Behavioral and neurophysiological evidence that lateral paracapsular GABAergic synapses in the basolateral amygdala contribute to the acquisition and extinction of fear learning

Abstract

The lateral/basolateral amygdala (BLA) is crucial to the acquisition and extinction of Pavlovian fear conditioning, and synaptic plasticity in this region is considered to be a neural correlate of learned fear. We recently reported that activation of BLA β3-adrenoreceptors (β3-ARs) selectively enhances lateral paracapsular (LPC) feed-forward GABAergic inhibition onto BLA pyramidal neurons, and that intra-BLA infusion of a β3-AR agonist reduces measures of unconditioned anxiety-like behavior. Here, we utilized a combination of behavioral and electrophysiological approaches to characterize the role of BLA LPCs in the acquisition of fear and extinction learning in adult male Long-Evans rats. We report that intra-BLA microinjection of β3-AR agonists (BRL37344 or SR58611A, 1μg/0.5μL/side) prior to training fear conditioning or extinction blocks the expression of these behaviors 24h later. Furthermore,ex vivo low-frequency stimulation of the external capsule (LFS; 1Hz, 15min), which engages LPC synapses, induces LTP of BLA fEPSPs, while application of a β3-AR agonist (SR58611A, 5μM) induces LTD of fEPSPs when combined with LFS. Interestingly, fEPSP LTP is not observed in recordings from fear conditioned animals, suggesting that fear learning may engage the same mechanisms that induce synaptic plasticity at this input. In support of this, we find that LFS produces LTD of inhibitory postsynaptic currents (iLTD) at LPC GABAergic synapses, and that this effect is also absent following fear conditioning. Taken together, these data provide preliminary evidence that modulation of LPC GABAergic synapses can influence the acquisition and extinction of fear learning and related synaptic plasticity in the BLA.

Keywords: Fear-potentiated startle; Inhibitory plasticity; Noradrenaline.

Figure 1

Intra-BLA microinjection the β3-AR agonist BRL37344 (1 µg/0.5 µL/side) prior to fear conditioning blocked fear-potentiated startle 24 hrs later, as evidenced by no change in startle amplitude in response to the CS-paired noise (a; p > 0.05). In contrast, animals who received vehicle microinjections before fear conditioning exhibited a CS-potentiated startle response (a; *p < 0.05). Accordingly, the percent change in startle amplitude was greater among vehicle-treated animals than BRL37344-treated animals (b; *p < 0.05). Similarly, the β3-AR agonist SR58611A (1 µg/0.5 µL/side) blocked the expression of fear-potentiated startle (c; p > 0.05), which remained unaffected in vehicle-treated controls (c; *p < 0.05). The percent change in startle response was likewise significantly increased in vehicle-treated animals relative to SR58611A-treated animals (d; *p < 0.05). Black circles represent microinjector tip locations (e).

Figure 2

Intra-BLA microinjection of the β3-AR agonist BRL37344 (1 µg/0.5 µL/side) before extinction training blocked the expression of extinction learning 24 hrs later. Animals in the vehicle group acquired fear conditioning normally (a; **p < 0.01), and this fear response was abolished following extinction training (a; p > 0.05). Accordingly, the percent change in startle response was significantly decreased following extinction (b; *p < 0.05). In contrast, intra-BLA microinjection of BRL37344 before to extinction training blocked the expression of extinction learning 24 hrs later (c; **p < 0.01). As such, the percent change in fear-potentiated startle was not significantly altered pre- vs post-extinction (d; p > 0.05). Black circles represent microinjector tip locations (e).

Figure 3

Low-frequency stimulation (LFS; 1 Hz, 15 min) of the external capsule lead to a significant increase in fEPSP amplitude in slices taken from naïve animals (a; 17.45% ± 3.73), and this plasticity was occluded by fear conditioning (a; 0.01% ± 3.989). LFS significantly decreased fEPSP amplitude when delivered after pretreatment with the β3-AR agonist SR58611A (5 µM) (a; 14.210% ± 3.36). Two-way repeated measures ANOVA revealed a significant interaction of time and group (a; *p < 0.05). Accordingly, one-way ANOVA revealed that the average percent change in fEPSP amplitude post-LFS was significantly different in each group (b; *p < 0.05, **p < 0.01, *** p < 0.001). Similarly, LFS of the external capsule resulted in significant depression of post-LFS IPSCs (c; 32.82% ± 6.93). However, this LTD was not observed in animals that had undergone fear conditioning (c; 4.32% ± 8.28). Two-way repeated measures ANOVA revealed a significant interaction of time and group (c; ***p < 0.001), and a paired t-test confirmed that the average percent change in eIPSC amplitude post-LFS was significantly reduced following fear-conditioning (d; *p < 0.05).