Dopamine is necessary for cue-dependent fear conditioning

Abstract

Dopamine (DA) is implicated in many behaviors, including motor function, cognition, and reward processing; however, the role of DA in fear processing remains equivocal. To examine the role of DA in fear-related learning, dopamine-deficient (DD) mice were tested in a fear-potentiated startle paradigm. DA synthesis can be restored in DD mice through administration of 3, 4-dihydroxy-l-phenylalanine (l-Dopa), thereby permitting the assessment of fear processing in either a DA-depleted or -replete state. Fear-potentiated startle was absent in DD mice but could be restored by l-Dopa administration immediately after fear conditioning. Selective viral-mediated restoration of DA synthesis within the ventral tegmental area fully restored fear learning in DD mice, and restoration of DA synthesis to DA neurons projecting to the basolateral amygdala restored short-term memory but not long-term memory or shock sensitization. We also demonstrate that the DA D(1) receptor (D(1)R) and D(2)-like receptors are necessary for cue-dependent fear learning. These findings indicate that DA acting on multiple receptor subtypes within multiple target regions facilitates the stabilization of fear memory.

Figure 1.

DA is critical for learning fear-potentiated startle. A, Acoustic startle response of control (n = 10; black squares) and DD (n = 10; white squares) mice to different sound intensities. Responses are reported in arbitrary units. B, Prepulse inhibition was tested at three different prepulse intensities in control (n = 10; black bars) and DD (n = 10; white bars) mice. *p < 0.05, repeated-measures ANOVA. C, Schematic illustrating the 7 d fear-potentiated startle paradigm. On baseline and test days, mice received 10 presentations of no-cue (40 ms duration presentation of a 105 dB startle pulse) and 10 presentations of cue trials (10 s light cue coterminating with the startle pulse) in pseudorandom order. On training days, mice received 10 pairings of the 10 s light cue that coterminated with 0.5 s duration, 0.2 mA footshock. Learning was assessed on test days as a percentage of potentiation on cue trials when compared with no-cue trials. D, DD mice (n = 10; white bars) given l-Dopa 3 h after training (Day 2, Day 4) failed to learn (Test 1, Test 2). However, when DD mice were injected immediately after training (Day 6), they displayed significant fear-potentiated startle (Test 3). *p < 0.05, repeated-measures ANOVA. E, Measurements of shock reactivity during training sessions (control, n = 10, black bars; DD, n = 10, white bars). Responses are reported in arbitrary units. F, Schematic illustrating the 3 d fear-potentiated startle paradigm used to determine the critical time period in which DA is important. All 30 cue–shock pairings were given on 1 training day, and DD mice were treated with l-Dopa immediately, 1 h, or 3 h posttraining. G, Only control (n = 8; solid black bars, C) and DD mice injected immediately after training (n = 7; vertical stripes, 0 h) exhibited fear-potentiated startle on the test day. This level of fear-potentiated startle was significantly higher than that seen in the DD mice given l-Dopa 1 h (n = 6; diagonal stripes) or 3 h (n = 6; white bars) after training. *p < 0.05 when compared with baseline, Fisher's post hoc. All values reported are means ± SEM.

Figure 2.

D₁R KO mice have significantly impaired learning. A, Acoustic startle response of D₁R WT (n = 9; black squares) and KO (n = 9; white squares) mice. B, Results of 7 d fear-potentiated startle paradigm with D₁R mice. D₁R WT mice (n = 9; solid black bars) but not D₁R KO mice (n = 9; white bars) displayed fear-potentiated startle by test day 3. **p < 0.01, comparing KO with WT, Fisher's post hoc. C, Measurements of shock reactivity. D₁R KO mice (n = 9; white bars) have higher responses to footshock than WT (n = 9; black bars). *p < 0.05, repeated-measures ANOVA. All values reported are means ± SEM. For startle responses and shock reactivity, numbers reported in arbitrary units.

Figure 3.

