAMP-2 by its proteolytic activity, thereby inhibiting neurotransmitter release from target cells.

3.2. Expression and purification of ITet

For preparation of ITet protein, the E. coli strain BL21 (DE3) carrying the pEX-ITet vector was grown, and expression of ITet was induced by IPTG. The cells were then harvested, sonicated, and centrifuged. The proteins were purified from the supernatant through DEAE ion-exchange, anti-FLAG affinity, and gel filtration chromatography. SDS-PAGE gave a single band corresponding to purified ITet with a molecular weight of approximately 100 kDa (Fig. 2A). The recovery of purified proteins was ∼200 μg from 1 liter of culture medium. We tested the binding activity of proteins toward human IL-2Rα by using ELISA. The binding was elevated with increasing concentrations of the protein (Fig. 2B). In addition, to test the proteolytic activity against VAMP-2, we incubated synaptosomes with different concentrations of ITet, and then evaluated the VAMP-2 and syntaxin-1A levels by Western blotting. The VAMP-2 band intensity was decreased in the presence of ITet in a dose-dependent manner, whereas the syntaxin-1A intensity was unaltered by ITet addition (Fig. 2C). These data show that purified proteins have both binding activity toward human IL-2Rα and proteolytic activity against VAMP-2.

Fig. 2.

Fig. 2

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Purification and characterization of purified ITet. (A) SDS-PAGE analysis of peak fractions from the column chromatography. Lane 1, supernatant of bacterial extract; lane 2, peak fraction from DEAE column; lane 3, peak fraction from anti-FLAG affinity gel; and lane 4, peak fraction from Superdex G-200 gel filtration column. Arrow indicates a single band corresponding to purified ITet with a molecular weight of ∼100 kDa. (B) Binding activity. Microtiter plates were coated with the extracellular domain of human IL-2Rα fused to an Fc fragment, and serially incubated with various doses of ITet, mouse anti-PE38 antibody, and HRP-conjugated anti-mouse IgG antibody (n = 4). Absorbance was detected at 450 nm. (C) Proteolytic activity. Synaptosomes were incubated with different concentrations of ITet. Proteins were analyzed by Western blotting with anti-VAMP-2 or anti-syntaxin-1A antibody. The intensity of the immunoreactive bands in the presence of ITet is shown as the ratio relative to the intensity of the bands in the absence of ITet (n = 6). *p < 0.05, **p < 0.01, significant differences from the control group without ITet.

3.3. Inducible and reversible behavioral change in the mutants after ITet injection

We investigated the in vivo actions of ITet by using the mutant mice that express human IL-2Rα in their striatal neurons bearing D2R (Sano et al., 2003). Unilateral immunotoxin injection into the dorsal striatum of D2R mutant mice is known to cause rotational behavior in the direction contralateral to the injected side because of ablation of the striatopallidal neurons (Sano et al., 2003). We injected ITet solution (50 μg/ml) or PBS unilaterally into the dorsal striatum by a stereotaxic approach (0.5 μl/site, 4 sites) and monitored the rotational behavior every 2 days (Fig. 3A). The ITet–injected mutant mice began to display contralateral rotations on day 2, and the rotation number increased on day 4 (p < 0.05). The number of rotations virtually reversed to the wild type level by day 8. However, the ITet–injected wild type mice exhibited no evident rotations in either direction. PBS injection did not cause rotations in either group of mice (Fig. 3B). Therefore, ITet injection induced a behavioral alteration in the mutants and this alteration gradually reversed to the normal level.

Fig. 3.

Fig. 3

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Rotational behavior. Mice received a unilateral injection of ITet (A) or PBS (B) into their striatum on day 0, and rotational behavior was monitored on day −1 (pre) and every 2 days after the injection. The number of contralateral or ipsilateral rotations during a 30-min test period was counted. Open circles, wild type mice (n = 5); and closed circles, mutant mice (n = 6). *p < 0.05, significant difference from the wild type mice.

