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Research Articles: Neurobiology of Disease
PRONOUNCED HYPERACTIVITY, COGNITIVE DYSFUNCTIONS AND BDNF
DYSREGULATION IN DOPAMINE TRANSPORTER KNOCKOUT RATS
Damiana Leo1, Ilya Sukhanov1,2, Francesca Zoratto3, Placido Illiano1, Lucia Caffino4, Fabrizio Sanna5,
Giulia Messa4, Marco Emanuele1, Alessandro Esposito1, Maria Dorofeikova2, Evgeny A. Budygin6,7,
Liudmila Mus1,2, Evgenia E. Efimova7, Marco Niello1, Stefano Espinoza1, Tatyana D. Sotnikova7, Marius
C. Hoener8, Gianni Laviola3, Fabio Fumagalli4, Walter Adriani3 and Raul R. Gainetdinov7,9
1Fondazione Istituto Italiano di Tecnologia, Neuroscience and Brain Technologies Department Via Morego, 30 16163,
Genoa, Italy.
2Pavlov First Saint Petersburg State Medical University, Valdman Institute of Pharmacology, St. Petersburg, Russia
3Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy
4Università degli Studi di Milano, Department of Pharmacological and Biomolecular Sciences, Via Balzaretti 9, 20133, Milan,
Italy
5University of Cagliari, Department of Biomedical Sciences, Cittadella Universitaria, 09042 Monserrato (CA), Italy
6Department of Neurobiology and Anatomy, Wake Forest School of Medicine, Winston- Salem, NC, USA
7St. Petersburg State University, Institute of Translational Biomedicine, Universitetskaya Emb. 7-9, 199034, St. Petersburg,
Russia
8Neuroscience Research, Roche Pharma Research and Early Development, Roche Innovation Center Basel, F. Hoffmann-
La Roche Ltd., CH-4070 Basel, Switzerland
9Skolkovo Institute of Science and Technology, 143025, Moscow, Russia
DOI: 10.1523/JNEUROSCI.1931-17.2018
Received: 10 July 2017
Revised: 7 January 2018
Accepted: 11 January 2018
Published: 18 January 2018
Author contributions: S.B.F. and M.v.H. designed research; S.B.F. and M.v.H. analyzed data; S.B.F., D.R.M., M.T.T., and
M.v.H. wrote the paper; D.R.M., M.T.T., and M.v.H. performed research.
Conflict of Interest: The authors declare no competing financial interests.
Funding and Disclosure: This research was supported by the Istituto Italiano di Tecnologia, NIH grant AA022449 (EAB),
by F. Hoffmann-La Roche Ltd (RRG), by the Russian Science Foundation grant 14-50-00069 (TDS, EEE, RRG) and partly
supported by EU Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 603016 (project MATRICS to
GL). In the last 3 years, RRG has consulted for the Orion Pharma and F. Hoffmann-La Roche Ltd. MCH is an employee of F
Hoffmann-La Roche. All views expressed herein are solely those of authors. The authors declare no conflict of interest.
Corresponding author: Raul R Gainetdinov, St. Petersburg State University, Institute of Translational Biomedicine,
Universitetskaya Emb. 7-9, 199034, St. Petersburg, Russia, Phone: +7 911 7316868 mail: gainetdinov.raul@gmail.com
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.1931-17.2018
Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this
article is published.
PRONOUNCED HYPERACTIVITY, COGNITIVE DYSFUNCTIONS AND BDNF 1
DYSREGULATION IN DOPAMINE TRANSPORTER KNOCKOUT RATS 2
3
Abbreviated title: DOPAMINERGIC DYSREGULATION IN DAT-KO RATS 4
5
Damiana Leo1,¥, PhD; Ilya Sukhanov1,2, PhD; Francesca Zoratto3, PhD; Placido Illiano1, $, PhD; 6
Lucia Caffino4, PhD; Fabrizio Sanna5, PhD; Giulia Messa4, MS; Marco Emanuele1, PhD; 7
Alessandro Esposito1, MS; Maria Dorofeikova2, MS,; Evgeny A. Budygin6,7, PhD; Liudmila 8
Mus1,2, PhD; Evgenia E. Efimova7, PhD; Marco Niello1§, MS; Stefano Espinoza1, PhD; Tatyana 9
D. Sotnikova7, PhD; Marius C. Hoener8, PhD; Gianni Laviola3, PhD; Fabio Fumagalli4, PhD; 10
Walter Adriani3, PhD and Raul R. Gainetdinov7,9, MD, PhD. 11
12
1.
Fondazione Istituto Italiano di Tecnologia, Neuroscience and Brain Technologies Department Via 13
Morego, 30 16163, Genoa, Italy. 14
2.
Pavlov First Saint Petersburg State Medical University, Valdman Institute of Pharmacology, St. 15
Petersburg, Russia 16
3.
Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Roma, Italy 17
4.
Università degli Studi di Milano, Department of Pharmacological and Biomolecular Sciences, Via 18
Balzaretti 9, 20133, Milan, Italy 19
5.
University of Cagliari, Department of Biomedical Sciences, Cittadella Universitaria, 09042 20
Monserrato (CA), Italy 21
6.
Department of Neurobiology and Anatomy, Wake Forest School of Medicine, Winston- Salem, NC, USA 22
7.
St. Petersburg State University, Institute of Translational Biomedicine, Universitetskaya Emb. 7-9, 23
199034, St. Petersburg, Russia 24
8.
Neuroscience Research, Roche Pharma Research and Early Development, Roche Innovation Center 25
Basel, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland 26
9.
Skolkovo Institute of Science and Technology, 143025, Moscow, Russia 27
$ Present address: University of Miami, Miller School of Medicine, 1095 NW 14th Terrace, Miami, FL 28
33136 29
§Present address: Medical University of Vienna, Center for Physiology and Pharmacology, Institute of 30
Pharmacology Währingerstr. 13a, 1090 Vienna, Austria 31
¥Present address: University of Mons, Neurosciences Unit, “Pentagone” wing 1A, Avenue du Champ de 32
Mars 6B - 7000 Mons, Belgium 33
34
Corresponding author: Raul R Gainetdinov, St. Petersburg State University, Institute of 35
Translational Biomedicine, Universitetskaya Emb. 7-9, 199034, St. Petersburg, Russia 36
Phone: +7 911 7316868 mail: gainetdinov.raul@gmail.com 37
38
Number of pages: 39 39
Number of figures: 5 40
Number of words (Abstract): 206 41
Number of words (Introduction): 582 42
Number of words (Discussion): 1475 43
44
Funding and Disclosure 45
This research was supported by the Istituto Italiano di Tecnologia, NIH grant AA022449 46
(EAB), by F. Hoffmann-La Roche Ltd (RRG), by the Russian Science Foundation grant 14-50-47
00069 (TDS, EEE, RRG) and partly supported by EU Seventh Framework Programme 48
(FP7/2007-2013) under grant agreement no. 603016 (project MATRICS to GL). In the last 3 49
years, RRG has consulted for the Orion Pharma and F. Hoffmann-La Roche Ltd. MCH is an 50
1
employee of F Hoffmann-La Roche. All views expressed herein are solely those of authors. The 51
authors declare no conflict of interest. 52
53
Abstract 54
55
Dopamine (DA) controls many vital physiological functions and is critically involved in several 56
neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder 57
(ADHD). The major function of the plasma membrane dopamine transporter (DAT) is the rapid 58
uptake of released DA into presynaptic nerve terminals leading to control of both the 59
extracellular levels of DA and the intracellular stores of DA. Here, we present a newly 60
developed strain of rats (DAT-knockout, DAT-KO rats) in which the gene encoding the DAT 61
has been disrupted by using zinc finger nuclease technology (ZFN). Male and female DAT-KO 62
rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate 63
pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and 64
concentrations are significantly increased, the total tissue content of DA is markedly decreased 65
demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of 66
DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial
Trace Amine-
67
Associated Receptor 1 (TAAR1) agonist
RO5203648
and haloperidol
. DAT-KO rats also 68
demonstrate a deficit in working memory and sensorimotor gating tests,
less propensity to develop
69
obsessive behaviors
and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats 70
could provide a novel translational model for human diseases involving aberrant DA function 71
and/or mutations affecting the DAT or related regulatory mechanisms. 72
73
74
75
76
2
77
Significance statement 78
79
Here, we present a newly developed strain of rats in which the gene encoding the
80
dopamine transporter (DAT) has been disrupted (DAT-KO rats). DAT-KO rats display
81
functional hyperdopaminergia accompanied with pronounced spontaneous locomotor
82
hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine,
83
methylphenidate, and a few other compounds exerting inhibitory action on dopamine-
84
dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working
85
memory and sensorimotor gating tests, less propensity to develop compulsive behaviors and strong
86
dysregulation in frontostriatal BDNF function. These observations highlight the key role of the
87
DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel
88
translational model for human diseases involving aberrant dopamine functions.
