Scopolamine infusion in BLA before the novel taste prevents AN

To test the role of muscarinic receptors in the BLA in the acquisition of AN, we performed intra-BLA SCOP or VEH infusions 20 min before novel taste presentation (S1). First, both the SCOP and VEH groups consumed similar amounts of fluid (water + saccharin) across the duration of the sessions, no main effect of Treatment (F(1,15) = 1.04, p > 0.32 or Treatment x Day interaction (F (4,60) = 2.12, p = 0.09) being observed (data not shown). This observation suggests that under our conditions, SCOP did not significantly affect thirst or motivation to drink. As for saccharin preference (PI, see methods), a significant interaction between Day and Group variables (F(4, 60) = 3.59, p < 0.05) was observed. On novel taste presentation day, SCOP and VEH groups consumed similar amounts of saccharine solution (PIs = 38.28 ± 6.6% vs. 41.38 ± 8.8% for VEH and SCOP, respectively, p > 0.78) suggesting that intra-BLA scopolamine infusions had no effect on taste perception or neophobic reaction (Fig. 2A-B). Tukey post hoc test showed that there were significant intragroup differences between S1 and S2 in the VEH group (PI´s = 38.28 ± 6.6% and 64.17 ± 6.0% for S1 and S2, respectively, p < 0.05) consistent with the phenomenon of AN. In the SCOP group however, no difference was observed between S1 and S2 (41.38 ± 8.8% vs. 28.67 ± 7.4% for S1 and S2, respectively, p > 0.73) suggesting that intra-BLA SCOP impaired the ability to form AN. In this group however AN was evident on the following day (i.e., S3) as the PI increased to 79.82 ± 8.8% compared to 28.67 ± 7.4% on the previous day (p < 0.05; see Fig. 2A-B). Together, these data suggest that intra-BLA SCOP infusions prior to novel taste intake prevents AN formation but does not affect the neophobia.

Fig. 2

A Effect of bilateral intra-BLA SCOP infusion 20 min before novel taste exposure on AN; *: p < 0.05 vs. S1 of same group; ##: p < 0.01 vs. S2 of same group. B Injector tips placements for rats included in A). C Bilateral intra-BLA SCOP infusion immediately after novel taste intake (S1) produced a marked decrease in taste preference on S2; **: p < 0.01 vs. S1 of same group. D Saccharin intake in mL in rats of the SCOP group of the experiment shown in C) from days 6 to 9 where they were exposed to saccharin only in the morning session; *: p < 0.05. E Fluid intake in mL of these same rats on day 10, where they had the choice between saccharin and water. ns: not statistically significant. F Injector tips placements for rats included in C)

Scopolamine infusion in BLA after novel taste intake produces conditioned taste avoidance

In our second experiment we assessed the effects of scopolamine infusions in the BLA immediately after novel taste intake. Here, surprisingly, when SCOP was infused immediately after novel taste consumption, animals showed a strong avoidance to the taste on the second presentation, two days after. A significant interaction was observed between the Day and Group variables (F(4,80) = 21.47, p < 0.0001; Fig. 2C). Post hoc Tukey test unveiled a significant increase in the PI in the VEH group (PI´s = 39.21 ± 5.3% and 57.65 ± 6.6% for S1 and S2, respectively, p < 0.01), consistent with AN in this group. In the SCOP group, however a significant decrease in the PI was observed from S1 to S2 (PI´s = 36.72 ± 5.9% and 6.72 ± 3.2%, respectively, p < 0.05; see Fig. 2C), suggesting that muscarinic receptors inhibition in the BLA posterior to novel taste consumption promoted avoidance to that taste. Subsequent sessions with free-choice of saccharin and water on the next three days (S3-S5) showed that this marked decrease in the PI was maintained although by S5, it was no longer significantly different from S1 (p = 0.14) possibly highlighting the start of an extinction process see Fig. 2C).

We further submitted this SCOP-induced taste avoidance to extinction by performing the next 4 drinking sessions (S6-S9) with all drinking burettes containing the saccharin solution and giving them the choice between saccharin and water again on the following day (S10). Indeed, although rats did not present preference compared to water on this day, they did not show avoidance to saccharin either (PI = 45.13 ± 9.0), indicating they underwent extinction, although perhaps not complete by this point (Fig. 2D-F). The fact that 4 additional “forced” saccharin drinking sessions were required underscore the robustness of the taste avoidance induced by intra-BLA SCOP infusion after taste intake.

Intra-BLA scopolamine-induced taste avoidance does not generalize to another novel taste

It has been demonstrated that under certain circumstances, animals conditioned to avoid saccharin initially generalize this avoidance to other novel tastants (Wu et al. 2021; Ramos et al. 2022). Given our observation that intra-BLA SCOP infusions in the BLA after novel taste consumption appeared to produce a clear conditioned taste avoidance, we next evaluated whether this pharmacologically induced avoidance was specific to the “conditioned” taste or if it could generalize to other novel tastes. For this, we presented rats with another different taste (0.15 M NaCl in tap water) that was not previously paired with intra-BLA SCOP infusions. When comparing taste preference for saccharin that was previously paired with intra-BLA SCOP infusions versus taste preference for NaCl, a significant Treatment x Taste interaction was observed (F(3,36) = 4.96, p < 0.01, Fig. 3). The neophobic reaction to saccharin in this set of experiments was exceptionally strong with a 15.8% PI when combining VEH and SCOP groups. This is possibly why we did not observe a significant drop in the PI on the second presentation in the SCOP group (12.7% on S2, p > 0.91). The VEH group on the other hand did present a significant AN on S2 (from 14.66% ± 3.2% on S1 to 48.11 ± 8.5% on S2, p < 0.01). Given our earlier observations presented on Fig. 2, as well as those from subsequent experiments (see Fig. 4) we attribute the lack of increase in the PI to a taste-avoidance inducing rather than an amnesic effect by SCOP.

