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. 2010 Sep 15;30(37):12288-300.
doi: 10.1523/JNEUROSCI.2655-10.2010.

Associative Learning and CA3-CA1 Synaptic Plasticity Are Impaired in D1R Null, Drd1a-/- Mice and in Hippocampal siRNA Silenced Drd1a Mice

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Associative Learning and CA3-CA1 Synaptic Plasticity Are Impaired in D1R Null, Drd1a-/- Mice and in Hippocampal siRNA Silenced Drd1a Mice

Oskar Ortiz et al. J Neurosci. .
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Abstract

Associative learning depends on multiple cortical and subcortical structures, including striatum, hippocampus, and amygdala. Both glutamatergic and dopaminergic neurotransmitter systems have been implicated in learning and memory consolidation. While the role of glutamate is well established, the role of dopamine and its receptors in these processes is less clear. In this study, we used two models of dopamine D(1) receptor (D(1)R, Drd1a) loss, D(1)R knock-out mice (Drd1a(-/-)) and mice with intrahippocampal injections of Drd1a-siRNA (small interfering RNA), to study the role of D(1)R in different models of learning, hippocampal long-term potentiation (LTP) and associated gene expression. D(1)R loss markedly reduced spatial learning, fear learning, and classical conditioning of the eyelid response, as well as the associated activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. These results provide the first experimental demonstration that D(1)R is required for trace eyeblink conditioning and associated changes in synaptic strength in hippocampus of behaving mice. Drd1a-siRNA mice were indistinguishable from Drd1a(-/-) mice in all experiments, indicating that hippocampal knockdown was as effective as global inactivation and that the observed effects are caused by loss of D(1)R and not by indirect developmental effects of Drd1a(-/-). Finally, in vivo LTP and LTP-induced expression of Egr1 in the hippocampus were significantly reduced in Drd1a(-/-) and Drd1a-siRNA, indicating an important role for D(1)R in these processes. Our data reveal a functional relationship between acquisition of associative learning, increase in synaptic strength at the CA3-CA1 synapse, and Egr1 induction in the hippocampus by demonstrating that all three are dramatically impaired when D(1)R is eliminated or reduced.