89
3
90
Introduction 91
Dopaminergic innervations are prominent in the brain and the dopamine (DA) system exerts 92
modulatory control of motivation, reward, cognition and locomotion (Carlsson, 1987; Gainetdinov, 93
2008). Concentration of DA in the synaptic cleft is the primary determinant of DA signaling 94
intensity. The key regulatory element of DA neurotransmission is the DAT, belonging to a family 95
of plasma membrane transporters of solute carrier family 6 (SLC6). The DAT controls levels of 96
extracellular DA and maintains DA stores by transporting released DA back into neurons. DAT is 97
the well-established target of many drugs of abuse and neurotoxins (Saunders et al., 2000; Kahlig et 98
al., 2006; Wheeler et al., 2015; Siciliano et al., 2016). Amphetamine (AMPH) and cocaine (COC) 99
are psychostimulants that are able to induce euphoria and hyperactivity by increasing extracellular 100
DA via interaction with the DAT. 101
The DAT knockout (KO) mouse model was generated more than 20 years ago by the groups of 102
Marc Caron (Giros et al., 1996) and George Uhl (Sora et al., 1998). DAT-KO mice display a 103
distinct behavioural phenotype: they are hyperactive, display certain cognitive deficits and have 104
sleep dysregulation (Gainetdinov et al., 1999; Spielewoy et al., 2000). The hyperdopaminergic 105
phenotype of DAT-KO mice has provided a simple model of hyperdopaminergic function in which 106
the effects of various pharmacological agents affecting DA related functions and behaviours have 107
been evaluated. In particular, they are extremely sensitive to D2 dopamine receptor blockers and 108
antipsychotics such as haloperidol and show a paradoxical inhibitory response to psychostimulants 109
AMPH and methylphenidate (MPH) (Gainetdinov et al., 1999; Spielewoy et al., 2000; Carboni et 110
al., 2001). 111
DAT-KO mice have provided important information on the pathological consequences of aberrant 112
DA function. DAT-KO mice are believed to best model Attention Deficit Hyperactive Disorders 113
(ADHD) endophenotypes by demonstrating spontaneous hyperactivity, deficits in cognitive tests 114
and anti-hyperkinetic responses to psychostimulants used in ADHD treatments (Gainetdinov and 115
4
Caron, 2000, 2001). At the same time, DAT-KO animals have provided numerous advances in 116
understanding the pathology and pharmacology of other dopamine-related brain disorders, such as 117
schizophrenia (Gainetdinov et al., 2001; Wong et al., 2012, 2015), bipolar disorder (Beaulieu et al., 118
2005, 2005), Parkinson’s disease (Cyr et al., 2003; Sotnikova et al., 2005) and addiction (Rocha et 119
al., 1998). Notably, several patients diagnosed with ADHD, bipolar disorder and parkinsonism 120
(Vaughan and Foster, 2013; Hansen et al., 2014) have rare coding variants of the SLC6A3 DAT 121
gene. Recently, DAT-KO mice were used to evaluate the efficacy of adenoviral therapy for 122
Dopamine Transporter Deficiency Syndrome (DTDS) (Illiano et al., 2017), a newly recognized 123
Parkinsonian-like condition with earlier hyperkinetic stage, whose symptomatology is directly 124
caused by an impaired DAT functioning due to loss-of-function mutations found in SLC6A3 DAT 125
gene (Kurian et al., 2009, 2011; Ng et al., 2014; Yildiz et al., 2017). 126
The recent progress in the development of gene editing approaches has made possible to perform 127
such studies in genetically altered rats. Beyond obvious advantages of rat models, such as larger 128
brain size for surgery and electrophysiological recordings as well as their closer physiological 129
similarity to humans, rats have a much wider repertoire of well-established behavioural approaches 130
to investigate cognitive functions that are critical for modelling neuropsychiatric conditions. 131
Here we present a new model of DAT deficiency, DAT-KO rats, generated by using zinc-finger 132
nucleases (ZFN) technology (Geurts et al., 2009; Brown et al., 2013) used for the elimination of the 133
DAT gene. The current study describes detailed neurochemical, behavioural, and pharmacological 134
characterization of this model that could open new perspectives in understanding pathology and 135
pharmacology of human diseases involving aberrant DA function and/or mutations affecting the 136
DAT or DAT-related regulatory mechanisms. 137
138
139
140
141
5
Materials and methods 142
143
Animals 144
ZFN design, construction, in vitro validation, microinjection and founder selection were performed 145
as previously described (Geurts et al., 2009; Carbery et al., 2010). The ZFN Target site was: 146
CTCATCAACCCGCCACAGAcaccaGTGGAGGCTCAAGAG in the Exon 2 of Slc6a3 gene 147
(NCBI Gene ID: 24898; Genomic NCBI Ref Seq: NC_005100.3; mRNA NCBI Ref Seq: 148
NM_012694.2). The knockout rat lines were created in the outbred Wistar Han background at 149
SAGE Labs. Adult littermate rats were housed by genotype in groups of 3 to 4 with free access to 150
tap water and standard pellet food. They were kept at 22°C and on a 12/12 h light/dark cycle (lights 151
on 0700–1900 h). All experiments were conducted in Istituto Italiano di Tecnologia, Genova, Italy,152
Università degli Studi di Milano, Milan, Italy and Istituto Superiore di Sanità, Rome, Italy 153
with approved animal protocols in full compliance with the Italian Ministry of Health (DL 116/92; 154
DL 111/94-B) and European Community (86/609/EEC) directives regulating animal research. All 155
efforts were made to minimize animal suffering and to reduce the number of animals used. Rats of 156
both sexes were used in all experiments excluding behavioral cognitive tests where only males were 157
investigated. 158
Genotyping was performed by PCR followed by enzymatic digestion with BtsI MutI (New England 159
Biolabs (Milan, Italy). 160
161
Real-Time PCR 162
Animals were sacrificed and PreFrontal Cortex (PFC), dorsolateral striatum (DLStr) and midbrain 163
were dissected and dissociated for 15 minutes at 37ºC with Pronase enzyme (Sigma) in Hank's 164
Balanced Salt Solution (HBSS, Invitrogen). Brain samples were triturated with three glass pipettes 165
of decreasing tip diameter and centrifuged at 900 rpm at room temperature for 5 minutes. To 166
6
remove excess debris, cell pellets were resuspended in HBSS and filtered through a 70 μm mesh 167
(BD Falcon, #352350). Cells-to-CT kit (Life Technology) was used to produce DNase I digested 168
cell lysates and perform cDNA synthesis, according to manufacturer's instructions. cDNAs were 169
used for Taqman singleplex PCR. To prevent false positive originating from genomic DNA, we 170
used negative control samples without reverse transcriptase. All reagents were supplied by Applied 171
Biosystems. PCR master mix contained 1x Taqman Universal PCR Master Mix, 1x Gene 172
Expression Assay mix, and 1μl cDNA for a total volume of 20μl. The following Gene Expression 173
Assays were used: Drd2 (Assay ID Rn01452984_m1), Drd1a (Assay ID Rn00569454_m1), TH 174
Assay ID Rn00562500_m1); Gapdh (Assay ID Rn01775763_g1) and Hprt (Assay ID 175
Rn01527840_m1). Samples were run in three replicates for each Gene Expression Assay. PCR 176
reactions were carried out on a 7900 Thermal Cycler (Applied Biosystems) with 40 cycles of 95ºC 177
for 15 seconds and 60 ºC for 1 minute. CT values for each gene were normalized to CT values for 178
GAPDH and HPRT to obtain a relative expression level for each replicate and the three replicates 179
were averaged together. 180
For BDNF analysis, an aliquot of total RNA of each sample (n=5 WT and n=5 DAT-KO rats) was 181
treated with DNase to avoid DNA contamination. RNA was analyzed by a TaqMan qRT-PCR 182
instrument (CFX384 real-time system, Bio-Rad Laboratories) using the iScriptTM one-step RT-183
PCR kit for probes (Bio-Rad Laboratories). Samples were run in 384-well formats in triplicate as 184
multiplexed reactions. Data were analyzed with the comparative threshold cycle (ΔΔCt) method 185
using 36B4 as reference gene. The primer efficiencies were experimentally set up for each couple of 186
primers. 187
Probes and primers for total BDNF, Creb, CaRF, Npas4 and 36B4 were purchased from Eurofins 188
MWG-Operon (Ebersberg, Germany) and their sequences are shown below: 189
- total BDNF: forward primer 5’-AAGTCTGCATTACATTCCTCGA-3’, reverse primer 5’ 190
GTTTTCTGAAAGAGGGACAGTTTAT-3’, probe 5’-TGTGGTTTGTTGCCGTTGCCAAG-3’; 191
- Creb: forward primer 5’-AGATTCTAGTGCCCAGCAAC-3’, reverse primer 5’-192
7
CTGTGCGAATCTGGTATGTTTG-3’, probe 5’-TGTTCAAGCTGCCTCTGGTGATGT-3’; 193
- Npas4: forward primer 5’-TCATTGACCCTGCTGACCAT-3’, reverse primer 5’-194
AAGCACCAGTTTGTTGCCTG-3’, probe 5’-TGATCGCCTTTTCCGTTGTC-3’; 195
- CaRF: forward primer 5’-GAGATGCACACACCATTCCA-3’, reverse primer 5’-196
GTGTTGGCTCATTGGGTTCT-3’, probe 5’-CAGCCATCCAGCTCTTGTTGAAGA-3’; 197
- 36B4: forward primer 5’-TTCCCACTGGCTGAAAAGGT-3’, reverse primer 5’-198
CGCAGCCGCAAATGC-3’, probe 5’-AAGGCCTTCCTGGCCGATCCATC-3’. 199
Probes and primers for BDNF exon IV and BDNF exon VI were purchased from Life Technologies 200
(Milan, Italy) (BDNF exon IV: ID Rn01484927_m1 and BDNF exon VI: ID Rn01484928_m1). 201
Thermal cycling was started with 10-min incubation at 50 °C (RNA retrotranscription) then 5 min 202
at 95 °C (TaqMan polymerase activation). After this, 39 PCR cycles were run. Each PCR cycle 203
consisted of heating the samples at 95 °C for 10 s to enable the melting process, then for 30 s at 204
60 °C for the annealing and extension reaction.
