In the 1990s, the model emerged that CS–US convergence in LA led to the Hebbian strengthening of CS synapses such that subsequent CS presentations excited LA cells more strongly, allowing them to elicit conditioned responses (CRs) via their projection to CeA.6,7,8,9 However, some aspects of this model were inconsistent with evidence available at the time. First, it was known that the most effective way to condition animals was to offset the start of the CS and US by 10–30 s with the two co-terminating, rather than presenting them simultaneously, as would be expected for a Hebbian mechanism.32 However, despite the persistence of the CS for tens of seconds, the CS responses of LA neurons were known to be transient: a brief burst of short-latency spikes followed by a return of FRs near baseline.33,34,35 Consistent with these observations, recent whole-cell recordings of BLA neurons in vivo revealed that prolonged auditory stimuli elicit synaptic potentials that rapidly become sub-threshold.36

Thus, by the time the US occurs, LA neurons are firing little, and synaptic inputs carrying CS information are no longer active. Yet, Hebbian plasticity should be contingent on N-methyl-D-aspartate (NMDA) receptor activity at the time of CS–US pairing. While the susceptibility of classically conditioned fear to NMDA receptor antagonists is consistent with this possibility,37,38,39 given the time course of LA activity and afferent inputs during the CS, it seems unlikely that NMDA receptors are engaged when the US is presented 20–30 s after CS onset.

Furthermore, fear conditioning was known to potentiate only the initial, short-lived response of LA neurons to the CS,33,34,35 raising the question of how such a transient CR could maintain CRs for tens of seconds. Despite these discrepant observations, the model lived on and it became natural to think of CS-triggered LA firing as sensory responses that automatically drive CRs. Soon, this tendency generalized to neurons in other amygdala nuclei and to appetitive conditioning paradigms.40,41,42,43,44,45

Yet, in the amygdala nuclei that are downstream of LA, like BL and CeA, CS-elicited firing parallels the time course of the associated CRs much more closely than in LA.44, 46 This raises the possibility that in between LA and its targets, there is a shift in the nature of the representation, from a sensory code to a behavioral response code. It seems to us that such a transformation is needed to account for the behavioral flexibility displayed by mammals when threatened. For instance, depending on threat proximity, rats will display freezing, fleeing, or attack.47 Models of fear learning solely based on the potentiated sensory responses of LA neurons cannot account for such flexibility.

If indeed the CS representation shifts from a sensory code in LA to a behavioral response code in downstream nuclei, given a CS–US contingency determined by the investigator, firing during the CS should be variable in targets of LA, depending on trial-to-trial variations in the subject’s “decisions”, even though the CS–US contingency is fixed. Lee et al.48 examined this question by contrasting the activity of BL neurons when rats did, or did not, produce the appropriate CR (Fig. 2). One tone (CS-R) predicted reward delivery while another (CS-N) did not. As a result of conditioning, a low proportion of projection cells (hereafter termed R-neurons) exhibited increased FRs during the CS-R. Lee et al.48 found that the CS-related firing of R-neurons varied strongly with conditioned responding: it was present when they emitted the conditioned approach behavior in response to the CS-R (Fig. 2a, blue) but was absent when rats omitted it (Fig. 2a, red). Furthermore, R-neurons responded to the CS-N when rats exhibited (in error) the CR during the CS-N. Therefore, the activity of R-neurons is only coincidentally related to the CSs’ sensory properties. They actually encode behavioral output (Fig. 2b, c). Presumably, amygdala nuclei downstream of LA contain multiple subsets of neurons that drive different aversive or appetitive behaviors via segregated projections to various subcortical sites.49,50,51,52,53

Fig. 2 Activity of BL neurons during CSs varies depending on behavior. Rats were presented with two CS, one of which predicted reward delivery (CS-R) and the second (CS-N), a neutral outcome. After rats learned to retrieve the liquid reward at the conclusion of the CS-R, the two CSs were presented in random order while recording BL neurons. a Activity of a principal BL cell during multiple presentations of the CS-R. Activity is depicted as a color-coded raster (top) and peri-CS histogram of neuronal discharges (bottom; solid and dashed lines are averages and SEM, respectively). Trials are grouped by behavior as indicated by the colored bar on the left of the rasters (blue, approach of water-port; red, no approach). Rasters and peristimulus histograms in b depict the activity of the same cell as in a, but aligned to onset of water-port approach instead of CS onset. Note higher variations in firing latency when activity is referenced to onset of CS-R than to approach behavior. c Raster and histogram show activity of the same cell when the rat spontaneously initiated approach of the water-port in the absence of CS. White dots in raster indicate time when rats left the water-port. Source: Ref. 48 Full size image

On the surface, the existence of BL cells that are preferentially active during specific behaviors (like R-neurons) appears inconsistent with the notion that amygdala activity inhibits behavioral engagement. A possible solution to this conundrum is that the ability of BL to influence behavior not only depends on the activation of a specific subset of behavior-coding neurons but also on the suppression of all others. Consistent with this possibility, in a mixed appetitive-aversive conditioning paradigm, only 7% of principal cells increased their FRs when the appetitive CS was presented; most (52%) were inhibited (S.C. Lee, A. Amir, D. Haufler, D. Paré, unpublished observations). Although excitatory CS responses tend to attract more attention, inhibitory responses actually prevail, consistent with our model.

So far, there were few opportunities to explore these questions because investigators have relied heavily on classical fear conditioning, where the range of possible behaviors is extremely limited. Clarifying what amygdala neurons code for will require behavioral paradigms that allow one to compare the activity of the same neurons when animals express a larger repertoire of spontaneous and learned behaviors under multiple reinforcement contingencies. Parallel measurements of autonomic responses, such as pupil dilation, gastric motility, as well as heart and respiration rate, would also be useful.