Figure 2. Targeted in vivo activation of mGluR5 in the BLA by itself initially causes indiscriminate fear, but it eventually leads to selective strengthening of cue-specific fear when combined with conditioning.

(a) Experimental protocol for discriminative auditory fear conditioning (10 CS⁺-US pairings were interleaved with 10 CS⁻ presentations during conditioning) using a weak US (0.4 mA) combined with in vivo infusion (1.0 μl per side) of saline (0.9% NaCl) followed by DHPG (50 µM of DHPG) into the BLA of the same animal. (b) Schematic coronal sections depicting cannula infusion sites for saline/DHPG. (c) Mean freezing levels 30 min after saline infusions on Day 3 showed no difference between the CS⁺ and CS⁻ during habituation (N = 8 rats, p>0.05). During testing 1 d after weak conditioning (Day 4), the CS⁺ did not evoke higher freezing relative to either the CS⁻ (p>0.05), or habituation (p>0.05). However, 30 min after DHPG infusion, the same animals exhibited significantly higher freezing to both CS⁺ and CS⁻ (*p<0.05), and the freezing levels were indistinguishable between CS⁺ and CS⁻ (Habituation, Day 4: p>0.05). Strikingly, subsequent conditioning in the presence of DHPG using the same weak US (Day 4, 0.4 mA) strengthened cue-specific fear. Thus, during testing 1 d after weak conditioning, the CS⁺ evoked higher freezing relative to the CS⁻ (Testing, Day 5: #, p<0.05), as well as CS⁺-evoked freezing 1 d after weak conditioning in saline (Testing, Day 4: *, p<0.05). (d) Mean freezing levels in the different spatial contexts immediately before the presentation of tones (pretone) during the same tests described in (c). Enhanced pretone freezing in the context was observed only immediately after DHPG infusion (Habituation, Day 4: *p<0.05). *p<0.05 between sessions in all graphs; #p<0.05 between CS⁺ and CS⁻ within sessions in all graphs.

DOI: http://dx.doi.org/10.7554/eLife.25665.004

Figure 2—figure supplement 1. Control experiments using the same discriminative auditory fear conditioning with a weak US combined with in vivo infusion (1.0 µl per side) of saline (0.9% NaCl) followed by a second episode of the same saline infusion into the BLA of the same animal did not cause the increase in indiscriminate fear and cue-specific fear seen with DHPG infusion (as reported in Figure 2).

‘Mean freezing levels 30 min after saline infusions on Day 3 showed no difference between the CS⁺ and CS⁻ during habituation (N = 7 rats, p>0.05). During testing 1 d after weak conditioning (Day 4), the CS⁺ did not evoke higher freezing relative to either the CS⁻ (p>0.05), or habituation (p>0.05). However, 30 min after the second saline infusion, the same animals exhibited significantly higher freezing specifically for CS⁺ and not for CS⁻ (*p<0.05) when habituated in the same conditioning context. The freezing levels were specific to CS⁺ (Habituation, Day 4: #p<0.05). Subsequently, a second episode of conditioning in the same animals in the presence of saline using the same weak US (Day 4, 0.4 mA) did not affect fear memory. Thus, during testing 1 d after weak conditioning, the CS⁺ did not evoke higher freezing relative to the CS⁻ (Testing, Day 5: #p<0.05), as well as CS⁺-evoked freezing 1 d after first weak conditioning in saline (Testing, Day 4: *p<0.05). (c) Mean freezing levels in the different spatial contexts immediately before the presentation of tones (pretone) during the same tests described in (b). There was no difference in pretone freezing to the context during any of the sessions (p>0.05). *p<0.05 between sessions in all graphs; #p<0.05 between CS⁺ and CS⁻ within sessions in all graphs.’.

DOI: http://dx.doi.org/10.7554/eLife.25665.006

Figure 2—figure supplement 2. Additional analysis of freezing behavior during differential conditioning after DHPG infusion on Day 4.

(a) A sample image showing the location of the infusion cannula. The white arrows denote the tip of the infusion cannula. (b) To assess the effect of mGluR activation on the freezing response during fear conditioning, the freezing response to the first CS+, CS- and the first pretone were analysed. These were compared to the freezing response to the last CS+, CS- and last pretone of the conditioning session. (c) The high level of indiscriminate freezing to CS+/CS-/pretone is visible not only at the beginning of the conditioning, but also at the end. Moreover, the levels of freezing at the end of conditioning show an overall increase for pretone as well as CS+/CS- (*p<0.05). Thus, although the conditioning session leads to enhanced freezing, this freezing continues to be indiscriminate.

DOI: http://dx.doi.org/10.7554/eLife.25665.008