D₂R KO mice have intact fear-potentiated startle. A, Acoustic startle response of D₂R WT (n = 8; black squares) and KO (n = 8; white squares) mice. B, Results of 7 d fear-potentiated startle paradigm with D₂R mice. Both WT (n = 8; black bars) and KO (n = 8; white bars) mice exhibited significant levels of fear-potentiated startle. C, Measurements of shock reactivity during training (WT, n = 8, black bars; KO, n = 8, white bars). D, WT and D₂R KO mice (n = 11 each) were subjected to the 3 d fear-potentiated startle paradigm. D₂R KO mice were administered eticlopride (0.5 mg/kg) before training and failed to express fear-potentiated startle on testing. **p < 0.01, KO versus WT, Fisher's post hoc. All values reported are mean ± SEM. For startle responses and shock reactivity, responses are reported in arbitrary units.

Figure 4.

Short-term memory and shock sensitization depend on DA. A, Design of the behavioral paradigm. On day 1, baseline startle responses were obtained. On day 2, mice received all 30 cue–shock pairings and were then put back in their home cage for 10 min before testing. B, Control mice (n = 10; black bars) have significantly greater shock sensitization and fear-potentiated startle when compared with DD (n = 10; white bars). *p < 0.05, Student's t test. C, WT (n = 7; black bars) and D₁R KO (n = 7; white bars) mice have intact shock sensitization. Only WT have fear-potentiated startle during the short-term memory test. *p < 0.05, KO versus WT, Student's t test. D, WT (n = 8; black bars) mice have significantly greater shock sensitization than D₂R KO (n = 8; white bars). Levels of fear-potentiated are similar between WT and D₂R KO mice. *p < 0.05, KO versus WT, Student's t test. All values reported are means ± SEM.

Figure 5.

Region-specific restoration of endogenous TH expression in DD mice. A, Schematic illustrating injection coordinates for BLA rescue experiments. DD (n = 7) and control (n = 7) mice were injected bilaterally into the BLA with CAV2–Cre vectors (0.5 μl/hemisphere). B, Schematic illustrating injection coordinates for VTA rescue experiments. AAV1–Cre–GFP was injected bilaterally (0.5 μl/hemisphere) into the VTA of DD (n = 7) and control (n = 10) mice. Figures adapted from Paxinos and Franklin (2001). C–E, Comparison of TH staining in coronal slice (4× magnification) showing ventral midbrain of virus-injected WT control, BLA-injected DD, and VTA-injected DD. C, TH immunohistochemistry in control midbrain demonstrates the presence of DA neurons in the VTA and substantia nigra pars compacta (indicated by arrow). D, BLA-rescued DD mice had a small number of TH-positive neurons in the VTA. Inset is a 40× magnification of the boxed region, showing TH expression in the soma and processes. E, VTA-rescued DD mice had TH expression predominately in the VTA. Note absence of TH staining in substantia nigra pars compacta (indicated by arrow). F–H, Coronal section (4× magnification) from WT virus-injected control, BLA-rescued, and VTA-rescued DD mice, showing TH expression in dorsal striatum and nucleus accumbens. F, WT virus-injected controls have TH expression throughout the entirety of the dorsal (indicated by arrow) and ventral striatum. G, There is no TH expression detected in the striatum of BLA-rescued DD mice. H, VTA-rescued DD mice have TH expression in the nucleus accumbens, with only a paucity of staining in the dorsal striatum (indicated by arrow). I–K, Coronal section (10× magnification) showing TH expression in the BLA of virus-injected WT control, BLA-rescued, and VTA-rescued DD mice.

Figure 6.

BLA-rescued DD mice have restored short-term memory, whereas VTA-rescued DD mice have fully restored learning. A, Virus-injected WT control (n = 7) and BLA-rescued DD (n = 7) mice were subjected to the 3 d fear-potentiated startle paradigm. Left, Shock sensitization is significantly lower in BLA-rescued mice. Middle, Short-term memory (STM) was restored to control levels in BLA-rescued mice. Right, Long-term memory (LTM), assessed 24 h after training, is absent in BLA-rescued mice. B, Results from WT control (n = 10) and VTA-rescued DD (n = 7) mice in the 3 d fear-potentiated startle paradigm. Left, Shock sensitization in VTA-rescued DD mice was not significantly different from control. Middle and Right, Levels of STM and LTM memory were the same as control in VTA-rescue mice. *p < 0.05, rescue versus control, Student's t test. All values reported are means ± SEM.