3.4. Transient reduction of striatal VAMP-2 level in the ITet-injected mutants

To assess the in vivo proteolytic activity of ITet, we performed unilateral intrastriatal injection of ITet or PBS into the striatum of mice, and then measured the striatal levels of VAMP-2 and GAPDH by Western blotting. The VAMP-2 band intensity was normalized by the GAPDH intensity, and the ratio of normalized VAMP-2/GAPDH value on the injected side relative to that on the non-injected side was calculated. The relative ratio on day 4 was markedly lower for the ITet-injected mutants than for the wild types injected with ITet (63.8%, p < 0.05), whereas the ratio on day 10 showed no difference between the two kinds of mice (Fig. 4A). Conversely, there was no significant change in the ratio between the PBS-injected wild type and mutant animals (Fig. 4B). The data suggest a cleavage of VAMP-2 in the mutant striatum by ITet, which explains the transient reduction in the striatal VAMP-2 level.

Fig. 4.

Fig. 4

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Striatal VAMP-2 level. Mice that received a unilateral intrastriatal injection of ITet (A) or PBS (B) were used for brain sectioning on day 4 or 10. The striatum was dissected from the ITet-injected (+) or non-injected (-) side, and homogenized. Proteins were analyzed by Western blotting with anti-VAMP-2 or anti-GAPDH antibody. Representative data of Western blotting obtained from ITet injection are shown and the bands for VAMP-2 (18 kDa) and GAPDH (37 kDa) are indicated. The intensity of the VAMP-2 band was normalized by the intensity of the GAPDH band in each sample. The normalized value on the injected side relative to that on the non-injected side was calculated. Open columns, wild type mice (n = 5); and closed columns, mutant mice (n = 6). *p < 0.05, significant difference from the wild type mice.

3.5. Temporary blockade of transmitter release in the ITet-injected mutants

To validate the influence of ITet action on transmitter release from the striatopallidal neurons, we measured the extracellular GABA level in the GP of unilaterally injected mice by using the microdialysis technique. GABA release was evoked by high K+, and the ratio of GABA level to the baseline value was calculated. The evoked GABA release on day 4 after ITet injection was significantly attenuated in the mutant mice as compared with that in the wild type mice (p < 0.05), whereas the release on day 11 showed no statistical difference between the two groups of mice (Fig. 5). PBS injection did not produce the transiently attenuated GABA release on day 4 in the mutants (data not shown). These results suggest a temporary blockade of GABA release from the striatopallidal terminals in the mutants caused by the ITet injection.

Fig. 5.

Fig. 5

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Microdialysis of GABA release in the GP. ITet was unilaterally injected into the striatum, and the microdialysis was performed on days −4 (pre), 4, and 11. Dialysis samples were fractionated at 20-min intervals for 1 hr, and ACSF containing high K+ was then perfused for 40 min. The dialysis buffer was then reversed to ACSF and washing was carried out for 1 hr. The concentration of GABA in each fraction was measured by using an HPLC-electrochemical detection system with precolumn amino acid derivation. The data are expressed as ratios to the mean of the three baseline values before high K+ stimulation. Open circles, wild type mice (n = 8); and closed circles, mutant mice (n = 7). *p < 0.05, significant difference from the wild type mice.

3.6. No histological damage in the ITet-injected brain

To explore whether ITet injection would cause cell damage in the mutant brain, we carried out histological staining on brain sections prepared from unilaterally injected mice. Neutral red staining of sections through the striatum did not show any cell loss on the ITet-injected side of the wild type or mutant mice (Fig. 6A). In situ hybridization with the D2R or D1R antisense probe indicated that the distribution and number of striatal cells containing these receptors were unaltered in the ITet-injected mutants (Fig. 6B). In addition, we stained the nigrostriatal dopamine neurons, which originate from the ventral midbrain (substantia nigra pars compacta) and innervate densely the dorsal striatum. Immunostaining with anti-TH antibody exhibited apparently normal morphology of dopamine neurons and synaptic terminals in the injected mutants (Fig. 6C). Therefore, there was no sign of damage to striatal cells and nerve terminals in the mutants after the ITet injection.

Fig. 6.

