Extinguishing trace fear engages the retrosplenial cortex rather than the amygdala

Highlights

• We test how the neural circuit for trace fear extinction differs from that of delay.

• Glutamate receptor blockade in the amygdala impairs delay but not trace extinction

• pERK is increased in the RSC and not the amygdala for trace extinction.

• Intra-RSC blockade of NMDA receptors impairs trace but not delay extinction.

• The RSC, instead of the amygdala, is required for complex trace fear extinction.

Abstract

Extinction learning underlies the treatment for a variety of anxiety disorders. Most of what is known about the neurobiology of extinction is based on standard “delay” fear conditioning, in which awareness is not required for learning. Little is known about how complex, explicit associations extinguish, however. “Trace” conditioning is considered to be a rodent model of explicit fear because it relies on both the cortex and hippocampus and requires explicit contingency awareness in humans. Here, we explore the neural circuit supporting trace fear extinction in order to better understand how complex memories extinguish. We first show that the amygdala is selectively involved in delay fear extinction; blocking intra-amygdala glutamate receptors disrupted delay, but not trace extinction. Further, ERK phosphorylation was increased in the amygdala after delay, but not trace extinction. We then identify the retrosplenial cortex (RSC) as a key structure supporting trace extinction. ERK phosphorylation was selectively increased in the RSC following trace extinction and blocking intra-RSC NMDA receptors impaired trace, but not delay extinction. These findings indicate that delay and trace extinction require different neural circuits; delay extinction requires plasticity in the amygdala whereas trace extinction requires the RSC. Anxiety disorders linked to explicit memory may therefore depend on cortical processes that have not been traditionally targeted by extinction studies based on delay fear.

Introduction

The ability to accurately predict and respond to danger signals is critical for an animal’s survival. Failure to react in the face of a cue that signals danger can result in harm or death. Conversely, it is also maladaptive for an animal to respond in a disproportionate manner to a nonthreatening cue. In humans, excessive responding to situations and cues that are poor predictors of danger may contribute to anxiety disorders (Davis, 2002, Rothbaum and Davis, 2003).

Anxiety disorders are often treated clinically through behavioral extinction in which the individual is exposed to the threatening stimulus in the absence of an aversive outcome (Barad, 2005, Foa, 2000, Rothbaum and Schwartz, 2002, Wolpe, 1969). Affective reactions to the stimulus are gradually reduced as the person learns that the cue does not predict danger. These exposure-based therapies can be modeled in rodents through fear conditioning and extinction training as a way to understand the neural mechanisms underlying anxiety reduction (Davis, 2002, Milad and Quirk, 2012).

To date, most of the research on the neural mechanisms of extinction learning comes from rodent studies that use delay fear conditioning to model anxiety (for review, see Milad & Quirk, 2012). In delay fear conditioning, an initially neutral conditional stimulus (CS), such as a white noise or tone, is presented contiguously with a naturally aversive unconditional stimulus (UCS), such as a foot shock. Delay fear can be acquired very rapidly and, in humans, can be learned and expressed without awareness of the stimulus relationship (Clark and Squire, 1998, Knight et al., 2006) making it a good model for basic, implicit fear memories. Delay fear extinction requires three critical brain structures: the infralimbic medial prefrontal cortex (IL), the hippocampus, and the amygdala (Sierra-Mercado, Padilla-Coreano, & Quirk, 2011). The hippocampus’ role in extinguishing fear to a discrete auditory CS is largely restricted to controlling the context specificity of extinction (Corcoran et al., 2005, Hobin et al., 2006) although more recent evidence points to a central role of the hippocampus in extinction when the most salient predictor of shock is the training context (Fischer et al., 2007, Huh et al., 2009, Radulovic and Tronson, 2010, Schimanski et al., 2002, Tronson et al., 2009, Vianna et al., 2001). In contrast to the hippocampus, both the IL and amygdala undergo plastic changes during the extinction of an auditory CS previously used in delay fear conditioning. This plasticity in IL and amygdala regions is believed to support the formation of a new extinction memory (Herry et al., 2010, Quirk and Mueller, 2008). Blocking neural activity or general plasticity in the IL (Burgos-Robles et al., 2007, Sierra-Mercado et al., 2011) or amygdala (Sierra-Mercado et al., 2011, Sotres-Bayon et al., 2007) is sufficient to disrupt the formation of extinction memory, usually tested the following day.

