P.1.l.025
Learning deficits and hippocampal neurogenesis in rats overexpressing the dopamine transporter Nadine Bernhardt1, Maike Kristin Lieser1, Alexander Garthe2, Christine Winter1,3 1
Klinik für Psychiatrie und Psychotherapie, Medizinische Fakultät Carl Gustav Carus, Technische Universität Dresden, Germany; 2 German Center for Neurodegenerative Diseases (DZNE) Dresden, Germany, Klinik für Psychiatrie und Psychotherapie, Charité Campus Mitte, Berlin, Germany
Background and Objective Tourette syndrome (TS) manifests with multiple sudden, repetitive, non-rhythmic movements or vocalizations known as tics. In addition to motor symptoms, TS patients have been found to exhibit cognitive impairments in procedural or habit learning correlated with tic severity [1,2]. Rats overexpressing the dopamine transporter (DAT-tg) have proven valid to study repetitive behaviour such as seen in TS [3]. DAT provides a rapid and efficient mechanism for reuptake of synaptic dopamine and is essential for the regulation of dopaminergic neurotransmission. Neurobiological studies show that for both, striatal- and hippocampal-forebrain circuits dopamine is necessary during acquisition and performance of learned behaviours. Thus the aim of the present study was to investigate cognitive functions, i.e. behavioural performance in tasks reflecting aspects of procedural and spatial learning in the DAT-tg model in combination with analysis of adult hippocampal proliferation and neurogenesis, processes known to contribute to some forms of hippocampus-dependent learning.
Methods
Behavioral Assessment
Adult DAT-tg rats (n=16) and littermate wildtype controls (n=11) were injected intraperitoneally with BrdU three times at 6h intervals to label the proportion of newly generated cells. Within a timeframe of 10-weeks post-injection animals underwent a set of learning tasks in addition to control experiments for sensorimotor function. Brains from all rats were consequently subjected to post-mortem histochemical analysis of adult neurogenesis including active proliferation, maintenance and neuronal integration in the subgranular zone of the dentate gyrus (DG). Analysis of variance were conducted to calculate significant differences between groups.
BrdU-Injection 3x50mg/kg
Handling
- MWM - RAM - RL - SCT - OF
Handling
72
IHC randomized order
Perfusion
100
- Ki67+ - BrdU+ - BrdU+ / NeuN+
150
PND
Fig. 1: Experimental design. Behavioural analysis following injections of BrdU (5-Bromo-2Deoxyuridine) for analysis of activity dependent neurogenesis. MWM = Morris water maze, RAM = radial arm maze, RL = reversal learning, OF = open field test, SCT = sucrose consumption test, IHC = Immunohistochemistry, Ki67 = endogenous marker for proliferating cells, PND = postnatal day
Results Reversal Learning
A
B
C
D Cued trial G
48% of DAT-tg animals do not learn to discriminate the correct arm and DAT-tg rats that reach criterion need significantly more trials compared to controls
No significant difference in performance during reversal
swim path
Strategies
Fig. 3 Comparison of animals reaching the criterion of correct discrimination over five consecutive trials within 25 trials. DAT-tg rats need significantly more trials in the discrimination stage (U (19) = 17, p = .022). Reversal stage did not significantly differ between genotypes (U (19) = 33.5, p = .356).
Hippocampal neurogenesis day 1
day 4
probe trial
Fig. 2:. A) DAT-tg rats showed reduced number of successful trials i.e. locating the platform (F(1, 25) = 181.2, p < 0.001) B) In line path length was significantly increased (F(1, 25) = 78.673, p < 0.001) C) Reduced performance was not explained by deficits in swimming (F(1,25) = .571, p > 0.05) and D) sensorimotor function during cued trials t(9) = .415, p = .688. E) During probe trial wt but not DAT-tg rats spent more time in the quadrant (wt: NW/NE t(10) = 3.452, p = .007; wt: NW/SE t(10) = 2.303, p = .047; all other p > 0.05) F) and crossed prior platform location more often than DATtg rats (U(27) = 1, p < 0.001). G) In all trials DAT-tg rats showed thigmotactic swimming close to the wall; wt rats improved strategies over trials towards allocentric navigation und used direct and indirect search strategies after platform removal.
DAT-tg rats do not learn position of hidden platform during acquisition trials
DAT-tg rats show mainly thigmotactic swimming
DAT-tg rats exhibit intact sensorimotor function and motivation to escape during cued platform trials
Sucrose consumption and open field
DAT-tg animals showed no significant difference in sucrose consumption compared to controls (U (26) = 51 p = .109).
DAT-tg animals show reduced explorative behavior; indicated by significant differences in velocity (t(19) = 7.203,p < .001), distance travelled (t(19) = 7.443, p < .001) and time spend close to the walls (t(19) = -3.253, p = .009)
B
A
wt
DAT-tg
Ki67
F
Probe trial
E
C
D
wt
DAT-tg
BrdU NeuN
Acquisition training
Morris water maze
Fig. 4: Analysis of adult hippocampal neurogenesis showed (A) a similar rate of active proliferation t(14) = 1.005, p = .332; but (C) a trend level reduction in BrdU cells t(13) = 1.456, p = .167 and a significant reduction in the proportion of BrdU+ neurons in DAT-tg rats compared to controls (t(13) = 2.389, p = .03). Representative images of (B) Ki67+ staining and (D) NeuN/BrdU double staining.
DAT-tg show no alterations in active proliferation DAT-tg rats exhibit a decrease in incorporation of newly generated neurons within DG circuitry
Conclusions and Outlook We conclude that DAT overexpression leads to profound alterations in the performance of learning tasks predominantly during initial acquisition. On the neurobiological level prominent changes in dopamine homeostasis within striatal and prefrontal circuitry in combination with the here reported changes in hippocampal neurogenesis might explain these cognitive impairments. Future work should be directed towards a further characterization of underlying mechanisms and possible treatment approaches to improve cognitive function in the DAT overexpression model of repetitive neuropsychiatric disorders.
References [1] Stebbins, GT et al. Neuropsychology 1995;9:329–37 [2] Marsh, R. et al. Arch Gen Psychiatry 2004;61:1259–68. [3] Hadar, R. et al. Scientific Reports 6, (2016).
Disclosure statement The authors declare that they have no conflict of interest
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