Figures

Figure 1.
Figure 1.
Experimental design for classical conditioning and LTP. Classical eyelid conditioning was achieved with a trace paradigm, using a tone as a CS. The loudspeaker was located 30 cm from the animal's head. Animals were implanted with bipolar stimulating electrodes on the left supraorbital nerve for US presentations. Eyelid conditioned responses were recorded with EMG electrodes implanted in the ipsilateral orbicularis oculi (O.O.) muscle. The top diagram illustrates that animals were also implanted with stimulating (St.) and recording (Rec.) electrodes to activate Schaffer collaterals and to record fEPSPs evoked at the pyramidal CA1 area of the right hippocampus and indicates the injection point for Drd1a-siRNA. A, Photomicrographs illustrating the location of stimulating and recording electrodes and lentivirus injection site. Scale bars, 200 μm. DG, Dentate gyrus; Sub., subiculum; D, dorsal; L, lateral; M, medial; V, ventral. B, The two sets of traces on the left illustrate the following: 1, a fEPSP evoked at the CA3–CA1 synapse; 2, an EMG recording evoked at the O.O. muscle by a single suprathreshold pulse presented to the supraorbital nerve. Both traces were collected during the ninth conditioning session of a control animal. Calibrations are as indicated.
Figure 2.
Figure 2.
Hippocampus-dependent learning is impaired in dopamine Drd1a−/− mice. Data show the mean values ± SEM. A, Progression of escape latency during the training phase in the Barnes maze. Drd1a−/− mice did not reduce escape latency at any time during the experiment (*p < 0.005). B, Immobility during the first day of training. WT and Drd1a−/− mice showed similar levels of immobility. C, Probe trial performed 3 d after the training phase. Histograms represent the time spent searching for the escape hole. Drd1a−/− mice did not reduce searching time during the probe trial (*p < 0.005). D, Escape latency during the relearning phase. For this test, the escape hole was located opposite to its position in the training phase (*p < 0.001). Statistics were determined with repeated-measures two-way ANOVA followed by Tukey's test for post hoc analysis (A, D) and with Student's t test (C).
Figure 3.
Figure 3.
Anxiety levels are similar in both genotypes, but Drd1a−/− mice are more sensitive to pain than WT mice. A, Anxiety-like behavior of Drd1a−/− and WT mice illustrated by the number of entries and percentage of total time (mean ± SEM) spent in the open arms of the elevated plus maze test. Drd1a−/− mice make more entries and spend more time in the open arms, indicating lower anxiety levels than the WT mice. *p < 0.05 versus WT mice. B, Pain sensitivity thresholds (in seconds, mean ± SEM) of mice in tail flick, hot plate, and plantar tests. Drd1a−/− mice exhibit lower pain thresholds than WT mice in all three tests, indicating higher pain sensitivity. *p < 0.05 versus WT mice; n = 8–10 animals.
Figure 4.
Figure 4.
Active avoidance performance is impaired in dopamine Drd1a−/− mice. Data shown are mean values ± SEM. A, Progression of active avoidance responses during the training phase. Drd1a−/− mice did not increase the number of avoidance responses during the training phase (*p < 0.001.). B, Time course of crossing latencies for WT and Drd1a−/− mice during the training phase. Drd1a−/− mice did not decrease escape latency at any point during training (*p < 0.001). C, Number of intertrial crosses. From day 3 on, there was no significant difference between WT and Drd1a−/− mice in the number of intertrial crosses. Statistics were performed with repeated-measures two-way ANOVA, followed by post hoc analysis with Tukey's test.
Figure 5.
Figure 5.
Performance in the passive avoidance test and fear conditioning are impaired in Drd1a−/− mice. Data show mean ± SEM. A, Thresholds for footshock responses. Increasing intensity footshocks were delivered to WT and Drd1a−/− mice, and the threshold for each listed behavior was determined. Thresholds for all three response behaviors were similar in the two genotypes. B, Avoidance response. Latency refers to the time spent in the light compartment before mice enter the dark compartment, which was paired with footshock in a single training trial. Drd1a−/− mice show partial impairment of passive avoidance at both 0.4 and 0.8 mA. C, Cued and contextual fear conditioning are impaired in Drd1a−/− mice. Freezing time was measured in contextual and cued fear conditioning trials 24 h after training. *p < 0.01 and **p < 0.001 versus wild type; #p < 0.01 and ##p < 0.001 versus Pre-shock (0 h). Statistics were determined by repeated-measures two-way ANOVA followed by post hoc analysis with Tukey's test.
Figure 6.
Figure 6.
