Dopamine D2R is Required for Hippocampal-dependent Memory and Plasticity at the CA3-CA1 Synapse

Abstract

Dopamine receptors play an important role in motivational, emotional, and motor responses. In addition, growing evidence suggests a key role of hippocampal dopamine receptors in learning and memory. It is well known that associative learning and synaptic plasticity of CA3-CA1 requires the dopamine D1 receptor (D1R). However, the specific role of the dopamine D2 receptor (D2R) on memory-related neuroplasticity processes is still undefined. Here, by using two models of D2R loss, D2R knockout mice (Drd2-/-) and mice with intrahippocampal injections of Drd2-small interfering RNA (Drd2-siRNA), we aimed to investigate how D2R is involved in learning and memory as well as in long-term potentiation of the hippocampus. Our studies revealed that the genetic inactivation of D2R impaired the spatial memory, associative learning, and the classical conditioning of eyelid responses. Similarly, deletion of D2R reduced the activity-dependent synaptic plasticity in the hippocampal CA1-CA3 synapse. Our results demonstrate the first direct evidence that D2R is essential in behaving mice for trace eye blink conditioning and associated changes in hippocampal synaptic strength. Taken together, these results indicate a key role of D2R in regulating hippocampal plasticity changes and, in consequence, acquisition and consolidation of spatial and associative forms of memory.

Keywords: Drd2−/−; hippocampal; learning; long-term potentiation; memory.

Figure 1

Drd2 inactivation delays spatial learning in Morris Water Maze. (A) The reduction in latency during training occurred much more slowly in Drd2^−/− mice compared to their WT littermates (_*P < 0.05). (B) Probe trial performed 3 days after the training phase. Histograms represent the time spent searching in the target quadrant. Drd2^−/− mice increased their time in the target quadrant slightly compared to the first day of training; this increase was significantly lower than that seen in WT mice (_*P < 0.001). In the probe trial on day 17, Drd2^−/− mice made significantly fewer crosses through the platform location site than WT mice (P = 0.006). (C) Latency to find the platform during reversal phase. Here, we placed the platform opposite to the original place in the training phase. Drd2^−/− mice show an impaired reversal learning performance. (_*P < 0.05). (D) Cued version of MWM did not show differences between groups. (E) Rotarod at constant speed did not show differences. However, motor coordination learning in acceleration condition is impaired in Drd2^−/− mice (P < 0.005). Data show the mean values ± SE. Repeated-measures two-way ANOVA followed by Tukey’s test for post hoc analysis (A, D) and with Student’s t test (B, C, E, F).

Figure 2

Drd2^−/− mice showed critically impaired associative avoidance responses. (A) Threshold responses to increasing intensity foot-shocks were similar in both genotypes (n = 10 mice per group). (B) Passive avoidance latency refers to the time spent in the light compartment before mice enter the dark compartment, which was paired with foot-shock in a single training trial. WT and Drd2^−/− mice increased their latency times at 1 and 24 h after the foot-shock. However, 1 h after the stimulus, the increase in latency was significantly (_*P < 0.05) smaller in Drd2^−/− mice than in WT mice. No difference was observed at 24 h. (C) Progression of avoidance responses during the training phase. WT mice quickly learn to avoid the shock while Drd2^−/− mice did not learn to avoid it during the entire 11-day training phase, indicating that these mice did not learn to associate the CS with the foot-shock (_*P < 0.001). (D) Number of escape responses during the training days. Drd2^−/− mice shown more escape responses during the entire training phase, indicating they respond almost only when the shock it is delivered (_*P < 0.001). (E) Time course of crossing latencies during the training phase _*P < 0.001 versus WT mice. (F) Number of inter-trial crosses. The number of inter-trial crosses was similar in both genotypes, indicating that Drd2^−/− mice have the same crossing ability as WT. Data show the mean values ± SE. Statistics were determined by repeated-measures two-way ANOVA followed by post hoc analysis with Tukey’s test. WT (n = 10) and Drd2^−/− (n = 10).

Figure 3

Drd2 inactivation does not affect emotional or motivational responses. (A) Open field results shown significant differences between groups in distance (cm) and velocity (cm\s; P < 0.005) whereas time in corner and center did not differ between genotypes. (B) NSF. Latency values to the first eat were similar between Drd2^−/− and WT mice. (C) Elevated-plus maze: anxiety-like behavior of Drd2^−/− and WT mice is illustrated by the number of entries and percentage of total time spent in the open arms of the elevated-plus maze. Drd2^−/− mice showed similar number of entries and time spent in the open arms. (D) Porsolt: WT and Drd2^−/− mice showed similar immobility time. Data shown are mean ± SE. Statistical values were determined by Student’s t test.

