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. 2018 Feb 21;38(8):1959-1972.
doi: 10.1523/JNEUROSCI.1931-17.2018. Epub 2018 Jan 18.

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats

Damiana Leo  1 , Ilya Sukhanov  1   2 , Francesca Zoratto  3 , Placido Illiano  1 , Lucia Caffino  4 , Fabrizio Sanna  5 , Giulia Messa  4 , Marco Emanuele  1 , Alessandro Esposito  1 , Mariia Dorofeikova  2 , Evgeny A Budygin  6   7 , Liudmila Mus  1   2 , Evgenia V Efimova  7 , Marco Niello  1 , Stefano Espinoza  1 , Tatyana D Sotnikova  7 , Marius C Hoener  8 , Giovanni Laviola  3 , Fabio Fumagalli  4 , Walter Adriani  3 , Raul R Gainetdinov  9   10
Affiliations

Pronounced Hyperactivity, Cognitive Dysfunctions, and BDNF Dysregulation in Dopamine Transporter Knock-out Rats

Damiana Leo et al. J Neurosci. .

Abstract

Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.

Keywords: ADHD; BDNF; dopamine; dopamine transporter; rat knock-out.

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Figures

Figure 1.
Figure 1.
Generation of DAT KO (KO) rats. A, DAT-KO rats were generated by using ZFN technology that produces a 5 bp deletion and an early stop codon. Green solid lines indicate exons 2 and 3, gray solid lines indicate introns; yellow boxes indicate DNA domains interaction sites; blue box indicates KO targeting site; red box indicates early stop codon generated after frameshift due to cleavage of 5 bp operated by ZFN. B, C, PCR primers were designed (black arrows). WT DNA contains BtsIMutI restriction enzyme site (blue brackets). WT-amplified DNA is fully digested into two low-molecular-weight bands (104 and 71 bp). Homozygote-mutated DNA loses digestion sites on both alleles, therefore resulting in only one final PCR product of 170 bp. Heterozygosis shows both digested DNA at lower molecular weight from the WT allele and mutated DNA at 170 bp. D, Western blot analysis of DAT expression in striatal samples of WT and DAT-KO rats. DAT-KO rats, in contrast to WT controls, show complete absence of DAT protein expression. Another representative blot is shown in Figure 1-1. E, Rats were bred following a HET–HET female breeding scheme. A total of 223 animals were born from 19 offspring and we obtained 62 DAT+/+ (26.46%), 102 DAT+/− (45.74%), and 59 DAT−/− (27.80%), numbers very close to the ratio (1:2:1) expected in the absence of prenatal and postnatal death. Infantile mortality was completely absent for all 3 genotypes followed for up to 4 months. F, Developmental phenotype. Both DAT-KO male and female rats develop normally but have lower weight compared with DAT-HET and WT rats (n = 10–20 per group). G, Analysis of food consumption for 3 d (ad libitum) revealed no difference between genotypes (males, n = 6–8 per genotype).
Figure 2.
Figure 2.
Recording of electrically stimulated DA efflux in striatal brain slices from DAT-KO rats. A, A single biphasic stimulus (400 μA, 1 ms) was used to evoke DA release in striatal brain slices from WT, DAT-HET, and DAT-KO rats. The average time to clear released DA was 1.3, 10, and 50 s respectively. Bottom, Color plot representing the voltammetric currents (encoded in color in the z-axis) plotted against the applied potential (y-axis) and time (x-axis). Top, Cyclic voltammogram identifying the detected analyte as DA. B, Kinetics of evoked DA release in DStr of DAT WT, DAT-HET, and DAT-KO. C, D, Effect of 3 μm COC on evoked DA release. COC had no effect on evoked DA overflow in DAT-KO rat but induced the well known increase in DA overflow in both WT and DAT-HET (C) COC-induced changes in extracellular DA half-life (D). E, Fluoxetine (10 μm) effect on evoked DA release. F, Tolcapone (10 μm) effect on DA release. G, Pargyline (10 μm) effect on evoked DA release (n = 5–6 per each group, *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 3.
Figure 3.
Neurochemical profile of striatal DA transmission in DAT-KO rats. A–C, Quantitative low perfusion rate microdialysis in freely moving rats showed an increased amount of extracellular DA (A) and both DA metabolites, DOPAC (B), and HVA (C) in the striatum of DAT-KO rats. Results are the mean ± SEM of six independent experiments. D, E, F, Effect of AMPH (2 mg/kg, i.p.) on extracellular DA levels measured by conventional microdialysis in the striatum of freely moving rats. Results are the mean ± SEM of six independent experiments. G, HPLC analysis on striatal samples showed a 13-fold decrease in total tissue DA levels, along with increased metabolites DOPAC and HVA levels (n = 5–6). H–J, Molecular profile of selected DA-related genes in striatal samples. H, TH mRNA expression is decreased in DAT-KO midbrain samples. I, TH protein levels in the striatum are decreased in DAT-KO rats. J, D1R mRNA levels are decreased in both DAT-HET and DAT-KO striatal samples. K, D2R mRNA levels are reduced in DAT-KO striatal samples. (n = 6; one-way ANOVA; *p < 0.05; **p < 0.01; ***p < 0.001).