Fig. 6

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Histology of the brain in the ITet-injected mice. Mice were injected unilaterally with ITet, and coronal sections through the striatum or the ventral midbrain were used for histological staining. (A) Neutral red staining. Light microscopic images of the ITet-injected (+) and noninjected (-) sides of the striatum in the wild type and mutant mice are shown. (B) In situ hybridization of D2R- and D1R-containing neurons in the mutant striatum. Sections were hybridized with digoxygenin-labeled riboprobe for a mouse D2R or D1R sequence. Lower panels show 6-fold magnified views of the dorsal region of the striatum. (C) Immunostaining of nigrostriatal dopamine neurons in the mutants. Sections through the ventral midbrain and striatum were immunohistochemically stained for TH. SNc, substantia nigra pars compacta; VTA, ventral tegmental area. Scale bars, 100 μm (A) and 200 μm (B, C).

4. Discussion

In the present study, we achieved the expression and purification of a new recombinant protein ITet consisting of the anti-Tac(Fv) monoclonal antibody variable region fused to TeTx-L. Purified ITet possessed both binding activity toward human IL-2Rα and proteolytic activity against VAMP-2. Unilateral injection of ITet into the dorsal striatum of the mutant mice expressing human IL-2Rα in neurons bearing D2R resulted in increased rotational behavior, and the rotations gradually returned to the normal level. ITet injection into the mutant striatum also induced a transient decrease in the striatal VAMP-2 level and in high K+-evoked GABA release in the GP. However, ITet injection caused no structural change in striatal cells and nerve terminals in the mutants. These results show that ITet acts on the striatal neurons bearing human IL-2Rα and transiently reduces VAMP-2 content, thereby inhibiting neurotransmitter release. Our ITet technology provides a powerful system for inducible and reversible control of synaptic transmission in specific neuronal types in transgenic animals.

Rotational behavior is induced by asymmetric basal ganglia output between both hemispheres (Pycok, 1980). The striatopallidal pathway is considered to be involved in the suppression of motor behavior in the standard model of basal ganglia circuitry (Alexander and Crutcher, 1990; DeLong, 1990; Parent and Hazzati, 1995; Gerfen and Wilson, 1996). Our previous study showed that unilateral injection of anti-Tac(Fv)-PE38 into the striatum of D2R mutant mice causes rotational behavior in the direction contralateral to the injected side because of elimination of the striatopallidal neurons (Sano et al., 2003). The striatopallidal elimination appears to increase the GP neural activity and then decrease the basal ganglia output activity, thereby resulting in hyperactivity of motor behavior. The transient behavioral influence of ITet injected into the mutant striatum resembled a consequence of striatopallidal elimination by the recombinant immunotoxin. Our results described herein support the role of the striatopallidal neurons in the suppression of motor behavior in the model of basal ganglia circuitry.

ITet treatment transiently reduced straital VAMP-2 level in the D2R mutant mice to 63.8% of the wild type control (Fig. 4A). In the striatum, spiny projection cell types including the straitonigral and striatopallidal neurons make up as much as 95% of the total number of striatal neurons, and these two projection cell types show approximately equal number (Gerfen and Wilson, 1996). These data suggest that ITet acted on the majority of the striatopallidal neurons containing D2R in the injected mutants to degrade VAMP-2. The reduction in striatal VAMP-2 level may interfere with the supply of the protein to synaptic terminals, resulting in the suppressed GABA release in the GP of the mutants.

Motor alteration in the ITet-injected mutants attained its peak on day 4, and recovery to the normal level occurred by day 8 (Fig. 3A). A previous study reported a reversible suppression of glutamatergic transmission in cerebellar granule cells by expression of a TeTx-L transgene by using the tetracycline-controlled system (Yamamoto et al., 2003). In that study, transgenic mice were administered orally tetracycline derivative doxycycline (DOX); and the transgene products in the cerebellum became detectable on day 3 and reached their maximal content on day 5. When DOX was withdrawn, the products became undetectable 14 days later. Although the time course of behavioral changes in response to DOX administration or withdrawal was not described in the previous study (Yamamoto et al., 2003), the behavioral alteration observed in our ITet-injected mutants appears to turn over more rapidly as compared to the TeTx-L expression under the tetracycline-controlled system. In addition, changing the injection dose of ITet or expression level of human IL-2Rα transgene may shift the turnover rate of the physiological influences of the ITet reaction.