205
206
Protein extraction and western blotting
207
Protein extraction and preparation of samples for Western blot analysis were performed as 208
described (Vecchio et al, 2017). Briefly, the tissues were dissected from freshly harvested brains. 209
Brain samples were mechanically homogenized in RIPA buffer (50 mmol/l Tris–HCl, pH 7.4/150 210
mmol/l NaCl/1% Nonidet P-40/0.5% sodium deoxycholate/0.1% SDS; Sigma) plus protease 211
inhibitor mixture (Roche 1873580), and protein concentration was measured using a BCA protein 212
assay kit (Thermo Scientific). Protein extracts (25 μg) were separated by 10% SDS/PAGE and 213
transferred to nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were immunostained 214
overnight at 4°C with the following primary antibodies: Gapdh (FL-335): sc-25778, Santa Cruz 215
Biotechnology, Inc., Heidelberg, Germany); DAT (C-20): sc-1433, Santa Cruz Biotechnology, Inc., 216
Heidelberg, Germany). After washing, the membranes were incubated for 2 hours at room 217
temperature with the appropriate secondary antibody (anti-mouse, anti-rabbit or anti-rat). Following 218
8
secondary antibody incubations, membranes were washed and finally incubated with ECL detection 219
reagent (Amersham RPN2232) for 5 minutes. 220
For BDNF analysis, medial prefrontal cortex (PFC) was dissected from a 2-mm section extending 221
from approximately bregma +5.16 to +3.24 (Paxinos and Watson, 2005) and dorsolateral striatum 222
(DLStr) was dissected from a 2-mm section immediately caudal to the PFC section from n=5 WT 223
and n=5 DAT-KO rats. Brain areas were immediately frozen on dry ice and stored at -80°C until 224
being processed for molecular analysis. Protein extraction was performed as previously described 225
(Caffino et al., 2017) with minor modifications. Briefly, PFC and DLStr were homogenized in a 226
Teflon-glass potter in cold 0.32 M sucrose buffer pH 7.4 containing 1 mM HEPES, 0.1 mM EGTA 227
and 0.1 mM PMSF, in presence of commercial cocktails of protease (Roche, Monza, Italy) and 228
phosphatase (Sigma-Aldrich, Milan, Italy) inhibitors and then sonicated. The homogenate of DLStr 229
was centrifuged at 800g for 5 min, the obtained supernatant was centrifuged at 13,000 g for 15 min 230
obtaining a pellet (P2) and a supernatant (S2), referred as the cytosolic fraction. P2 pellet was 231
resuspended in buffer containing 150 mM KCl and 1% Triton X-100 and centrifuged at 100,000 x g 232
for 1 h. The resulting supernatant, referred as Triton X-100 soluble fraction (TSF), was stored at -233
20°C; the pellet, referred as PSD or Triton X-100 insoluble fraction (TIF), was homogenized in a 234
glass–glass potter in 20 mM HEPES, protease and phosphatase inhibitors and stored at -20°C in 235
presence of 30% glycerol. Total protein content was measured according to the Bradford Protein 236
Assay procedure (Bio-Rad, Milan, Italy) using bovine serum albumin as calibration standard. Equal 237
amounts of protein were run under reducing conditions on the criterion TGX precast gels (Bio-Rad 238
Laboratories, Milan, Italy) and then electrophoretically transferred onto nitrocellulose membranes 239
(Bio-Rad, Milan, Italy). Blots were blocked one hour at room temperature with 10% non-fat dry 240
milk in TBS + 0.1% Tween-20 buffer, incubated with antibodies against the phosphorylated forms 241
of the proteins and then stripped and reprobed with the antibodies against corresponding total 242
proteins. The conditions of the primary antibodies were the following: mBDNF (1:1000, Icosagen, 243
San Francisco CA, USA); anti phospho trkB Y706 (1:1000, Santa Cruz Biotechnology, Santa Cruz, 244
9
CA, USA); anti total trkB (1:750, Santa Cruz Biotechnology); anti phospho CaMKII T286 (1:2000, 245
Thermo Scientific, Waltham, MA, USA); anti total CaMKII (1:5000, Chemicon, Temecula, CA, 246
USA); anti total PSD95 (1:4000, Cell Signaling Technology) and anti β-Actin (1:10000, Sigma-247
Aldrich, Milano, Italy). Results were standardized using β-actin as the control protein, which was 248
detected by evaluating the band density at 43 kDa. Immunocomplexes were visualized by 249
chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories) and analyzed 250
using the Image Lab software from Bio-Rad Laboratories. The activation of the proteins 251
investigated were expressed as a ratio between the phosphorylated and the respective total forms. 252
253
In vivo microdialysis 254
In vivo brain microdialysis was performed in the right dorsal striatum (DStr) of freely moving rats 255
(Carboni et al., 2001; Budygin et al., 2004) using concentric microdialysis probes (2 mm membrane 256
length cut off 6000 Da; CMA-11, CMA/Microdialysis, Solna, Sweden). Stereotaxic coordinates for 257
the position of the probes were chosen according to the atlas of Franklin and Paxinos (1997) and are 258
relative to the bregma: AP 1.0; L 3.0; DV -6.6. Prior to fixation in stereotaxic apparatus, the animals 259
were anesthetized with an oxygen/isoflurane mixture. The probes were implanted in the brain 260
vertically through a small drilled aperture in the scull and fixed with dental cement. During 261
implantation into the brain and for 1 h afterward, the dialysis probes were perfused with artificial 262
cerebrospinal fluid (aCSF) (NaCl 147 mM, KCl 2.7 mM, CaCl2 1.2 mM, MgCl2 0.85 mM; CMA 263
Microdialysis). 1 h after the operation, the animals were returned to their home cages. 264
Approximately 24 h after surgery, the dialysis probes were connected to a syringe pump and 265
perfused with the aCSF at 1.0 μl/min for 60 min (equilibration period). To reliably determine the 266
basal extracellular DA levels in the striatum of freely moving rats a quantitative “low perfusion” 267
rate microdialysis experiment was conducted (Gainetdinov et al., 2003). The perfusate was 268
collected at a perfusion rate of 0.1 μl/min every 90 min over a 6 h period into collection tubes 269
10
containing 2 μl of 1 M perchloric acid. To determine effect of AMPH (3 mg/kg, i.p.) on the 270
extracellular DA level a “conventional” microdialysis approach was used. Dialysis probes were 271
connected to a syringe pump and perfused with aCSF at 1 ul/min for at least 60 min for 272
equilibration. Then perfusate was collected every 20 min, for 1 hour before injection and 2 hours 273
after injection of AMPH. 274
275
HPLC 276
HPLC measurements were carried out as described before (Leo et al., 2014). Brain tissues were 277
dissected from WT, KO and HET rats and homogenized in 40 volumes of 0.1 M HClO4. Following 278
centrifugation and filtration, the samples were analyzed by HPLC as described below. The protocol 279
for sample preparation for the HPLC determination of tissue monoamines and their metabolites was 280
performed as previously described. Measurements of DA, 5-hydroxytryptamine (5-HT) and 281
metabolites in tissue samples and DA in microdialysis samples were performed by HPLC with 282
electrochemical detection (ALEXYS LC-EC system, Antec Leyden BV, Netherlands) with a 0.7-283
mm glass carbon electrode (Antec; VT-03). The system was equipped with a reverse-phase column 284
(3-μm particles, ALB-215 C18, 1x150 mm, Antec) at a flow rate of 200 μl/min. The mobile phase 285
contained 50 mM H3PO4, 50 mM citric acid, 8 mM KCl, 0.1 mM EDTA, 400 mg/l octanesulfonic 286
acid sodium salt and 10% (vol/vol) methanol, pH 3.9. The sensitivity of this method permitted the 287
detection of fmol DA. Dialyzate samples (11 μl) were injected into HPLC without any additional 288
purification. 289
290
Fast Scan Cyclic Voltammetry (FSCV) 291
The brains were sectioned in cold carboxygenated artificial cerebrospinal fluid (aCSF) (126 mM 292
NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 25 mM NaHCO3, 2.4 mM CaCl2, 11 mM D-glucose, 1.2 293
mM MgCl2) on a VT1000S vibrating microtome (Leica Microsystems, Nussloch, Germany) at a 294
thickness of 300 μm. Coronal slices containing the dorsal striatum were allowed to recover for at 295
11
least 1 h at room temperature in carboxygenated aCSF. For recordings, slices were superfused with 296
32 °C carboxygenated aCSF at a flow rate of 1 ml/min. FSCV recordings started 20 min after 297
transfer to the slice chamber. Carbon fiber electrodes (7 μm diameter, Goodfellow, Huntingdon, 298
England) were made as previously described ( Kawagoe et al, 1993; Kuhr and Wightman, 1986a). 299
The carbon fibers were trimmed with a scalpel to 80-120 μm under a microscope (Nikon) A carbon 300
fiber microelectrode was inserted into the slice and a twisted bipolar stimulating electrode (Plastics 301
One, Roanoke, VA) was placed on the surface of the brain slice ~200 μm away. The potential of the 302
working electrode was held at -0.4 V and scanned to +1.3 V and back at 300 V/s. Axonal DA 303
release in the striatum was evoked by a single biphasic electrical pulse (1 ms long, 400 μA) every 2 304
min through a stimulus isolator (AM-system, Carlsborg, WA). Data were filtered to reduce noise. 305
Oxidation and reduction peaks were observed at ~ +0.65 V and -0.2 V (vs. Ag/AgCl reference) 306
identifying DA as the released chemical. Electrodes were calibrated in a flow injection system using 307
1 μM DA (Sigma Aldrich, St. Louis, MO, USA). 308
FSCV kinetic analysis 309
There were several established criteria for choosing which DA signals to use for analysis, two of 310
these being that there should be no confounding electrical artefacts to interfere with the DA traces, 311
and no pH shifts during recordings, which allow for a flat baseline before stimulation and provide 312
the most uncontaminated DA dynamics as possible, Next, a 10:1 signal to noise ratio was used in 313
order to guarantee that the actual signal was separated from background. All of these criteria 314
insured accuracy in analysis. One to two recordings from each experimental group were excluded 315
from analysis based on these criteria. 316
Data analysis was performed using Demon Voltammetry software described (Yorgason et al., 317