While the neural mechanisms supporting delay fear extinction have received substantial recent attention, far less is understood about the extinction of more complex associations that may better relate to explicit memory in humans. This is important because anxiety disorders can involve both implicit and explicit associations (Brewin, 2001, Rothbaum and Davis, 2003). One way to investigate the neural basis of explicit memory extinction is to use trace fear conditioning. In trace fear conditioning, the CS and UCS are separated by an empty period of time, called the trace interval. Temporal separation of the two cues makes the association slightly more difficult to learn but significantly alters the circuitry and attentional mechanisms required for acquisition. Whereas delay fear can be acquired without awareness and relies largely on subcortical structures (particularly the amygdala), trace fear conditioning requires awareness of the CS–UCS contingency and relies on hippocampal and cortical participation for acquisition (Gilmartin and Helmstetter, 2010, Gilmartin et al., 2012, Han et al., 2003, Knight et al., 2006, Quinn et al., 2002) in addition to the amygdala (Gilmartin et al., 2012, Kwapis et al., 2011). Trace conditioning shares a number of important characteristics with human declarative memory. First, as with explicit memory in humans, trace fear conditioning involves learning a relatively complex relationship between multiple stimuli. Second, explicit awareness of the CS–UCS contingency is necessary for human participants to learn trace fear (Knight et al., 2006, Weike et al., 2007). Finally, trace fear conditioning involves structures known to participate in declarative memory, including the hippocampus and cortex (Gilmartin and Helmstetter, 2010, Gilmartin et al., 2012, Han et al., 2003, Quinn et al., 2002, Squire, 1992). Trace fear conditioning is therefore a particularly good paradigm for modeling explicit fear memory in rodents.

Despite the clear value of trace fear conditioning as a model of fear memory, few studies have investigated extinction after this training procedure (Abumaria et al., 2011, Kaczorowski et al., 2012). To date, no study has systematically investigated how the circuitry supporting trace extinction differs from the established circuit that supports delay fear extinction. Delay and trace conditioning rely on different structures for acquisition, however, so it is feasible that the circuits required for extinction are also distinct. Structures such as the PL, which is required for trace fear acquisition (Gilmartin & Helmstetter, 2010), and the retrosplenial cortex (RSC), which is involved in contextual and relational associations (Aggleton, 2010, Cooper et al., 2001, Corcoran et al., 2011, Haijima and Ichitani, 2008, Katche et al., 2013, Katche et al., 2013, Keene and Bucci, 2008a, Keene and Bucci, 2008b, Robinson et al., 2011), for instance, may supplement or take over the roles of the amygdala, IL, and hippocampus in the extinction of trace fear. The RSC is particularly suitable for supporting explicit associations, as it plays a well-documented role in supporting autobiographical, relational, and spatial memory in humans (Maddock, 1999, Maguire, 2001, Rosenbaum et al., 2004, Steinvorth et al., 2006, Svoboda et al., 2006). Whether the RSC plays a role in trace extinction, however, is unknown. Characterizing the neural circuit that underlies trace fear extinction is an important step towards a comprehensive understanding of anxiety reduction in humans.

Here, we tested whether the circuitry supporting trace fear extinction is the same or different from that of delay extinction. First, we tested whether the amygdala is necessary for trace extinction, as it is with delay. We then measured the phosphorylation of extracellular regulated kinase (pERK) in a number of candidate brain structures in order to identify regions that undergo extinction-related plasticity following trace fear extinction. One region of interest, the retrosplenial cortex, showed elevated pERK following trace, but not delay extinction, suggesting that this region is selectively involved in the extinction of trace fear. In our final study, we directly tested whether the RSC is required for trace, but not delay fear extinction. Together, our results demonstrate that trace fear extinction relies on a different neural circuit than delay extinction.