A recent study reported another system for inducible and reversible silencing of synaptic transmission in selected neurons by using chemical induction of dimerization of modified synaptic proteins (Karpova et al., 2005). In this system, some synaptic proteins are fused to FK506 binding protein (FKBP) or FKBP-rapamycin binding protein and expressed in target cells. The cells are treated with derivatives of FK506 or rapamycin, which induce dimerization of the same or different types of fusion proteins and thereby inhibit synaptic transmission. Indeed, VAMP-2/FKBP fusion protein was expressed in Purkinje cells in transgenic mice, and the i.c.v. injection of dimerizer temporally impaired motor learning and performance on the rotarod task. The behavioral change in the transgenic mice initiated on day 1 after the injection and restored until day 3. This approach appears to show more rapid turnover rate as compared to our ITet technology. However, the reaction mechanism of dimerized synaptic proteins to block neurotransmission remains unclear. One possibility to explain this mechanism is that the dimerized proteins replace endogenous proteins through dominant-negative actions. Therefore, the efficacy of this approach may require a high expression level of fusion proteins in target neurons. In contrast, our ITet technology is based on the property of the recombinant protein consisting of anti-Tac(Fv) fused to TeTx-L. ITet selectively binds on the target cells bearing human IL-2Rα at low dose because of the high affinity binding of anti-Tac(Fv) region to the receptor (Chaudharry et al., 1989; Batra et al., 1990; Kreitman et al., 1994). Then, incorporated TeTx-L cleaves directly VAMP-2, resulting in the inhibition of synaptic transmitter release. Moreover, the ITet technology is applicable for some previously established transgenic strains that express IL-2Rα for IMCT (Watanabe et al., 1998; Kaneko, et al. 2000; Hikida et al., 2003; Yasoshima et al., 2005). One transgenic strain is used for two strategies that include selective elimination of target cell types and selective suppression of synaptic transmission in the same cell types.

In conclusion, we created a novel system for inducible and reversible suppression of synaptic transmission in target neurons genetically engineered to express human IL-2Rα in transgenic animals. Here, we applied ITet technology for functional analysis of the striatopallidal neurons containing D2R in the dorsal striatum. This technology can be used for studying the role of target neurons in other brain regions by converting the coordinates of the stereotaxic injection. Our new system will contribute to a clearer understanding of the neural circuit mechanisms that mediate a variety of brain functions and some pathological states of neurological and neuropsychiatric diseases.

Supplementary Material

Supplementary

Supplementary material 1. Distribution of ITet in the dorsal striatum after stereotaxic injection. Mice were injected with ITet unilaterally into the striatum, and coronal sections were used for immunostaining with anti-FLAG monoclonal antibody. Light microscopic images of the ITet-injected and non-injected sides of the striatum are shown. Immunoreactive area in the injected side is indicated by the dotted line. Scale bar, 500 μm.

Supplementary material 2. Location of the microdialysis probes in the GP. After the microdialysis experiments, the placement sites of the probes were determined by histological examination with neutral red staining. Lines in the plates represent locations of the dialysis probes. Number indicates the distance (mm) from bregma in the anteroposterior plane.

Acknowledgments

This work was supported by a grant-in aid from Core Research for Evolutional Science and Technology, Japan Science and Technology Cooperation. We thank Dr. I. Pastan for providing the anti-Tac(Fv)-PE38 expression vector, Dr. J. Gogos for providing the tetanus toxin light chain cDNA, Drs. A. Morozov and Y. Yasoshima for valuable discussion, and T. Minakawa, M. Kikuchi, and N. Sato for their technical support in conducting the animal experiments.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary

Supplementary material 1. Distribution of ITet in the dorsal striatum after stereotaxic injection. Mice were injected with ITet unilaterally into the striatum, and coronal sections were used for immunostaining with anti-FLAG monoclonal antibody. Light microscopic images of the ITet-injected and non-injected sides of the striatum are shown. Immunoreactive area in the injected side is indicated by the dotted line. Scale bar, 500 μm.

Supplementary material 2. Location of the microdialysis probes in the GP. After the microdialysis experiments, the placement sites of the probes were determined by histological examination with neutral red staining. Lines in the plates represent locations of the dialysis probes. Number indicates the distance (mm) from bregma in the anteroposterior plane.

NIHMS70051-supplement-Supplementary.tif (2.3MB, tif)

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