2011a). Briefly, computations were based on user-defined positions on current traces for baseline 318
(Pre-Stim cursor), peak (Peak Cursor) and return to baseline (Post-Stim cursor) positions. Half-life 319
values were determined from exponential fit curves based on Peak cursor and Post-Stim cursor 320
positions using a least squares constrained exponential fit algorithm (National Instruments, Milan, 321
12
Italy) (Yorgason et al., 2011a). These measurements were performed on individual traces within 322
each experiment. These numbers were then averaged within each experimental group (WT, KO and 323
KO treated) and reported as mean ± SEM. Half-life is considered to be a reliable measure for 324
evaluating changes in striatal DA clearance in vivo and in vitro. This parameter accurately 325
distinguishes differences in clearance rate similar to other established measures (Yorgason et al, 326
2011). 327
328
Locomotor activity in a novel environment 329
Locomotor activity was evaluated as described before (Sukhanov et al., 2014) by using an 330
automated Omnitech Digiscan apparatus (AccuScan Instruments, USA) under illuminated 331
conditions. The apparatus included four open field monitors, each consisting of a set of 16 light 332
beams arrayed in the horizontal X and Y axes. The hardware detected beams broken by the animal, 333
allowing the software to determine the location of the rat in the cage. Cages were divided into four 334
compartments (20 cm x 20 cm). Animals were tested individually for defined periods with 5-min 335
intervals. The total distance traveled was measured and expressed in terms of centimeters traveled 336
by the animal. In addition, vertical activity as expressed in terms of the number of beam breaks. All 337
rats were habituated to the test room for at least 1 hour prior to testing. Effects of drugs on 338
locomotor activity in a novel environment were tested 30 min after placement of animals into 339
locomotor activity monitor. d-AMPH hydrochloride, MPH hydrochloride, haloperidol (Sigma–340
Aldrich, Co., St. Louis, MO) and RO5203648 (F. Hoffmann-La Roche Ltd., Basel, Switzerland) 341
were dissolved as described (Gainetdinov et al, 1999; Spielewoy et al, 2000; Revel et al., 2012). 342
For all behavioral experiments, drugs were administered intraperitoneally (i.p.) in a volume of 1 343
ml/kg. All solutions were made fresh daily. 344
345
24-hour spontaneous locomotor activity in a home cage 346
13
Rats were continuously monitored for spontaneous home-cage locomotor activity by means of an 347
automatic device equipped with small passive infrared sensors placed on a standard rack over the 348
top of each home-cage (ActiviScope system; TechnoSmart, Rome, Italy). These sensors (20 Hz) 349
detected any movement of rats: scores were automatically divided into 60-min intervals. 350
351
Y maze spontaneous alteration test 352
To measure spontaneous alternation behavior and exploratory activity, a white plastic material 353
Y-maze with arms 40 cm (long) by 6 cm (wide) with 13 cm walls was used. Each animal was 354
tested in a single 8-min session, during which the animal was placed in the central platform and 355
allowed free exploration of the maze. Spontaneous alternation, expressed as a percentage, refers 356
to ratio of arm choices differing from the previous two choices to the total number of arm 357
entries. 358
359
Schedule-induced polydipsia 360
Apparatus 361
The experiments were carried out in five standard operant conditioning chambers for rats, with 362
interior dimensions 31 cm x 27 cm x 33 cm located in sound-attenuating and light-proof 363
cubicles. Each cubicle was equipped with an electric fan that provided background white noise 364
during the experimental sessions. The right wall of each chamber contained three metal panels 365
with built-in appliances. Each chamber was illuminated by houselights (25 W) installed at the 366
top of the central panel was turned on during experiments. The pellets tray was situated 2 cm 367
from floor also at the central panel. A water bottle was fitted at the right panel such a way that 368
the drinking spout of the bottle was obtainable for rats, approximately 3 cm from the floor. The 369
chambers inputs were connected to the operating PC equipped with Med-PC software through 370
MED interface (MED Associates, East Fairfield, VT, USA). The volume of water consumed 371
14
during each session was measured by weighting of the bottle at the start and at the end of the 372
session. 373
Polydipsia induced by fixed-time 60 seconds schedule of reinforcement 374
Rats were habituated to the operant chambers and to pellet feeding within daily magazine 375
training sessions. During the sessions the rats were placed in the chambers with 15 pellets in the 376
food trough for 30 minutes. The next day after successful magazine training (all animals had 377
eaten all pellets), the rats were given daily one-hour sessions (6 days a week) with fixed-time 60 378
seconds (FT 60 sec) schedule of food reinforcement (i.e food pellets were delivered every 60 379
seconds, independently of an animal’s behaviour). Within the experimental sessions the rats had 380
free access to the drinking spouts of the bottles filled with fresh tap water. When the rats had 381
acquired stable adjunctive drinking behaviour (i.e. the volume of consumed water did not 382
change more than 10 % within two consecutive days), three test sessions were performed. 383
During each test session volume of consumed water, was assessed. 384
385
Startle and PPI test 386
Startle and Pre-Pulse Inhibition (PPI) testing were performed as described (Frau et al., 2016). The 387
apparatus used for detection of startle reflexes (Med Associates, St Albans, VT, USA) consists of 388
four standard cages placed in sound-attenuated chambers with fan ventilation. Each cage consists of 389
a Plexiglas cylinder of 9 cm diameter, mounted on a piezoelectric accelerometric platform 390
connected to an analogue-digital converter. Two separate speakers convey background noise and 391
acoustic bursts. Both speakers and startle cages are connected to a main PC, which detects and 392
analyzes all chamber variables with specific software. Before each testing session, acoustic stimuli 393
and mechanical responses were calibrated. 394
The test begins with a 5-min acclimatization period, with a 70-dB background white noise, which 395
continues for the entire session. The acclimatization period is followed by three blocks, each 396
15
consisting of a sequence of trials: the first and the third block consist of five pulse-alone trials of 397
115 dB. The second block consists of a pseudorandom sequence of 50 trials, including 12 pulse-398
alone trials, 30 trials of pulse preceded by 74, 78, or 82 dB pre-pulses (10 for each level of pre-pulse 399
loudness), and 8 no-pulse trials, where only the background noise is delivered. Inter-trial intervals 400
(i.e., the time between two consecutive trials) are randomly selected between 10 and 15 s. The % 401
PPI was calculated using the following formula: 100 - (mean startle amplitude for pre-pulse pulse 402
trials/ mean startle amplitude for pulse alone trials) x 100. 403
404
405
Experimental Design and Statistical Analysis 406
All the data are expressed as the mean ± SEM and the statistical analysis was performed with the 407
software SPSS 21.0 and SigmaPlot 12.5 and GrapPad Prism 6. Sample sizes are determined by 408
intrinsic variation of the data set. All sample sizes are indicated in the figure legends. Population 409
genotype distribution was analyzed by Chi-square test. Two-way ANOVA was used when 410
comparing two variables (genotypes and drugs). Bonferroni test, Dunnet’s test and t-test were used 411
for post-hoc comparisons depending on the experiments. One-way ANOVA test was used for 412
multiple group comparisons followed by Tukey’s or Bonferroni posthoc test. Student’s unpaired 413
two-tailed t-test was used when two groups were compared. P<0.05 was predetermined as the 414
threshold for statistical significance. 415
416
16
417
Results 418
Generation of DAT-KO rats 419
Slc6a3 (DAT) KO rats were generated by Sigma Advanced Genetic Engineering Labs (SAGE®
420
Labs; Sigma–Aldrich Co., St. Louis, MO). DAT-KO rats were created by using ZFN technology 421
that produces a 5 bp mutation and an early stop codon (Fig. 1A). The targeted DNA to be deleted 422
(Slc6a3 exon 2) contained a specific nucleotide sequence substrate for restriction enzyme BtsIMutI. 423
According to our genetically modified model, disruption of this specific sequence causes the loss of 424
restriction site for BtsIMutI enzyme, resulting in wild-type rats (WT) maintaining all restriction 425
sites on both alleles while heterozygous (DAT-HET) rats bear one truncated allele and an intact 426
one. DAT-KO rats lose both restriction enzyme sites as predicted. (Fig. 1B). PCR primers were 427
designed according to protocol and amplified DNA was digested as shown (Fig. 1B and C). In 428
DAT-KO rats no detection of digestion product was observed, due to the loss of restriction site on 429
both alleles. DAT-HET rats show both the digested DNA at lower molecular weight and mutated 430
DNA, while WT DNA amplicon was completely digested by BtsIMutI, thus resulting in one single 431
band at lower molecular weight (Fig. 1B and C). We also verified the absence of DAT protein in 432
KO animals in western blot from striatal tissue (Fig. 1D and extended data Fig. 1-1). 433
While there are reasonable concerns for potential off-targeting effects of gene editing nucleases 434
(including ZFN) technologies, there are little chances for these events in DAT-KO rats. First, 435
the ZFN designs were generated using a proprietary algorithm by bioinformatics team at SAGE 436
Laboratories that screens each design for the top 20 most homologous sequences, as well as for 437
repeat elements, SNPs, and splice variants. In addition, the ZFN used in this study contains 438
specifically engineered obligate heterodimer FokI cleavage domains that help guard against off-439