Fig. 3

A Behavioral procedures for the experiment aimed at assessing the possibility of generalization of the SCOP-induced taste avoidance. B Taste avoidance observed on S2 after post-S1 bilateral intra-BLA SCOP infusion did not extend to another novel taste (NaCl); *: p < 0.05 vs. S2-VEH. C Injector tips for rats included in B)

Fig. 4

Effect of intra-BLA SCOP infusion immediately after S2 (A) or S5 (C). B-D injector tip placements for rats included in A and C, respectively

When comparing the PI for the second NaCl presentation (N2), no difference was observed between groups (PI N2 = 30.11 ± 7.2% vs. 30.73 ± 8.7%, for VEH and SCOP, respectively, Sidak post hoc test p > 0.72; Fig. 3). This suggests that post-taste intra-BLA SCOP infusions produces a conditioned taste avoidance that is specific and does not generalize to other novel tastes.

Saccharin pre-exposition for several days does not prevent intra-BLA scopolamine-induced taste avoidance

We assessed whether this taste avoidance-producing effect was dependent upon the novelty of the taste stimulus. Under a one-bottle experimental paradigm, in which rats are presented with saccharin only without the option to drink plain water, AN has been described as a graded learning task in which animals usually reach asymptotic performance by the 4th or 5th taste presentation (Rodriguez-Ortiz et al. 2005; Morin et al. 2011). We therefore performed two additional experiments, the first with intra-BLA SCOP infusions after AN retrieval (post-S2) and the second with the same treatment administered after familiar taste intake (post-S5). In the first experiment, the data obtained showed a significant interaction between Day and Treatment (F(4, 56) = 23.44, p < 0.0001). Post hoc Tukey test detected a marked decrease in the PI in the SCOP group from S2 to S3 (PI = 58.60 ± 7.7% and 5.89 ± 2.4% SCOP p < 0.001; Fig. 4A-B), suggesting that muscarinic receptors inhibition in the BLA after AN retrieval promoted avoidance to the taste on the third presentation. In the second experiment, the data yielded a significant interaction between Day and Group (F(5, 75) = 7.374, p < 0.0001). Post hoc Tukey test showed a pronounced decrease in PI in SCOP group from S5 to S6 (PIs = 61.83 ± 7.3 vs. 16.35 ± 8.4%, p < 0.05; Fig. 4C-D). Therefore, although a weak latent inhibition (LI) appears to have occurred in the post-S5 experiment (the PI of S6 was comparable to that of S1, p > 0.89) our data show that even when rats were previously familiarized with the taste, infusions of SCOP in the BLA after its consumption produced conditioned taste avoidance.

M1/M3, but not M2 antagonism in BLA following novel taste produces a moderate avoidance to the taste on subsequent encounters

We next assessed the effect of administering specific muscarinic receptors antagonists in the BLA after novel taste intake. First, M1-type receptor antagonist PZP was bilaterally infused in the BLA immediately after novel taste intake and subsequent taste intake was compared to their VEH counterparts. A significant interaction between Treatment and Day variables F(5, 90) = 2.54, p < 0.05) was observed. Post hoc analysis unveiled that VEH animals did not reach a significant increase in their PI by S2 but did so by S3 (PI´s = 43.47 ± 7.7% and 72.86 ± 7.4% for S1 and S3, respectively, p < 0.001; Fig. 5A-B). This increase, however, was not observed in the PZP group: S1 and S3 PIs = 40.41 ± 6.6% and 44.42 ± 8.5%, respectively, p > 0.99, indicating that AN did not occur in these rats (see Fig. 5A-B).

Fig. 5

Effect of bilateral intra-BLA PZP (A-B), 4-DAMP (C-D) and MET (E–F) immediately after novel saccharin intake on AN. *: p < 0.05 vs. S1 of same group; ***: p < 0.001 vs. S1 of same group; #: p < 0.05 MET + VEH S1 vs. S2

Then we evaluated the effect of bilateral, intra-BLA infusion of M3 receptor antagonist 4-DAMP after novel taste. Here again, we observed a significant interaction between Day and Treatment variables F(4, 64) = 4.63, p < 0.01). Tukey post hoc tests showed that VEH had a significant IP increase from S1 to S2 (PI’s = 34.74 ± 5.0% and 63.76 ± 8.7%, respectively (n = 9), p < 0.05; Fig. 5 C-D). This increase was not observed in the 4-DAMP-infused group (PIs = 46.71% ± 8.7% vs. 32.77 ± 8.1, for S1 and S2, respectively, p > 0.74) again indicating that AN did not occur in this group (see Fig. 5 C-D).

Finally, we tested the effect of inhibiting presynaptic M2 receptors by infusing MET intra-BLA after S1. Data showed that there was no interaction between Day and Drug F(4,64) = 1.53, p > 0.20. There was, however, a main effect of Day (F(4,64), = 8.80, p < 0.0001; Fig. 5 E–F), indicating safe taste memory formation regardless of treatment.