Figure 4.
Figure 4.
Basal and drug-modified locomotor activity of DAT-KO rats. A, Locomotor activity of adult animals was recorded in home cages for 24 h. B, D, Total distance traveled and vertical activity of animals in different ages were assessed using an automated Omnitech Digiscan locomotor activity chambers for 2 h. C, E, Total distance and vertical activity of 4-month-old animals were evaluated for 4 h. The results are expressed as the mean ± SEM. * and #p < 0.001 (Bonferroni's test) relative to the corresponding WT or DAT-HET groups (n = 6–20 per group). F–I, Drug effects on vertical activity and total distance traveled of DAT-KO rats. Before drug administration, the rats were habituated to the activity monitor for 30 min. After drug injection, the locomotor activity of the animals was recorded for additional 70 min or 90 min (haloperidol). Total distance covered (F, G) and the vertical activity (H, I) counts for 60 min (from 40–100 min) for all drugs (F, H) or 90 min (from 30–120 min) for haloperidol (G, I) were used for subsequent analysis. The results are expressed as the mean ± SEM. *p < 0.05 (Dunnett's test, t test), relative to the corresponding saline-treated control groups. n = 6–19 per group, with exception of DAT-HET rats treated with haloperidol, for which n = 4.
Figure 5.
Figure 5.
Cognitive dysfunction and altered BDNF transmission in DAT-KO rats. A, Spontaneous alternation test. To measure spontaneous alternation behavior and exploratory activity, a white plastic material Y-maze with arms 40 cm (long) by 6 cm (wide) with 13 cm walls was used. Each animal was tested in a single 8 min session during which the animal was placed in the central platform and allowed free exploration of the maze. Spontaneous alternation, expressed as a percentage, refers to ratio of arm choices differing from the previous two choices to the total number of arm entries. The percentage of alternation observed is strongly reduced in DAT-KO animals. Values are expressed as mean ± SEM of n = 8 rats/group. One-way ANOVA followed by Bonferroni's test. *p < 0.05, with respect to WT and DAT-HET rats. B, Schedule-induced polydipsia induced by fixed-time 60 s schedule of reinforcement. The polydipsia induced by FT 60 s schedule of reinforcement was successfully established in WT and DAT-HET, but not in DAT-KO rats. After the rank transformation, data were subjected to one-way ANOVA, which demonstrated a significant effect of genotype. Post hoc analyses (Bonferroni's test) revealed that WT rats consumed more water than DAT-HET and DAT-KO rats, whereas DAT-HET rats consumed more than DAT-KO rats (p < 0.05). Values are expressed as mean ± SEM (n = 9–18). C, D, Startle response (C) and PPI of the startle response (D). The day of the test, rats were transferred into the experimental room under environmentally controlled conditions (sound proof and red dim lights) and, after 30 min of habituation, were positioned in the apparatus and startle response and prepulse inhibition assessed (see Materials and Methods). Values are expressed as mean ± SEM of n = 8 rats/group. Block1, Block2, and Block3 indicate the three consecutive sequences of stimuli of the test. PP1, PP2, and PP3 indicate the three different intensities of the prepulse stimulus. Two-way ANOVA followed by Bonferroni's test. *p < 0.05, **p < 0.01, ***p < 0.001 with respect to WT rats. E, Total BDNF and BDNF exon IV mRNA levels are reduced in the PFC of DAT-KO rats, whereas no changes were observed in the mRNA levels of BDNF exon VI. F, Analysis of the transcription factors involved in the modulation of BDNF exon IV revealed a significant reduction in the expression of Npas4 and CaRF and no changes in Creb mRNA levels. G, H, Activation of αCaMKII is reduced in the PFC homogenate of DAT-KO rats. The transcriptional changes of BDNF were paralleled by a reduction in the mBDNF levels and accompanied by reduced activation of trkB in the PFC homogenate of DAT-KO rats. H, Original immunoblots used to generate data presented in G. W-WT, K-KO rats. I, K, In the DLStr, mBDNF levels are increased in the homogenate and in the cytosol but reduced in the postsynaptic density. J, K, trkB activation and PSD-95 levels are reduced in the postsynaptic density of DLStr of DAT-KO rats. Data are presented as percentage of WT levels. K, Original immunoblots used to generate data presented in I and J. W, WT; K, KO.
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