targeting by increasing specificity to cut at only the desired site. In any case, founder animals were 440
bred back to WT Wistar Han rats (Charles River, France) for 6 generations. that should be sufficient 441
17
to minimize chances of any potential unintended off-target effects that may have occurred even with 442
rigorous screening in the ZFN design and construction phase. Assuming hypothetical, unlinked off-443
target modifications will be diluted through breeding, an indirect way to detect potential off-target 444
events could be to compare phenotypically early-generation to later-generation homozygotes. The 445
lack of difference in phenotypes implies the absence of off-target events. Animals starting from the 446
3rd generation of backcrossing were used in neurochemical and locomotor activity studies. For all 447
other experiments we used animals following the 6th generation of backcrossing. No difference in 448
the locomotor hyperactivity of early-generation and later generation homozygotes was observed. 449
The Wistar Han DAT-KO rat colony was kept under HET-HET breeding. Surprisingly, DAT-KO 450
rats do not show a propensity to premature death as DAT-KO mice (Giros et al., 1996), although it 451
might depend on the genetic background, as for example in F1 D2/B6 hybrid mice no such lethality 452
was present (Morice et al., 2004). They are viable as the WT controls and population genotype 453
distribution at age 4 months was not significantly different (Chi-square=1,7; DF=2; p=0.4275, Chi-454
square test) from expected Mendelian distribution 1:2:1, resulting from HET/HET mating (Fig. 1E). 455
Analysis of body weight from birth up to adulthood (4 months) revealed a major effect of genotype 456
in both male (Fig. 1F; 2-way ANOVA; F interaction = 58,40; p=0.0001) and female (Fig. 1F; 2-way 457
ANOVA; F interaction = 11,11; p=0.0001) populations with DAT-KO animals showing lower 458
weights compared to HET and WT siblings. At the same time no difference in food intake was 459
found between genotypes (Fig. 1G). 460
461
Neurochemical characterization of striatal DA transmission in DAT-KO rats 462
Analysis of DA dynamics by Fast Scan Cyclic Voltammetry 463
In order to evaluate consequences of DAT gene deletion on the extracellular dynamics of DA we 464
employed Fast Scan Cyclic Voltammetry (FSCV) technique on brain slices (Jones et al., 1998). 465
First, we detected the kinetics of evoked DA release and clearance following single pulse (400 μA, 466
18
1 ms, biphasic) stimulation in striatal slices prepared from DAT-KO, DAT-HET and WT 467
littermates. The oxidation peak occurred at approximately +0.6 V and the reduction peak at 468
approximately -0.2 V (Fig. 2A, insert), consistent with the electrochemical characteristics of DA 469
(Bradaia et al., 2009; Leo et al., 2014; Jones et al., 2006; John and Jones, 2007). The maximal 470
amplitude of DA overflow evoked by single pulses in the dorsal striatum was not significantly 471
different between genotypes but the clearance of released DA was markedly prolonged in DAT-472
HET and DAT-KO rats (Fig. 2A, B, C). We evaluated the uptake kinetics from an exponential fit 473
curve using a least squares-constrained exponential fit algorithm (Yorgason et al., 2011) and 474
quantified the half-life parameters for an estimation of DA uptake rates. Under basal conditions, the 475
time to clear released DA was 1.3, 10 and 50 seconds respectively in WT, DAT-HET and DAT-KO 476
rats (Fig. 2B). 477
Next, we evaluated the effect of cocaine on evoked DA release and clearance. As might be 478
expected, application of 3 PM cocaine progressively prolonged an amplitude of striatal DA outflow 479
and clearance (John and Jones, 2007) in WT and DAT-HET, but not in DAT-KO rats (Fig. 2C, D). 480
Further, in order to test whether serotonin transporter (SERT) could provide extracellular DA 481
clearance via promiscuous uptake, we applied 10 PM fluoxetine, a selective SERT blocker. As 482
presented (Fig. 2E), fluoxetine did not affect DA clearance in any of the genotypes. To evaluate if 483
DA metabolizing enzymes can contribute to the clearance of DA in the absence of DAT we then 484
tested the effect of the inhibition of Catechol-O-methyl-transferase (COMT) by 10 PM tolcapone 485
(Fig. 2F) and monoamine oxidase (MAO) by 10 PM pargyline (Fig. 2G). COMT inhibition did not 486
alter the kinetics of DA clearance in any genotype whereas inhibition of MAO did not affect 487
released DA half-life in WT or DAT-HET animals but prolonged it in DAT-KO rats (Fig. 2F, G). 488
489
Analysis of extracellular DA levels by in vivo microdialysis 490
To evaluate the consequences of disrupted DA clearance on the basal extracellular DA levels, we 491
applied a quantitative low perfusion rate microdialysis approach to freely moving animals that, 492
19
unlike conventional microdialysis, provides a true measure of extracellular neurotransmitter 493
concentrations (Bradaia et al., 2009; Leo et al., 2014). As expected, DAT-KO and DAT-HET rats 494
showed an increased amount of basal extracellular DA levels in the striatum with about 8-fold and 495
3-fold increase over WT levels, respectively (Fig. 3A). Levels of DA metabolites DOPAC and 496
HVA were elevated in DAT-KO rats only. Furthermore, to assess effects of AMPH on striatal DA 497
release, we administered AMPH and monitored the extracellular levels of DA in the striatum of 498
freely moving rats by a conventional microdialysis. As expected, AMPH produced a genotype-499
dependent effect (Fig. 3D, E, F). AMPH induced significant increase in extracellular DA in WT and 500
to a lesser degree in DAT-HET rats. At the same time, no effect was found in animals lacking the 501
DAT. 502
503
Striatal tissue DA, 5-HT and metabolites 504
We next evaluated the total tissue content of monoamines and their metabolites in the striatum (Fig. 505
3G). Like in DAT-KO mice (Jones et al., 1998), we observed a pronounced reduction in total tissue 506
DA levels in DAT-KO rats (around 13-fold; Fig. 3G) compared to control and HET animals. At the 507
same time, the levels of an intraneuronal DA metabolite DOPAC and predominantly extracellular 508
DA metabolite HVA, appeared to be significantly increased in DAT-KO rats. Similar to DAT-KO 509
mice (Jones et al., 1998), striatal 5-HT levels were decreased in DAT-KO rats with no changes 510
observed in 5-HIAA levels. 511
Expression profile of selected DA-related genes in striatal samples 512
To evaluate the consequences of remarkable changes in the extracellular DA due to absence of re-513
uptake process, we evaluated by real time PCR the expression of genes critical for DA homeostasis 514
in the midbrain and striatum and TH protein levels in the striatum by western blot. In the midbrain 515
of DAT-KO rats, TH mRNA was decreased (Fig. 3H), with even more pronounced decrease of TH 516
protein levels in the striatum (Fig. 3I). Similar decrease was reported previously in DAT-KO mice; 517
20
(Jaber et al., 1999). DAT-KO rats, like mice (Giros et al., 1996) also showed decreased levels of 518
both dopamine D1 (D1R) and dopamine D2 (D2R) receptors in the striatum in response to 519
persistently elevated extracellular DA (Fig. 3J and K, respectively). 520
521
Behavioral Phenotyping of DAT-KO rats 522
Locomotor activity tests 523
It is well known, that alterations in DAT function cause pronounced changes in locomotor behavior 524
by influencing the dopaminergic tone in the basal ganglia (Giros et al., 1996). We evaluated the 525
locomotor activity of mutant rats in home cages during 24 hours and observed a significantly 526
altered pattern of motor activity in DAT-KO animals that varied according to the light/dark cycle, 527
increasing significantly during the night compared to WT and HET siblings (Fig. 4A). Locomotor 528
activity of DAT rats was also assessed in a novel environment in illuminated conditions using 529
activity chambers. Total distance traveled and vertical locomotor activity counts were recorded in 530
1.5, 2.0, 2.5, 3.0 and 4.0 months-old animals (Fig. 4B, C). In each tested age, total distance traveled 531
by DAT-KO rats was highly elevated comparing WT and DAT-HET animals, with a characteristic 532
to DAT-KO mice perseverative locomotor pattern of activity (Ralph et al., 2001). DAT-KO rats 533
also displayed prominent vertical activity at all tested ages (Fig. 4D, E). 534
Next, we evaluated effects of compounds that are known to suppress hyperactivity of DAT-KO 535
mice (Gainetdinov, 2008). Among them, the most noticeable are psychostimulants AMPH and 536
MPH used in the treatment of ADHD (Gainetdinov et al., 1999). Administration of AMPH (1, 2, 3, 537
and 4 mg/kg i.p.) to DAT-KO animals had a paradoxical calming effect in hyperactive DAT-KO 538
animals with the most significant effect starting from 2 mg/kg AMPH (Fig. 4F and H). In WT and 539
HET rats, treatment with this psychostimulant produced a significant increase in locomotor activity 540
(Fig. 4F and H). Similar results were obtained with MPH (Ritalin ®), drug of choice in the 541
treatment of ADHD. Administration of 1.5, 2.5 and 5 mg/kg MPH dose-dependently reduced the 542
21
hyperlocomotion in DAT-KO animals (Fig. 4F and H) while inducing a strong increase in WT and 543
DAT-HET rats. Recent studies have revealed that Trace Amine-Associated Receptor 1 (TAAR1) 544
can regulate the DA system and affect dopamine-related behaviors. As it has been shown previously 545
in DAT-KO mice (Revel et al., 2011), the partial TAAR1 agonist, RO5203648, effectively reduced 546
the hyperlocomotor behavior of DAT-KO rats without significant effect in control and DAT-HET 547
animals (Fig. 4F and H). We finally demonstrated that the administration of the typical 548
antipsychotic drug, haloperidol (0.5 mg/kg, s.c.), potently reversed hyperlocomotion in the DAT 549
mutant rat model as it does in DAT-KO mice (Gainetdinov et al., 1999). Haloperidol administration 550
also reduced the locomotor activity of WT and DAT-HET rats (Fig. 4G and I) 551
