The involvement of amygdala neurons and amygdaloid dopaminergic and glutamatergic receptors in the acquisition and reinstatement of fear-potentiated startle in rats.

摘要

The basolateral amygdala (BLA) contains NMDA, AMPA, and dopamine (DA) D1 and D2 receptors and neurobiochemical events within the amygdala mediate conditioned-fear-learning (CFL) and fear expression. Long-term potentiation (LTP) occurs in the amygdale during Pavlovian fear conditioning and is associated with fear-memory storage. CFL depends on NMDA, AMPA and dopaminergic receptor-mediated processes and enhanced amygdaloidal synaptic transmission facilitates fear-memory retrieval and makes the expression of conditioned fear possible. Since mesoamygdaloid DA receptors contribute to CFL and fear expression, Experiment 1A and 1D examined the impact of intra-BLA infusion of the DA D1 and D2 receptor antagonists SCH 23390 and raclopride L-tartrate on the acquisition of FPS in rats. Rats of the Wistar strain were bilaterally implanted with guide cannulae positioned 1.0 mm above the medial portion of the BLA. Approximately fourteen days later rats were assessed for baseline acoustic startle responding and assigned to drug-treatment groups. Forty-eight hours later rats were infused with either saline or the appropriate dopaminergic antagonist. The intra-BLA infusions occurred before five fear conditioning and testing blocks and were conducted to see if antagonism of DA receptors would prevent FPS acquisition. Retention testing for FPS took place forty-eight hours later. The results demonstrated that blockade of amygdaloid DA D1 and D2 receptors prevented the acquisition of FPS. The pretraining intra-BLA infusions of either raclopride or SCH 23390 disrupted the formation of long-term conditioned fear memories as rats treated with these DA antagonists failed to exhibit FPS on the retention test. Thus, the deficits in FPS displayed by SCH 23390 and raclopride-treated rats are likely due to the impact of these DA antagonists on associative learning and fear-memory consolidation processes. Experiment 2 demonstrated that fear-extinguished rats exposed to unsignalled footshocks displayed a reinstatement of FPS, but the exact neurobiochemicaI events involved in FPS reinstatement have not been elucidated. In contrast, fear-extinguished rats that received no unsignalled footshocks exhibited no FPS during final testing. Since unsignalled footshocks produced robust FPS reinstatement, Experiments 3A to 3D independently examined the effects of NMOA, AMPA, and DA D2 and D1 receptor antagonists on this phenomenon. Over a period of ten days, rats with cannulae targeting the BLA were base lined, fear-conditioned, pretested, fear-extinguished and then infused with either raclopride L-tartrate (8.0µg, 4.0 µg and 2.0µg), SCH 23390 (4.0 µg), (±)-2-amino-5-phosphonopentanoic acid {(APS); 2.5 µg and 1.25 µg}, 6-Cyno-7-nitroquinoxaline-2,3-dione disodium {(CNQX); 5.0 µg and 2.5 µg), or phosphate buffered saline (PBS) before exposure to five unsignalled footshocks. FPS reinstatement was assessed 24 hours later. Results from these experiments demonstrate that PBS-infused rats showed FPS reinstatement, whereas rats infused with AP5, CNQX, or the two higher doses of raclopride failed to exhibit FPS reinstatement. Intra-BLA SCH 23390 infusions did not appear to disrupt the reinstatement of FPS in Experiment 3B, however obstructed guide cannulae may have affected these results. In Experiment 3C, intra-BLA AP5 infusions made just before unsignalled footshock presentation, prevented rats from exhibiting FPS reinstatement during final testing. A similar effect on FPS reinstatement was produced by CNQX application to the BLA of rats in Experiment 3D. The overall findings of Experiment 3 suggest that DA D2 receptor antagonism and the glutamatergic receptor antagonists (AP5 and CNQX) impaired amygdaloid fear-memory reconsolidation and retrieval processes by preventing the re-excitation of neurons and pathways that had become established during fear-conditioning. It is speculated that these drugs may have interfered with excitatory synaptic transmission processes and neurobiological intracellular cascades within the amygdala and thus prevented FPS reinstatement from occurring. Two expression-control experiments (Experiments 4 and 5), revealed that the observed blockade of FPS reinstatement in Experiment 3 could not be attributed to the drugs simply blocking fear expression since infusion of raclopride, AP5, or CNQX into the BLA of non-extinguished rats 24 hours before final testing did not prevent rats from expressing FPS. Electrical stimulation (ES) of the human amygdala and temporal lobe region produces emotionally charged memory flash-backs and behaviours indicative of a central fear-state. ES of the rat amygdala is known to elevate acoustic startle amplitudes and enhance emotionality in rats and kindling of the rat amygdala exaggerates FPS and produces a variety of autonomic and behavioural fear responses. In rats, conditioned fear and LTP are induced by ES of amygdaloid afferents so it is possible that electrical excitation of BLA neurons can trigger FPS reinstatement. Experiments 7A and 7B were conducted to test the hypothesis that ES of BLA neurons can restore FPS responding in fear-extinguished rats. Thus, rats with bipolar electrodes implanted unilaterally in the BLA were baselined, fear conditioned, pretested and then assigned to one of five groups matched on FPS. Rats then received fear-extinction or no-extinction training, followed 48 hours later by either 100 unsignalled electrical stimulations of the BLA or no stimulation. Experimental groups included; [Extinction + Stimulation (N=12), Extinction + No stimulation (N=12), No Extinction + No stimulation (N= 12), No Extinction + Stimulation (N=11) and Extinction + Stimulation out of context (N=12)]. FPS reinstatement was assessed 24 hours later and BLA AD-current threshold were recorded four days after FPS reinstatement testing was completed. Experiment 7A demonstrated that all experimental groups exhibited FPS except the Extinction + No stimulation control group which displayed a robust extinction effect. Most importantly, the Extinction + Stimulation group exhibited statistically significant FPS reinstatement after ES of the amygdala. Experiment 7B demonstrated that ES of the amygdala in a context different to the startle apparatus also produced FPS reinstatement in fear-extinguished rats. However, the magnitude of this FPS reinstatement effect was much smaller than that obtained when rats received ES of the amygdala in the startle testing apparatus. The overall finding that ES of the BLA causes FPS reinstatement in fear-extinguished rats suggests that ES of the BLA resensitised fear-memory systems and restored FPS responding. This effect was likely achieved by exciting amygdaloid neurons and pathways containing the memory-trace of the CS-UCS association originally established during Pavlovian fear conditioning. Experiments 8 to 10 used the same protocol as Experiment 7A and investigated whether ES of other brain regions that share reciprocal connections with the amygdala would trigger FPS reinstatement in fear-extinguished rats. Rats in Experiments 8 to 10 had bipolar electrodes unilaterally implanted in the perirhinal/insular cortex (PRh/IC), dorsal periaqueductal gray (dPAG), ventral tegmental area (VTA) and ventral periaqueductal gray (vPAG). Experiment 8 revealed that ES of the PRh/IC produced a positive trend towards FPS reinstatement. Experiment 9 examined the impact of dPAG and VTA ES on FPS reinstatement. This experiment demonstrated that VTA but not dPAG ES produced a FPS reinstatement effect. This finding seems to support the research evidence that highlights the importance of mesoamygdaloid systems in mediating conditioned fear and stress responses. Experiment 10 assessed the impact of vPAG ES on FPS reinstatement and discovered that stimulation of this region did not trigger a restoration of FPS in fear-extinguished rats but it did seem to elevate overall acoustic startle responding during final testing, This finding would seem to indicate that ES of the vPAG likely increased contextual fear but not cue specific fear since acoustic startle amplitudes during both the noise-alone and the CS + noise conditions were elevated, The key finding that ES of the amygdala produces robust FPS reinstatement provides further proof that the amygdala and its afferent and efferent neural circuits are essential for fear-memory reconsolidation and conditioned fear expression and reinstatement.