Figure 4

Drd2 inactivation reduces fear conditioning but does not alter fear extinction. (A) After the shock, Drd2^−/− mice spent significantly (_*P < 0.01) less time freezing than their WT littermates, and 24 h after shock, freezing times were still significantly lower in Drd2^−/− mice, although the magnitude of the difference was reduced (_*P < 0.01). (B) Freezing time after 3 foot-shocks separated by 2 min to reach similar freezing times in both genotypes. (C) Daily freezing time in the context without foot-shock. The extinction curve was similar for both groups of mice, except on the last day, when Drd2^−/− mice had higher values indicating lower extinction. Data shown are mean ± SE. Statistics were performed with repeated-measures 2-way ANOVA, followed by post hoc analysis with Tukey’s test.

Figure 5

Efficient in vitro and in vivo silencing of Drd2 by siRNA. (A) Drastic reduction of Drd2 protein expression in STHdh⁺/Hdh⁺ cells in vitro after infection with DrD2-siRNA constructs (P = 0.014). (B) Drd2 mRNA levels in striatum 48 h after injection of Drd2-siRNAs mixture. mRNA levels were determined by RT-PCR, normalized to Actin, and expressed as a percentage of Drd2 mRNA expression in LV-GFP-injected animals. Injection of Drd2-siRNAs specifically decreased Drd2 mRNA expression. (C) D₂R protein levels were decreased 48 h after injection of siRNAs. D₂R protein levels were decreased after injection of siRNAs P = 0.0049, and levels in Drd2^−/− mice were P = 0.021, Student’s t test. (D) Photomicrograph of a coronal brain section that shows the lentiviral infection in the CA1 layer of the hippocampus of WT mice after injection with lenti-GFP particles (2 μL). (E) Highly magnified image of the infected pyramidal cells from panels D, E. Particles have infected a large portion of the dorsal hippocampus, and are spreading through the pyramidal CA1 cell layer. Scale bar: 100 μm.

Figure 6

LTP evoked at the CA3-CA1 synapse is decreased in behaving Drd2^−/− and in Drd2-siRNA mice. (A) Input–output curves collected from the 4 experimental groups. Stimulus consisted of single pulses presented at increasing intensities in 20 μA steps. Collected data was best adjusted by sigmoid curves (r ≥ 0.984; _*P < 0.001; n = 3 animals per group), with no significant (P = 0.067) differences between groups. (B) Paired-pulse facilitation of fEPSPs recorded in the CA1 area following stimulation of Schaffer collaterals. The data shown are mean ± SE slopes of the second fEPSP expressed as a percentage of the first of the six (10, 20, 40, 100, 200, and 500 ms) interpulse intervals. The four groups of mice presented a significant (_*P < 0.05) paired-pulse facilitation at short (10–40 ms) interpulse intervals, but no significant differences (P = 0.181) between groups. (C) At the top are illustrated representative fEPSPs recorded from WT and Drd2^−/− animals before (baseline), and 5 min (1) and 24 h (2) after HFS. Graphs illustrate the time course of changes in fEPSPs (mean ± SE) following HFS stimulation of the Schaffer collaterals. The HFS train was presented after 15 min of baseline recordings, at the time marked by the dashed line. fEPSPs are given as a percentage of the baseline (100%) slope. WT mice (white circles) presented a significantly larger LTP than Drd2^−/− (black circles) animals (_*P < 0.001). (D) Same analysis as in C for WT-Sham and Drd2-siRNA groups. Here again, the control group presented a significantly larger LTP than Drd2-siRNA mice (_*P < 0.05).

Figure 7

Learning-dependent changes in CA3-CA1 synaptic strength are impaired in Drd2^−/− and Drd2-siRNA mice. (A, B) From top to bottom are illustrated the conditioning paradigm, representative EMG and hippocampal recordings collected during paired CS-US presentations for WT and Drd2^−/− mice (A) and for WT-Sham and Drd2-siRNA mice (B). The moment of stimulus presentation at Schaffer collaterals (St.) is indicated, as is the time of delivery of CS (dashed line). Data shown were collected during the ninth conditioning session. (C, D) Percentage of eyelid CRs reached by the four experimental groups. The acquisition curve presented by the WT (white circles) group was significantly larger than values reached by the Drd2^−/− (black circles) group (C; _*P < 0.05). The acquisition curve of the WT-Sham(white squares) group was also significantly larger than that presented by Drd2-siRNA (black squares) animals (D; _*P < 0.05). (E, F). Evolution of fEPSPs evoked at the CA3-CA1 synapse across conditioning for WT and Drd2^−/− mice (E) and for WT-Sham and Drd2-siRNA animals (F). fEPSP slopes are expressed as the percentage of fEPSP slope values collected during habituation sessions for each group. Differences in fEPSP slopes between WT and Drd2^−/− groups were statistically significant at the indicated sessions (E; _*P < 0.05), indicating that activity-dependent synaptic plasticity was severely impaired in both Drd2^−/− mice. No significant differences were found between the WT-Sham and Drd2-siRNA groups (P = 0.154).