The basolateral amygdala complex and perirhinal cortex represent focal and peripheral states of information processing in rats

Reviewer #1 (Public review):

Summary:

This study advances the lab's growing body of evidence exploring higher-order learning and its neural mechanisms. They recently found that NMDA receptor activity in the perirhinal cortex was necessary for integrating stimulus-stimulus associations with stimulus-shock associations (mediated learning) to produce preconditioned fear, but it was not necessary for forming stimulus-shock associations. On the other hand, basolateral amygdala NMDA receptor activity is required for forming stimulus-shock memories. Based on these facts, the authors assessed: (1) why the perirhinal cortex is necessary for mediated learning but not direct fear learning, and (2) the determinants of perirhinal cortex versus basolateral amygdala necessity for forming direct versus indirect fear memories. The authors used standard sensory preconditioning and variants designed to manipulate the novelty and temporal relationship between stimuli and shock and, therefore, the attentional state under which associative information might be processed. Under experimental conditions where information would presumably be processed primarily in the periphery of attention (temporal distance between stimulus/shock or stimulus pre-exposure), perirhinal cortex NMDA receptor activation was required for learning indirect associations. On the other hand, when information would likely be processed in focal attention (novel stimulus contiguous with shock), basolateral amygdala NMDA activity was required for learning direct associations. Together, the findings indicate that the perirhinal cortex and basolateral amygdala subserve peripheral and focal attention, respectively. The authors provide support for their conclusions using careful, hypothesis-driven experimental design, rigorous methods, and integrating their findings with the relevant literature on learning theory, information processing, and neurobiology. Therefore, this work will be highly interesting to several fields.

Strengths:

(1) The experiments were carefully constructed and designed to test hypotheses that were rooted in the lab's previous work, in addition to established learning theory and information processing background literature.

(2) There are clear predictions and alternative outcomes. The provided table does an excellent job of condensing and enhancing the readability of a large amount of data.

(3) In a broad sense, attention states are a component of nearly every behavioral experiment. Therefore, identifying their engagement by dissociable brain areas and under different learning conditions is an important area of research.

(4) The authors clearly note where they replicated their own findings, report full statistical measures, effect sizes, and confidence intervals, indicating the level of scientific rigor.

(5) The findings raise questions for future experiments that will further test the authors' hypotheses; this is well discussed.

Weaknesses:

As a reader, it is difficult to interpret how first-order fear could be impaired while preconditioned fear is intact; it requires a bit of "reading between the lines".

Reviewer #2 (Public review):

Summary:

This paper continues the authors' research on the roles of the basolateral amygdala (BLA) and the perirhinal cortex (PRh) in sensory preconditioning (SPC) and second-order conditioning (SOC). In this manuscript, the authors explore how prior exposure to stimuli may influence which regions are necessary for conditioning to the second-order cue (S2). The authors perform a series of experiments which first confirm prior results shown by the author - that NMDA receptors in the PRh are necessary in SPC during conditioning of the first-order cue (S1) with shock to allow for freezing to S2 at test; and that NMDA receptors in the BLA are necessary for S1 conditioning during the S1-shock pairings. The authors then set out to test the hypothesis that the PRh encodes associations in a peripheral state of attention, whereas the BLA encodes associations in a focal state of attention, similar to the A1 and A2 states in Wagner's theory of SOP. To do this, they show that BLA is necessary for conditioning to S2 when the S2 is first exposed during a serial compound procedure - S2-S1-shock. To determine whether pre-exposure of S2 will shift S2 to a peripheral focal state, the authors run a design in which S2-S1 presentations are given prior to the serial compound phase. The authors show that this restores NMDA receptor activity within the PRh as necessary for the fear response to S2 at test. They then test whether the presence of S1 during the serial compound conditioning allows the PRh to support the fear responses to S2 by introducing a delay conditioning paradigm in which S1 is no longer present. The authors find that PRh is no longer required and suggest that this is due to S2 remaining in the primary focal state.

Strengths:

As with their earlier work, the authors have performed a rigorous series of experiments to better understand the roles of the BLA and PRh in the learning of first- and second-order stimuli. The experiments are well-designed and clearly presented, and the results show definitive differences in functionality between the PRh and BLA. The first experiment confirms earlier findings from the lab (and others), and the authors then build on their previous work to more deeply reveal how these regions differ in how they encode associations between stimuli. The authors have done a commendable job of pursuing these questions.

Table 1 is an excellent way to highlight the results and provide the reader with a quick look-up table of the findings.

Weaknesses:

The authors have attempted to resolve the question of the roles of the PRh and BLA in SPC and SOC, which the authors have explored in previous papers. Laudably, the authors have produced substantial results indicating how these two regions function in the learning of first- and second-order cues, providing an opportunity to narrow in on possible theories for their functionality. Yet the authors have framed this experiment in terms of an attentional framework and have argued that the results support this particular framework and hypothesis - that the PRh encodes peripheral and the BLA encodes focal states of learning. This certainly seems like a viable and exciting hypothesis, yet I don't see why the results have been completely framed and interpreted this way. It seems to me that there are still some alternative interpretations that are plausible and should be included in the paper.

Reviewer #3 (Public review):

Summary:

This manuscript presents a series of experiments that further investigate the roles of the BLA and PRH in sensory preconditioning, with a particular focus on understanding their differential involvement in the association of S1 and S2 with shock.

Strengths:

The motivation for the study is clearly articulated, and the experimental designs are thoughtfully constructed. I especially appreciate the inclusion of Table 1, which makes the designs easy to follow. The results are clearly presented, and the statistical analyses are rigorous. My comments below mainly concern areas where the writing could be improved to help readers more easily grasp the logic behind the experiments.

Weaknesses:

(1) Lines 56-58: The two previous findings should be more clearly summarized. Specifically, it's unclear whether the "mediated S2-shock" association occurred during Stage 2 or Stage 3. I assume the authors mean Stage 2, but Stage 2 alone would not yet involve "fear of S2," making this expression a bit confusing.

(2) Line 61: The phrase "Pavlovian fear conditioning" is ambiguous in this context. I assume it refers to S1-shock or S2-shock conditioning. If so, it would be clearer to state this explicitly.

(3) Regarding the distinction between having or not having Stage 1 S2-S1 pairings, is "novel vs. familiar" the most accurate way to frame this? This terminology could be misleading, especially since one might wonder why S2 couldn't just be presented alone on Stage 1 if novelty is the critical factor. Would "outcome relevance" or "predictability" be more appropriate descriptors? If the authors choose to retain the "novel vs. familiar" framing, I suggest providing a clear explanation of this rationale before introducing the predictions around Line 118.

(4) Line 121: This statement should refer to S1, not S2.

(5) Line 124: This one should refer to S2, not S1.

(6) Additionally, the rationale for Experiment 4 is not introduced before the Results section. While it is understandable that Experiment 4 functions as a follow-up to Experiment 3, it would be helpful to briefly explain the reasoning behind its inclusion.