siRNA-mediated Drd1a silencing in vitro and in vivo. A, Drastic reduction of Drd1a mRNA expression in HEK cells in vitro after infection with Drd1a-siRNA constructs. mRNA levels were determined by quantitative RT-PCR 48 h after infection and normalized to Gapdh mRNA levels. *p < 0.05; **p < 0.01; ***p < 0.005; one-way ANOVA. B, Drd1a and Drd2 mRNA levels in NAc 48 h after intra-accumbal injection of a mixture of all three Drd1a-siRNAs. mRNA levels were determined by RT-PCR, normalized to Gapdh, and expressed as a percentage of Drd1a mRNA expression in Lv-GFP-injected animals. Injection of Drd1a-siRNAs specifically decreased Drd1a mRNA expression. C, D1R protein expression in hippocampus 48 h after injection of Drd1a-siRNAs. D1R protein levels were decreased ∼75%. ***p < 0.001, Student's t test. D, Photomicrograph of a coronal brain section illustrating the spread of lentiviral infection in the CA1 layer of the hippocampus of WT mice injected with 2 μl of Lenti-GFP particles. E, High-magnification image of infected pyramidal cells illustrated in D. Particles infect most of the dorsal hippocampus spreading through pyramidal CA1 cell layer. Scale bar, 100 μm.
Figure 7.
Figure 7.
Input–output curves, paired-pulse facilitation, and LTP induction in the CA1 area in wild type, Drd1a−/−, WT-GFP, and Drd1a-siRNA. A, Representative example of an input–output curve collected from a Drd1a−/− mouse. Stimulus consisted of paired (S1, S2) pulses (40 ms interpulse interval) presented at increasing intensities in 20 μA steps. Note the sigmoid shape of S1 + S2 value. B, The four experimental groups presented similar sigmoid curves for S1 + S2 values (n = 10 animals per group). Representative sample records collected from the four experimental groups are illustrated on the right. C, Paired-pulse facilitation of fEPSPs recorded in the CA1 area after stimulation of Schaffer collaterals. The data shown are mean ± SEM. slopes of the second fEPSP expressed as a percentage of the first for the six interpulse intervals. No significant differences were observed between the four experimental groups. Extracellular fEPSP paired traces were collected from a representative Drd1a-siRNA animal at the indicated interstimulus intervals. D, Top, Representative fEPSPs recorded from WT and Drd1a−/− animals before (baseline), and 15–30 min (1) and 24 h (2) after HFS. Graphs illustrate the time course of changes in fEPSPs (mean ± SEM) after HFS stimulation of the Schaffer collaterals. The HFS train was presented after 15 min of baseline recordings, at the time indicated by the dashed line. fEPSP slopes are expressed as a percentage of the baseline (100%) slope. WT mice exhibited significantly greater LTP than Drd1a−/− mice (*p < 0.001). E, Same analysis as in D for WT-GFP and Drd1a-siRNA groups. Here again, the control group (WT-GFP) presented significantly larger LTP than Drd1a-siRNA mice (*p < 0.01). St., Stimulating electrode.
Figure 8.
Figure 8.
Evolution of CA3–CA1 synaptic field potentials and learning curves for WT, Drd1a−/−, WT-GFP, and Drd1a-siRNA. A, B, Top to bottom, the conditioning paradigm, representative EMG, and representative hippocampal recordings during paired CS–US presentations for WT and Drd1a−/− mice (A) and for WT-GFP and Drd1a-siRNA mice (B). The time of stimulus presentation at Schaffer collaterals (St. Hipp.) is indicated, as are the times of delivery of CS (dashed line) and US. Data shown were collected during the ninth conditioning session. 3× Thr., Three times threshold. C, D, Percentage of eyelid CRs reached by the four experimental groups. The acquisition curve presented by the WT group (open circles) was significantly larger than values reached by the Drd1a−/− group (C, filled circles; *p < 0.001). Similarly, the acquisition curve of the WT-GFP group was also significantly larger than that presented by Drd1a-siRNA animals (D; *p < 0.001). E, F, Evolution of fEPSPs evoked at the CA3–CA1 synapse across conditioning for WT and Drd1a−/− mice (E) and for WT-GFP and Drd1a-siRNA animals (F). fEPSP slope is expressed as a percentage of fEPSP slope during habituation for each group. Differences in fEPSP slopes between WT and Drd1a−/− groups were statistically significant from the 4th to the 10th conditioning sessions (E; *p < 0.006), as well as between WT-GFP and Drd1a-siRNA animals (F; *p < 0.001), indicating that activity-dependent synaptic plasticity was severely impaired in both Drd1a−/− and Drd1a-siRNA mice. St., Stimulating electrode.
Figure 9.
Figure 9.
D1R is required for activity-induced Egr1 in CA1 pyramidal cells after HFS of Schaffer collaterals. Photomicrographs of the CA1 pyramidal cell layer of the hippocampus from coronal brain slices showing immunohistochemistry for the Egr1 transcription factor are presented. A–D, Egr1 expression under basal conditions for WT, Drd1a−/−, WT-GFP, and Drd1a-siRNA mice, respectively. E–H, Expression 24 h after in vivo HFS of the Schaffer collaterals in the hippocampus of WT, Drd1a−/−, WT-GFP, and Drd1a-siRNA mice, respectively. Scale bar, 50 μm.

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