The role of amygdala nuclei in the expression of auditory signaled two-way active avoidance in rats

Using a two-way signaled active avoidance (2-AA) learning procedure, where rats were trained in a shuttle box to avoid a footshock signaled by an auditory stimulus, we tested the contributions of the lateral (LA), basal (B), and central (CE) nuclei of the amygdala to the expression of instrumental active avoidance conditioned responses (CRs). Discrete or combined lesions of the LA and B, performed after the rats had reached an asymptotic level of avoidance performance, produced deficits in the CR, whereas CE lesions had minimal effect. Fiber-sparing excitotoxic lesions of the LA/B produced by infusions of N-methyl-d-aspartate (NMDA) also impaired avoidance performance, confirming that neurons in the LA/B are involved in mediating avoidance CRs. In a final series of experiments, bilateral electrolytic lesions of the CE were performed on a subgroup of animals that failed to acquire the avoidance CR after 3 d of training. CE lesions led to an immediate rescue of avoidance learning, suggesting that activity in CE was inhibiting the instrumental CR. Taken together, these results indicate that the LA and B are essential for the performance of a 2-AA response. The CE is not required, and may in fact constrain the instrumental avoidance response by mediating the generation of competing Pavlovian responses, such as freezing.Early studies of the neural basis of fear often employed avoidance conditioning procedures where fear was assessed by measuring instrumental responses that reduced exposure to aversive stimuli (e.g., Weiskrantz 1956; Goddard 1964; Sarter and Markowitsch 1985; Gabriel and Sparenborg 1986). Despite much research, studies of avoidance failed to yield a coherent view of the brain mechanisms of fear. In some studies, a region such as the amygdala would be found to be essential and in other studies would not. In contrast, rapid progress in understanding the neural basis of fear and fear learning was made when researchers turned to the use of Pavlovian fear conditioning (Kapp et al. 1984, 1992; LeDoux et al. 1984; Davis 1992; LeDoux 1992; Cain and Ledoux 2008a). It is now well established from such studies that specific nuclei and subnuclei of the amygdala are essential for the acquisition and storage of Pavlovian associative memories about threatening situations (LeDoux 2000; Fanselow and Gale 2003; Maren 2003; Maren and Quirk 2004; Schafe et al. 2005; Davis 2006).Several factors probably contributed to the fact that Pavlovian conditioning succeeded where avoidance conditioning struggled. First, avoidance conditioning has long been viewed as a two-stage learning process (Mowrer and Lamoreaux 1946; Miller 1948b; McAllister and McAllister 1971; Levis 1989; Cain and LeDoux 2008b). In avoidance learning, the subject initially undergoes Pavlovian conditioning and forms an association between the shock and cues in the apparatus. The shock is an unconditioned stimulus (US) and the cues are conditioned stimuli (CS). Subsequently, the subject learns the instrumental response to avoid the shock. Further, the “fear” aroused by the presence of the CS motivates learning of the instrumental response. Fear reduction associated with successful avoidance has even been proposed to be the event that reinforces avoidance learning (e.g., Miller 1948b; McAllister and McAllister 1971; Cain and LeDoux 2007). Given that Pavlovian conditioning is the initial stage of avoidance conditioning, as well as the source of the “fear” in this paradigm, it would be more constructive to study the brain mechanisms of fear through studies of Pavlovian conditioning rather than through paradigms where Pavlovian and instrumental conditioning are intermixed. Second, avoidance conditioning was studied in a variety of ways, but it was not as well appreciated at the time as it is today; that subtle differences in the way tasks are structured can have dramatic effects on the brain mechanisms required to perform the task. There was also less of an appreciation for the detailed organization of circuits in areas such as the amygdala. Thus, some avoidance studies examined the effects of removal of the entire amygdala or multiple subdivisions (for review, see Sarter and Markowitsch 1985). Finally, fear conditioning studies typically involved a discrete CS, usually a tone, which could be tracked from sensory processing areas of the auditory system to specific amygdala nuclei that process the CS, form the CS–US association, and control the expression of defense responses mediated by specific motor outputs. In contrast, studies of avoidance conditioning often involved diffuse cues, and the instrumental responses used to indirectly measure fear were complex and not easily mapped onto neural circuits.Despite the lack of progress in understanding the neural basis of avoidance responses, this behavioral paradigm has clinical relevance. For example, avoidance behaviors provide an effective means of dealing with fear in anticipation of a harmful event. When information is successfully used to avoid harm, not only is the harmful event prevented, but also the fear arousal, anxiety, and stress associated with such events; (Solomon and Wynne 1954; Kamin et al. 1963). Because avoidance is such a successful strategy to cope with danger, it is used extensively by patients with fear-related disorders to reduce their exposure to fear- or anxiety-provoking situations. Pathological avoidance is, in fact, a hallmark of anxiety disorders: In avoiding fear and anxiety, patients often fail to perform normal daily activities (Mineka and Zinbarg 2006).We are revisiting the circuits of avoidance conditioning from the perspective of having detailed knowledge of the circuit of the first stage of avoidance, Pavlovian conditioning. To most effectively take advantage of Pavlovian conditioning findings, we have designed an avoidance task that uses a tone and a shock. Rats were trained to shuttle back and forth in a runway in order to avoid shock under the direction of a tone. That is, the subjects could avoid a shock if they performed a shuttle response when the tone was on, but received a shock if they stayed in the same place (two-way signaled active avoidance, 2-AA). While the amygdala has been implicated in 2-AA (for review, see Sarter and Markowitsch 1985), the exact amygdala nuclei and their interrelation in a circuit are poorly understood.We focused on the role of amygdala areas that have been studied extensively in fear conditioning: the lateral (LA), basal (B), and central (CE) nuclei. The LA is widely thought to be the locus of plasticity and storage of the CS–US association, and is an essential part of the fear conditioning circuitry. The basal amygdala, which receives inputs from the LA (Pitkänen 2000), is not normally required for the acquisition and expression of fear conditioning (Amorapanth et al. 2000; Nader et al. 2001), although it may contribute under some circumstances (Goosens and Maren 2001; Anglada-Figueroa and Quirk 2005). The B is also required for the use of the CS in the motivation and reinforcement of responses in other aversive instrumental tasks (Killcross et al. 1997; Amorapanth et al. 2000). The CE, through connections to hypothalamic and brainstem areas (Pitkänen 2000), is required for the expression of Pavlovian fear responses (Kapp et al. 1979, 1992; LeDoux et al. 1988; Hitchcock and Davis 1991) but not for the motivation or reinforcement of aversive instrumental responses (Amorapanth et al. 2000; LeDoux et al. 2009). We thus hypothesized that damage to the LA or B, but not to the CE, would interfere with the performance of signaled active avoidance.