552
Cognitive tests 553
To evaluate the consequences of DAT-deficiency related hyperdopaminergia on cognitive abilities 554
of rats we first employed Y-maze task. The performance of the rats in the Y-maze task is expressed 555
as a percentage and refers to ratio of arm choices differing from the previous two choices to the 556
total number of arm entries. DAT-KO rats alternated between the arms of the maze significantly 557
less than WT and HET rats (F(2,24)=11.944, P=0.0003) indicating deficiency in working memory 558
(Fig. 5A). Importantly, analysis of food consumption (ad libitum) revealed no difference in food 559
intake between genotypes (Fig. 1G). 560
Schedule-induced polydipsia is excessive drinking that induced by a particular sub-optimal 561
schedule of food reinforcement is considered to model compulsive behaviors in rats (Alonso et al., 562
2015). The polydipsia induced by FT 60 sec schedule of reinforcement was successfully established 563
in WT (15.9±1.7 ml) and DAT-HET (10.4±1.3 ml), but not in DAT-KO rats (2.5±0.7 ml) (Fig. 5B). 564
Following the rank transformation, the data were subjected to one-way ANOVA which 565
demonstrated significant effect of genotype (F(2,42)=23,078, P=0.00001). Post hoc analysis 566
(Bonferroni’s test) revealed that WT consumed more water than DAT-HET and both WT and DAT-567
HET more than DAT-KO rats. 568
22
Finally, DAT-KO rats showed significant difference also in the startle response and pre-pulse 569
inhibition test, used to assess sensorimotor gating functioning (Fig. 5C and D). DAT-KO rats 570
showed an increased startle response amplitude and impaired pre-pulse inhibition compared to WT 571
and DAT-HET rats. Two-way ANOVAs revealed significant differences both in startle response (F 572
(2,42) = 7.35, P = 0.01) and pre-pulse inhibition (F (2,42) = 21.11, P = 0.001) (for significance level 573
of single points see Fig. 5C and D). No differences were found between WT and DAT-HET rats (all 574
P > 0.05). 575
576
Analysis of BDNF transmission in DAT-KO rats 577
In an attempt to investigate the putative mechanisms responsible for the cognitive impairment in 578
DAT-KO rats, we evaluated the pattern of BDNF expression and related signaling in the rat 579
prefrontal cortex (Fig 5E-H). Fig. 5E shows that, in the homogenate, total BDNF mRNA levels are 580
reduced in the prefrontal cortex of DAT-KO rats (-12%, t(8)= 3.2; p= 0.013). In order to get further 581
insights into the complex organization of the BDNF gene, we investigated the modulation of several 582
transcripts whose expression is driven by separate promoters (Aid et al. 2007). We focused our 583
analysis on BDNF exon IV, the most abundant transcript whose transcription is activity-dependent, 584
and exon VI, primarily targeted to dendrites (Pruunsild et al., 2011). Interestingly, the analysis of 585
these different BDNF transcripts revealed that such decrease could be ascribed to BDNF exon IV (-586
20%, t(8)= 4.43; p=0.002) since no changes were seen in BDNF exon VI (+2%, t(8)= 0.26; p=0.80; 587
Fig. 5E). We then restricted our analysis to the regulation of promoter responsible for the 588
transcription of BDNF exon IV, by investigating its main transcription factors. We found reduced 589
gene expression of the calcium responsive factor (CaRF) (-21%, t(8)= 4.15; p=0.003) and neuronal 590
Per-Arnt-Sim domain protein 4 (Npas4) (-40%, t(8)= 5.41; p=0.0006) with no changes in cAMP 591
responsive element binding protein (Creb) (Fig. 5F). Since the reduction of CaRF mRNA levels 592
may suggest defective processes mediating calcium influx, we measured the activity of 593
Ca2+/calmodulin-dependent protein kinase II (DCaMKII), known to be activated by calcium. As 594
23
shown in Fig. 5G and H, the activation of DCaMKII (as reflected by the ratio between the 595
phosphorylated and the respective total form) is significantly reduced in the prefrontal cortex of 596
DAT KO rats (-30%, t(8)=3.30; p=0.011). We then measured mBDNF protein levels in the 597
prefrontal cortex and found it reduced (-30%, t(8)=3.12; p=0.014). Similarly, we found a significant 598
reduction in the activation of the high affinity BDNF receptor trkB (-30%, t(8)=2.38; p=0.044; Fig. 599
5G and H). 600
We then measured mBDNF expression and signaling in the dorsolateral striatum. Fig. 5I and K 601
shows that lack of DAT induces the subcellular redistribution of the neurotrophin in this brain 602
region, as mBDNF is increased in the cytosol of dorsolateral striatum while reduced in the post-603
synaptic density (homogenate: +143%, t(7)=9.56; p<0.0001; cytosol: +78%, t(7)=2.59; p=0.036; post-604
synaptic density: -20%, t(8)=2.53; p=0.035). Additionally, in the post-synaptic density of the 605
dorsolateral striatum, we found reduced expression and phosphorylation of the high affinity BDNF 606
receptor, trkB (-15%, t(8)=2.38; p=0.045; Fig. 5J and K) suggesting a reduced BDNF-mediated 607
signaling in DAT-KO rats. As the interaction between dopaminergic and glutamatergic systems in 608
the striatum are important for cognitive processing, we examined the expression of a critical 609
determinant of glutamate transmission, PSD-95, as an index of glutamate spine density, and found a 610
reduced expression of PSD-95 in the striatum of DAT-KO rats compared to WT counterparts (-611
21%, t(8) 2.57; p=0.033; Fig. 5J and K). 612
613
614
Discussion 615
The involvement of DA neurotransmission in several physiological and pathological conditions has 616
led to intense studies on animal models of dopaminergic dysfunction. One such model, DAT-KO 617
mice, has provided numerous advances in understanding the role of DA in these disorders (Giros et 618
al., 1996; Gainetdinov, 2008). Here we describe the rat KO model of DAT deficiency that provides 619
certain advantages compared to the mouse model. Rat models offer many powerful advantages: the 620
24
larger size means larger tissues and samples that can lead to a reduction in the number of animals 621
required for a study. Moreover, the larger size of the rat allows surgeries that are much easier to 622
perform, particularly in small brain areas. Rats are particularly far superior in comparison to mice 623
when it comes to behavioural assays. They perform much more reliably and robustly than mice, and 624
mouse behavioural assays typically require larger cohort sizes than those needed for rats due to 625
increased variability. 626
627
Viability of DAT-KO rats 628
DAT-KO rats are viable and demonstrate the expected Mendelian ratio from birth up to 4 months of 629
age. By comparison, DAT-KO mice showed a continuous decrease in survival rate starting from 4 630
weeks of age with about 35% of mutants demonstrating neurological dysfunctions and death due to 631
postsynaptic neurodegeneration (Cyr et al., 2003) although it might depend on genetic background 632
of mice (Morice et al., 2004). Like mutant mice (Bossé et al., 1997), DAT-KO rats develop 633
normally but weigh less than HET and WT control animals, an effect independent from food intake. 634
635
Dysregulation of striatal DA transmission in DAT-KO rats 636
To evaluate the neurochemical consequences of the absence of DAT on striatal DA transmission, 637
we first measured real time extracellular DA dynamics following evoked release by using FSCV. 638
As in DAT-KO mice (Jones et al., 1998), DA persists for a protracted period in the synaptic cleft in 639
DAT-HET and DAT-KO rats. The clearance of extracellular DA was about 40-fold slower in the 640
striatum of DAT-KO compared to WT controls while decreased about 2-fold in DAT-HET rats. 641
Extracellular half-life of evoked DA in the striatal slices of DAT-KO animals was not altered by 642
cocaine, fluoxetine or COMT inhibitor tolcapone. At the same time it was somewhat prolonged by 643
MAO inhibitor pargyline, indicating that while diffusion is the major mechanism available to 644
eliminate DA from the extracellular space in the absence of DAT (Jones et al., 1998), there is some 645
minor contribution of MAO in DA clearance as well (Benoit-Marand et al., 2000). As in DAT-KO 646
25
mice (Jones et al., 1998), prolonged extracellular lifetime of released DA resulted in about 3 and 7-647
fold increase in striatal basal extracellular levels of DA in DAT-HET and DAT-KO rats, 648
respectively. At the same time, like in DAT-KO mice, total tissue content of striatal DA was 649
reduced 13-fold, indicating the critical contribution of DAT-mediated transport of DA in 650
maintenance of intraneuronal stores of DA (Jones et al., 1998). The only neurochemical difference 651
detected was a significant increase in total tissue concentration of DOPAC, presumably indicating a 652
more prominent role of MAO in metabolism of DA in rats in comparison to mice. As for cocaine in 653
FSCV experiments, AMPH effects on striatal extracellular DA levels was absent in DAT-KO and 654
reduced in DAT-HET rats, indicating the critical role of DAT in the effects of this psychostimulant. 655
The absence of DAT and the persistent elevation of DA in the synaptic cleft produce profound 656
variation in key DA-related genes. Like in DAT-KO mice (Giros et al., 1996) striatal mRNA levels 657
of the two major DA receptors, D1 (D1R) and D2 (D2R) are significantly decreased. At the same 658
time, midbrain mRNA and striatal protein levels of the rate-limiting enzyme for DA biosynthesis, 659
TH were decreased in DAT-KO rats supporting previous observations in DAT-KO mice (Jaber et 660
al., 1999). 661
662
Hyperactivity of DAT-KO rats 663
The most prominent phenotype of DAT-KO rats is the hyperlocomotion determined by an increased 664
DA tone in basal ganglia. DAT-KO rats showed an intense locomotor activity both in a new 665
environment and in the home cages. The hyperlocomotion of homozygous KO rats persists during 666
the lifespan, and as already seen in DAT-KO mice, is greater than the effect produced by DAT 667
blocker drugs (Giros et al., 1996). As in DAT-KO mice, AMPH and MPH significantly reduced 668
