Deletion of Stk11 and Fos in mouse BLA projection neurons alters intrinsic excitability and impairs formation of long-term aversive memory

Abstract

27Conditioned taste aversion (CTA) is a form of one-trial learning dependent on basolateral 28 amygdala projection neurons (BLApn). Its underlying cellular and molecular mechanisms 29 are poorly understood, however. We used RNAseq from BLApn to identify learning-30 related changes in Stk11, a kinase with well-studied roles in growth, metabolism and 31 development, but not previously implicated in learning. Deletion of Stk11 restricted to 32 BLApn completely blocks memory when occurring prior to training, but not following it, 33 despite altering neither BLApn-dependent encoding of taste palatability in gustatory 34 cortex, nor transcriptional activation of BLApn during training. Deletion of Stk11 in BLApn 35 also increases their intrinsic excitability. Conversely, BLApn activated by CTA to express 36 the immediate early gene Fos had reduced excitability. BLApn knockout of Fos also 37 increased excitability and impaired learning. These data suggest that Stk11 and Fos 38 expression play key roles in CTA long-term memory formation, perhaps by modulating 39 the intrinsic excitability of BLApn. (149 words) 40 41 103 104 Results 105CTA long-term memory requires BLA transcription 106 In order to determine whether CTA requires new RNA transcription within the BLA, we 107 inhibited transcription by injecting Actinomycin-D (1 µl, 50 ng, bilaterally, Figure 1), a 108 widely used RNA polymerase 2 inhibitor (Alberini, 2009), into the BLA 20 min prior to CTA 109 training, and tested memory 48 hours later. As a control, a separate group of mice 110 received vehicle injection (1 µl of PBS, bilaterally). CTA training consisted of 30 min of 111 access to 0.5% saccharin followed by an intraperitoneal injection of 0.15M LiCl, 2% body 112 weight; (Figure 1-figure supplement 1). A two-way ANOVA comparing vehicle and 113 actinomycin-D injected mice before and after training revealed significant training and 114 129 analysis revealed significant reduction in the consumption of saccharin (CTA vs. Test) for 130 the vehicle group, indicating impairment of learning for the actinomycin-D treated group 131 compared to control mice. As a convergent measure, we also assessed the strength of 132 CTA memory by calculating the relative consumption of saccharin during the test day to 133 that consumed on the training day (Neseliler et al., 2011). The differences between the 134 groups were large (23% in the vehicle group vs. 80 % for the actinomycin D group) and 135 significant. Meanwhile, actinomycin-treated mice were neither impaired in their ability to 136 detect the palatability of saccharin, nor in their drinking behavior-consumption of 137 saccharin during CTA training was similar for the two groups, as was consumption of 138 water 8 hours after the test ( Figure 1E), suggesting that these nonspecific effects cannot 139 account for the memory impairment. Thus, BLA transcription is essential for CTA memory 140 formation. These results extend prior work showing the importance of BLA protein 141 synthesis for CTA memory (Josselyn et al., 2...

Perturbation of amygdala-cortical projections reduces ensemble coherence of palatability coding in gustatory cortex

“…Previous studies have reported robust effects of similar perturbations on palatabilityguided behavior (CTA learning and taste neophobia; Gallo et al, 1992;Lin et al, 2009;Lin and Reilly, 2012;Lavi et al, 2018;Levitan et al, 2020). In our hands, however, the impact of BLA→GCx was less complete, and some GC neurons even gained palatability responses in perturbed trials ( Figure 8B).…”

“…Palatability-relatedness in the firing of some single neurons was not utterly eliminated by our input manipulation; in some cases it was even enhanced. This fact is perhaps somewhat surprising given the well-known importance of amygdala for emotion processing (e.g., Quirk et al, 1995;Schoenbaum et al, 1998;LeDoux, 2000;Wang et al, 2005;Wassum and Izquierdo, 2015;Beyeler et al, 2018), and findings suggesting that BLA-GC circuitry is vital for palatability-related behavior (CTA learning and taste neophobia; Gallo et al, 1992;Lin and Reilly, 2012;Levitan et al, 2020). Our recent data suggest a possible explanation, however: as previously discussed, the emergence of Late-epoch palatability coding is revealed, using single-trial analyses involving Hidden Markov Modeling (HMM), to be a sudden transition into a new ensemble state, in which firing-rate changes occur simultaneously in multiple GC neurons (Jones et al, 2007;Miller and Katz, 2010;Sadacca et al, 2016); it is this sudden transition itself that directly drives behavior (Mukherjee et al, 2019).…”

Intrinsic excitability in layer IV-VI anterior insula to basolateral amygdala projection neurons encodes the confidence of taste valence

“…By virtue of its extensive network of connectivity, this elongated cortical structure has been shown to integrate sensory, emotional, motivational, and cognitive brain centers through distinct mechanisms. For example, deletions of either Fos or Stk11 in BLA-aIC neurons, alter intrinsic properties at the aIC, and impair CTA acquisition (Levitan et al, 2020). Furthermore, approach behaviors in social decision-making are modulated by subjective and sex-specific affective states that regulate cell-type-specific changes in intrinsic properties at IC terminals receiving hypothalamic input (Rogers-Carter et al, 2018, 2019; Rieger et al, 2022).…”