locomotor activity of DAT-KO rats while inducing the well-known increase in DAT-HET and WT 669
animals (Gainetdinov et al., 1999). The psychostimulatory actions of AMPH and MPH are 670
primarily dependent on a direct interaction of these compounds with DAT, leading to elevated DA 671
(Fumagalli et al., 1998; Chen et al., 2006; Thomsen et al., 2009). Because DAT-KO rats lack the 672
26
major target of AMPH action, the “calming” effect of psychostimulants suggests the involvement of 673
a DAT-independent mechanism of action, possibly involving other monoamine transporters. 674
Interestingly, we observed an inhibitory action of MPH at lower (about 10-fold) doses in rats in 675
comparison to mice (Gainetdinov et al., 1999) suggesting significant differences in 676
pharmacodynamic or pharmacokinetic properties of this drug between these species. Hyperactivity 677
and paradoxical inhibitory effects of psychostimulants used in the treatment of ADHD, could be a 678
first indication of face and predictive validity of DAT-KO rats as an improved animal model for this 679
disorder. Moreover, the ability of the partial TAAR1 agonist, RO5203648, to modulate the 680
locomotor behaviour of DAT-KO rats supports the potential of TAAR1 as a target for therapeutic 681
intervention in DA-related disorders. Previous data showed that activation of TAAR1 strongly 682
regulates DA transmission and reduces DA-dependent behavioural effects of psychostimulants as 683
well as hyperactivity of DAT-KO mice (Bradaia et al., 2009; Revel et al., 2011; Leo et al., 2014) 684
likely via an interaction of TAAR1 with D2R or with different neurotransmitter pathways, such as 685
the glutamatergic (Espinoza et al., 2015). 686
687
DAT-KO rats show DA-related cognitive deficits 688
As an indication of DA-related cognitive deficits, DAT-KO rats, like DAT-KO mice, show an 689
impaired sensorimotor gating measured as reduced pre-pulse inhibition (PPI) of the acoustic startle 690
reflex (Ralph et al., 2001). Notably, DA hyperactivation, induced by pharmacological interventions 691
or genetic manipulation of DAT, causes PPI deficits in experimental animals similar to those 692
observed in psychiatric disorders, such as schizophrenia and ADHD (Braff et al., 2001; Swerdlow 693
et al., 2001); (Yamashita et al., 2006; Arime et al., 2012). DAT-KO rats show also a decreased Y-694
maze spontaneous alternation that indicated an impaired working memory function. Since intact 695
dopaminergic signalling in the prefrontal cortex seems to be required for correct mnemonic function 696
(Goldman-Rakic, 1996; Kesner and Rogers, 2004), we can postulate that absence of DA reuptake 697
could impair the retention of short term memory information in DAT-KO rats. At least some of 698
27
these dopaminergic effects may involve D1 receptors (Lidow et al., 1991; Goldman-Rakic et al., 699
1992; Gaspar et al., 1995). 700
Schedule-induced polydipsia (SIP) is an adjunctive model in which animals exhibit exaggerated 701
drinking (polydipsia) when presented with food pellets under a fixed-time schedule. SIP is one of 702
the best established tests of compulsive behaviours in rats that commonly used to model obsessive 703
compulsive disorder (OCD) (Alonso et al., 2015). The dopaminergic neurotransmission plays an 704
important role in the development and maintenance of SIP (for review see Moreno and Flores, 705
2012). The lack of SIP development in DAT-KO animals is in concordance with several reports 706
showing that AMPH is able dose-dependently attenuate SIP (Sanger, 1977; Flores and Pellón, 707
1997). Interestingly, that ADHD and OCD are often considered as disease antipodes with the 708
significant difference in brain biochemistry (Carlsson, 2000) and our results indicating a deficit in 709
the development of compulsive behaviour in DAT-KO rats might further support the use of these 710
mutant animals as a valuable model related to ADHD. 711
712
Frontostriatal BDNF dysregulation in DAT-KO rats 713
In an attempt to find a molecular correlate of the above shown cognitive deficit, we focused 714
our attention on the neurotrophin BDNF, which plays a role in working memory (Egan et al., 2003) 715
and whose expression was found to be developmentally dysregulated in the prefrontal cortex of 716
DAT-KO mice (Fumagalli et al., 2003). We found that DAT deletion in rats reduces total BDNF 717
mRNA levels in the prefrontal cortex. Further, the analysis of the main different transcripts 718
produced by the activation of the different promoters at 5’-UTR revealed that only BDNF exon IV 719
is reduced. Since this transcript is tightly dependent on neuronal activity we suggested that, in these 720
rats, activity-dependent gene expression is impaired. Moreover, the analysis of the transcription 721
factors involved in the modulation of BDNF exon IV showed that the expression of the neuronal 722
transcription factor NPAS 4, an immediate early gene selectively induced by neuronal activity (Sun 723
and Lin, 2016), is reduced as well. Notably, the expression of calcium responsive factor CaRF, 724
28
which also regulates the BDNF exon IV expression, is reduced suggesting reduced calcium influx, 725
an evidence corroborated by the reduced activation of DCaMKII, a well-known intracellular 726
calcium sensor required for CaRF activity (Zheng et al., 2011). Given that both calcium influx and 727
DCaMKII activation often represent the first stimulus for activity-dependent gene expression in 728
neurons (Fields et al., 2005), we suggested defective calcium influx as a potential mechanism for 729
the impairment in the cortical activity-dependent gene expression in the prefrontal cortex of DAT 730
KO rats. Changes in BDNF mRNA levels were accompanied by a down-regulation of BDNF 731
protein levels, suggesting that DAT deletion regulates also the translation of the neurotrophin. Of 732
note, we also found reduced activation of the high affinity BDNF receptor trkB, suggesting that, in 733
the prefrontal cortex of DAT KO rats, the BDNF-mediated transmission is impaired, an effect that 734
may contribute to the impaired working memory function (Galloway et al., 2008). 735
Our analyses also revealed that the removal of DAT altered the BDNF system also in the 736
dorsolateral striatum, where it results in a redistribution of the neurotrophin. In the whole 737
homogenate, we found increased expression of BDNF leading to the activation of trkB and its 738
downstream intracellular signalling; however, the removal of DAT has led to increased BDNF 739
expression in the cytosol while reducing its expression, together with its downstream signalling, in 740
the post-synaptic density. This observation may have a relevant and functional effect, given the 741
well-established role of BDNF in the control of glutamatergic spine density (Young et al., 2015) 742
and since reduced spine density was reported in striatal medium spiny neurons of DAT-KO mice 743
(Berlanga et al., 2011). In fact, in line with the herein shown reduced BDNF expression, we also 744
found a decreased expression of the marker of glutamatergic spines PSD-95, as previously observed 745
in DAT-KO mice (Yao et al., 2004), suggesting potential structural rearrangements of glutamate 746
neurons in the dorsolateral striatum. 747
748
Conclusions 749
29
Taken together, these observations indicate that DAT-KO rats represent an improved model of 750
persistently increased dopaminergic transmission, that is presumably involved, at least in part, in 751
endophenotypes of such disorders as schizophrenia, bipolar disorder, ADHD and Huntington’s 752
disease (Carlsson, 1987; Howes and Kapur, 2009; Gardoni and Bellone, 2015; Bonvicini et al., 753
2016; Vengeliene et al., 2017). These rats have obvious advantages over mouse models by having 754
increased survival rate, a larger size of the brain allowing investigations of smaller brain regions 755
and being closer physiologically to humans. Since behavioural repertoire of rats is significantly 756
more complex in comparison to mice, mutant rats could be particularly useful to evaluate the effects 757
of novel therapeutics on cognitive functions. 758
759
760
Figure Legends 761
Figure 1. Generation of DAT knockout (KO) rats. A) DAT-KO rats were generated by using zinc 762
finger nuclease (ZFN) technology that produces a 5 bp deletion and an early stop codon. Green 763
solid lines indicate exons 2 and 3, grey solid lines indicate introns; yellow boxes indicate DNA 764
domains interaction sites; blue box indicates KO targeting site; red box indicate early stop codon 765
generated after frameshift due to cleavage of 5 base pairs operated by ZFN. B and C) PCR primers 766
were designed (black arrows). Wild-type (WT) DNA contains BtsIMutI restriction enzyme site 767
(blue brackets). WT amplified DNA is fully digested into two low molecular weight bands (104 bp 768
and 71 bp). Homozygote mutated DNA loses digestion sites on both alleles, therefore resulting in 769
only one final PCR product of 170 bp. Heterozygosis shows both digested DNA at lower molecular 770
weight from the WT allele and mutated DNA at 170 bp. D) Western Blot analysis of DAT 771
expression in striatal samples of WT and DAT-KO rats. DAT-KO rats in contrast to WT controls 772
show complete absence of DAT protein expression. Please see also another representative blot as 773
extended data Figure 1-1. E) Rats were bred following heterozygous (HET) male/HET female 774
breeding scheme. 223 animals were born from 19 offsprings and we obtained 62 DAT+/+ (26.46%), 775
30
102 DAT+/- (45.74%) and 59 DAT-/- (27.80%), numbers very close to the ratio (1:2:1) expected in 776
the absence of pre- and postnatal death. Infantile mortality was completely absent for all three 777
genotypes followed for up to 4 months. F) Developmental phenotype. Both DAT-KO male and 778
female rats develop normally but have lower weight in comparison to DAT-HET and WT rats 779
(n=10-20 per group). G) Analysis of food consumption for 3 days (ad libitum) revealed no 780
difference between genotypes (males, n=6-8 per genotype). 781
782
Figure 2. Recording of electrically stimulated DA efflux in striatal brain slices from DAT-KO rats. 783
A) A single biphasic stimulus (400 μA, 1 ms) was used to evoke DA release in striatal brain slices 784
from WT, DAT-HET and DAT-KO rats. The average time to clear released DA is 1.3; 10 and 50 785
second respectively. Lower panel. Color plot represent the voltammetric currents (encoded in color 786
in the z axis) plotted against the applied potential (y axis) and time (x axis). Upper Panel. Cyclic 787
voltammogram identifies the detected analyte as DA. B) Kinetics of evoked DA release in dorsal 788
striatum of DAT WT, DAT-HET and DAT-KO. C and D) Effect of 3μM cocaine on evoked DA 789
release. Cocaine had no effect on evoked DA overflow in DAT-KO rat but induced the well-known 790
increase in DA overflow in both WT and DAT-HET (C) Cocaine-induced changes in extracellular 791
DA half-life (D). E) Fluoxetine (10μM) effect on evoked DA release. F) Tolcapone (10 μM) 792
effect on DA release. G) Pargyline (10 μM) effect on evoked DA release (n=5-6 per each group, *- 793
p<0.05; **- p<0.01 ***p<0.001). 794
Figure 3. Neurochemical profile of striatal DA transmission in DAT-KO rats. A-C) Quantitative 795
low perfusion rate microdialysis in freely moving rats showed an increased amount of extracellular 796
DA (A) and both DA metabolites, DOPAC (B) and HVA (C) in the striatum of DAT-KO rats. 797
Results are the mean ± SEM of six independent experiments. E-F) Effect of AMPH (2 mg/kg, i.p.) 798
on extracellular DA levels measured by conventional microdialysis in the striatum of freely moving 799
rats. Results are the mean ± SEM of six independent experiments. G) HPLC analysis on striatal 800
samples showed 13-fold decrease in total tissue DA levels along with increased metabolites 801
31
DOPAC and HVA levels (n=5-6). H-J) Molecular profile of selected DA related genes in striatal 802
samples. H) TH mRNA expression is decreased in DAT-KO midbrain samples I) TH protein levels 803
in the striatum are decreased in DAT-KO rats. J) D1R mRNA levels are decreased in both DAT-804
HET and DAT-KO striatal samples. K) D2R mRNA levels are reduced in DAT-KO striatal 805
samples. (n=6; one-way ANOVA; *- p<0.05; **- p<0.01 ***p<0.001). 806
807
Figure 4. Basal and drug modified locomotor activity of DAT-KO rats. A) Locomotor activity of 808
adult animals was recorded in home cages for 24 hours. B, D) Total distance travelled and vertical 809
activity of animals in different ages were assessed using an automated Omnitech Digiscan 810
locomotor activity chambers for 2 hours. C, E) Additionally, total distance and vertical activity of 4 811
month-old animals were evaluated for 4 hours. The results are expressed as the mean ± SEM. * and 812
# P < 0.001 (Bonferroni’s test), relative to the corresponding WT or DAT-HET groups. N = 6–20 813
per group. F-I) Drug effects on vertical activity and total distance traveled of DAT-KO rats. Before 814
drug administration, the rats were habituated to the activity monitor for 30 min. After drug 815
injection, the locomotor activity of the animals was recorded for additional 70 min or 90 min 816
(haloperidol). Total distance covered (F, G) and the vertical activity (H, I) counts for 60 min (from 817
40 min to 100 min) for all drugs (F, H) or 90 min (from 30 min to 120 min) for haloperidol (G, I) 818
were used for subsequent analysis. The results are expressed as the mean ± SEM. * P < 0.05 819
(Dunnet’s test, t-test), relative to the corresponding saline-treated control groups. N = 6–19 per 820
group, with exception of DAT-HET rats treated with haloperidol (n=4). 821
Figure 5. Cognitive dysfunction and altered BDNF transmission in DAT-KO rats. A) Spontaneous 822
alternation test. To measure spontaneous alternation behavior and exploratory activity, a white 823
plastic material Y-maze with arms 40 cm (long) by 6 cm (wide) with 13 cm walls was used. Each 824
animal was tested in a single 8-min session, during the course of which the animal was placed in the 825
central platform and allowed free exploration of the maze. Spontaneous alternation, expressed as a
826
percentage, refers to ratio of arm choices differing from the previous two choices to the total 827
32
number of arm entries. The percentage of alternation observed is strongly reduced in DAT-KO 828
animals. Values are expressed as Mean ± SEM of 8 rats/group. One-way ANOVA followed by 829
Bonferroni’s test. *P < 0.05, with respect to WT and DAT-HET rats. B) Schedule-induced 830
polydipsia induced by fixed-time 60 seconds schedule of reinforcement. The polydipsia induced by 831
FT 60 sec schedule of reinforcement was successfully established in WT and DAT-HET, but not in 832
DAT-KO rats. Following the rank transformation, the data were subjected to one-way ANOVA 833
which demonstrated significant effect of genotype. Post hoc analyses (Bonferroni’s test) revealed 834
that WT consumed more water than DAT-HET and DAT-KO whereas DAT-HET more than DAT-835
KO rats (P<0.05). Values are expressed as Mean ± SEM (n=9-18). C) Startle response and D) Pre-836
pulse inhibition of the startle response. The day of the test, rats were transferred in the experimental 837
room under environmentally controlled conditions (sound proof and red dim lights) and after 30 838
min of habituation were positioned in the apparatus and startle response and pre-pulse inhibition 839
assessed (see methods section). Values are expressed as Mean ± SEM of 8 rats/group. Block1, 840
Block2 and Block3 indicate the three consecutive sequences of stimuli of the test. PP1, PP2 and 841
PP3 indicate the three different intensities of the pre-pulse stimulus. Two-way ANOVA followed by 842
Bonferroni’s test. *P < 0.05, **P < 0.01, ***P < 0.001 with respect to WT rats. E) Total BDNF and 843
BDNF exon IV mRNA levels are reduced in the prefrontal cortex (PFC) of DAT-KO rats, while no 844
changes were observed in the mRNA levels of BDNF exon VI. F) The analysis of the transcription 845
factors involved in the modulation of BDNF exon IV revealed a significant reduction in the 846
expression of Npas4 and CaRF and no changes in Creb mRNA levels. G and H) Activation of the 847
DCaMKII is reduced in the PFC homogenate of DAT-KO rats. The trascriptional changes of BDNF 848
were paralleled by a reduction in the mature form of BDNF (mBDNF) levels and accompanied by 849
reduced activation of trkB in the PFC homogenate of DAT-KO rats. G) Original immunoblots used 850
to generate the data presented in I) and J). W-WT, K-KO rats. I and K) In the dorsolateral striatum 851
(DLStr), mBDNF levels are increased in the homogenate and in the cytosol while reduced in the 852
post-synaptic density. J and K) trkB activation and PSD-95 levels are reduced in the post-synaptic 853
33
density of DLStr of DAT-KO rats. Data are presented as % of WT levels. K) Original immunoblots 854
used to generate the data presented in I) and J). W-WT, K-KO rats. 855
Extended Data Figure 1-1. Full uncropped immunoblot referring to expression of DAT in DAT 856
mutant rats (Figure 1 in the manuscript). Western Blot analysis on striatal samples of WT, DAT-857
HET and DAT-KO rats. DAT-KO rats, unlike DAT-HET and WT rats show complete absence of 858
DAT protein. 859
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1078
1079
EF
0 5 10 15
0
100
200
300
400
500 WT
HET
KO
weeks
0 5 10 15
0
100
200
300
weeks
Males Females
A CB
DAT
70 KDa
KO
WT
C
DWT WT WT WT
KO KO KO KO
L
Food consumption (g)
Body weight (g)
0
5
10
15
20
25
30
35
WT HET KO
G
Genotype distribution
Total = 223
Actin
A B
C
-0,6
-0,4
-0,2
0
0,2
0,4
0,6
0,8
1
-600 -400 -200 0 20 0 400 600 800 1000 12 00
Cyclic Voltammogram
(nA vs V)
WT HET KO
0
10
20
30
40
half life (sec)
***
***
**
DEF
Tolcapone 10μM
KO
0
10
20
30
+++-- -
+++-- -
half life (sec)
0
10
20
30
40
*
*
Fluoxetine 10μM
0
10
20
30
40
KO
+++-- -
KO
HET
WT
G
Pargyline 10μM
+++-- -
0
10
20
30 **
Cocaine 3μM
Normalized current (nA)
+++-- -
0
0.5
1
1.5
2
* *
Cocaine 3μM
-0.4
-0.4
0.6
060
Time (s)
1μM
5 s
-0.4
-0.4
0.6
010
37
-24
0
WT KO
-0.4
-0.4
0.6
015
HET
25
-16
0
18
-12
0
KO
HET
WT
Fig.3
D
Time (min)
DA, % of baseline
HVA
0
100
200
300
400
C
***
**
B
DOPAC
***
**
0
100
200
300
020406080100120
0
50
100
150
200
250
300
350
400
450
WT VEH
WT AMPH
**
**
**
Time (min)
DA, % of baseline
KO VEH
KO AMPH
0 20 40 60 80 100 120
0
50
100
150
200
250
300
350
400
450
**
E
Time (min)
DA, % of baseline
HET VEH
HETAMPH
0 20 40 60 80 100 120
0
50
100
150
200
250
300
350
400
450
****
*
**
A
DA
0
10
20
30
40 WT
HET
KO
Extracellular Levels (nM)
***
** **
**
**
**
**
**
Striatal tissue levels (ng/mg wet tissue)
0
3
6
9
12
15
18
***
***
** **
**
DA DOPAC HVA 5HIAA 5HT
** **
WT
HET
KO
F
G
HIJ K
Striatal TH Protein
(Relative Protein levels)
Midbrain TH RNA
(Relative mRNA levels)
Striatal D1R
(Relative mRNA levels)
Striatal D2R
(Relative mRNA levels)
0
0.5
1
1.5
2
0
0.5
1
1.5
0
0.5
1
1.5
2
0
0.5
1
1.5
WT WT WT WTHET HET HET HETKO KO KO KO
FG
HI
ABD
Prepulse Inhibition
% of PPI
PP1 PP2 PP3
FGH
WT HET KO
Volume of consumed water (ml)
C
E
mRNA levels (% of WT)
0
50
100
150
Total
BDNF
BDNF
Exon IV
BDNF
Exon VI
***
PFC
***
0
50
100
150
Creb Npas4 CaRF
**
PFC
0
50
100
150
pCaMKII/
CaMKII
mBDNF pTrkB/
Trkb
***
Protein levels (% of WT)
PFC
0
100
200
300
Protein levels (% of WT)
mBDNF
Homogenate
mBDNF
Cytosol
mBDNF
PSD
*
*
***
DLStr
0
50
100
150 DLStr
pTrkB/
Trkb
PSD-95
Protein levels (% of WT)
**
I
100
80
60
40
20
0
% of Alternation
WT HET KO
5
10
15
20
**
0
500
1000
1500
2000
2500
Startle Amplitude
Startle Response
WT HET KO
*** **
Block1 Block2 Block3 0
20
40
60
80
100
***
JK
*
0
*
*
*** ***
mRNA levels (% of WT)
WK
Prefrontal Cortex Homogenate
pDCaMKII
T286
DCaMKII
EActin
pTrkB
Y706
TrkB
EActin
mBDNF
DLStr Homogenate
WWWWKKKK
WKWWWWKKKK
WKW W W WKKKK
EActin
mBDNF
DLStr Postsynaptic Density
WKW W W WKKKK
pTrkB
Y706
TrkB
EActin
mBDNF
EActin
mBDNF
DLStr Cytosol
WKW W W WKKKK
DLStr Postsynaptic Density
EActin
PSD-95
WKW W W WKKKK
