Which classical conditioning process is associated with the development of phobias?

4.4 Organization of fear memory associations

Pavlovian fear conditioning involves establishment of associations between neural representations of learning events (Konorski, 1967). The organization of associations developed during a learning experience puzzled early learning theorists (Hull, 1943; Konorski, 1967; Rizley & Rescorla, 1972). Typically, the strength of proposed associations is assessed using various post-training manipulations (Gewirtz & Davis, 2000). In this approach, especially second-order conditioning procedures, compared to first-order learning protocols, were found to be useful, offering a tool for analyzing the structure of associations and the number of possible associations (Gewirtz & Davis, 2000). In second-order conditioning, the CS2 may become associated with the CS1, forming a CS2–CS1 association. Alternatively, the CS2 may become associated with the US, establishing a CS2–US association, or with the fear response, producing a CS2–response memory. Manipulating the value of the CS memory reveals the structure of existing associations. For example, extinction of responding to CS1 resulting in attenuation of responding to CS2 suggests that during second-order learning, a CS2–CS1 association is developed (Gewirtz & Davis, 2000). In contrast, if extinction of responding to CS1 has no effect on memory for CS2, the existence of CS2–US or CS2–response associations would be more plausible (Rizley & Rescorla, 1972). Some authors have proposed to use this approach in analyzing the structure of Pavlovian fear conditioning in the amygdala (Gewirtz & Davis, 2000). We used this methodology, combining extinction procedures with reconsolidation protocols (Debiec et al., 2006, 2010; Diaz-Mataix et al., 2011; Doyere et al., 2007). We found that when two distinct auditory CSs are paired with the same US, exposure to one of these CSs followed by the intra-LA microinfusions of a reconsolidation blocker results in a selective disruption of responding to this CS (Debiec et al., 2010; Doyere et al., 2007). However, an exposure to the shared US followed by the disruption of reconsolidation affects responding to both CSs (Debiec et al., 2010). These findings suggest that in our protocol, each distinct CS has a distinct representation in the LA, although each of these representations is associated with a shared element (representation of the US). However, if these same distinct CSs are used in a second-order conditioning protocol, the post-CS1 disruption of reconsolidation in the LA or extinction of CS1 both affect freezing responding to the CS2 (Debiec et al., 2006). This demonstrates that the same representation of the auditory CS, depending on the learning conditions, forms distinct associations. In another series of experiments, we used two distinct auditory CSs, each paired with a distinct US (either electric foot- or eyelid shock) (Debiec et al., 2010; Diaz-Mataix et al., 2011). Using reconsolidation protocols, we found that exposure to one of the USs followed by the pharmacological disruption of reconsolidation in the LA selectively affects responding to the CS that was paired with this US, leaving responding to the other US intact (Debiec et al., 2010; Diaz-Mataix et al., 2011). These findings were paralleled by the extinction experiments as described in the previous section (Diaz-Mataix et al., 2011). US-selective character of reconsolidation processes suggests that the amygdala distinguishes between these USs and encodes their specific sensory values.

Our findings demonstrate that reconsolidation protocols in combination with extinction procedures provide a powerful tool to gain insights into the architecture of fear memories in the LA.

4.11.8.5 Memory Modulation by the Amygdala

Pavlovian fear conditioning is an implicit form of learning and memory. However, during most emotional experiences, including fear conditioning, explicit or declarative memories are also formed (LeDoux, 2000). These occur through the operation of the medial temporal lobe memory system involving the hippocampus and related cortical areas (Milner et al., 1998; Eichenbaum, 2000). The role of the hippocampus in the explicit memory of an emotional experience is much the same as its role in other kinds of experiences, with one important exception. During fearful or emotionally arousing experiences, the amygdala activates neuromodulatory systems in the brain and hormonal systems in the body via its projections to the hypothalamus, which can drive the hypothalamic-pituitary-adrenal (HPA) axis. Neurohormones released by these systems can, in turn, feed back to modulate the function of forebrain structures such as the hippocampus and serve to enhance the storage of the memory in these regions (McGaugh, 2000). The primary support for this model in animals comes from studies of inhibitory avoidance learning, a type of passive avoidance learning where the animal must learn not to enter a chamber in which it has previously received shock. In this paradigm, various pharmacological manipulations of the amygdala that affect neurotransmitter or neurohormonal systems modulate the strength of the memory. For example, immediate posttraining blockade of adrenergic or glucocorticoid receptors in the amygdala impairs memory retention of inhibitory avoidance, while facilitation of these systems in the amygdala enhances acquisition and memory storage (McGaugh et al., 1993; McGaugh, 2000). The exact subnuclei in the amygdala that are critical for memory modulation remain unknown, as are the areas of the brain where these amygdala projections influence memory storage. Candidate areas include the hippocampus and entorhinal and parietal cortices (Izquierdo et al., 1997). Indeed, it would be interesting to know whether the changes in unit activity or the activation of intracellular signaling cascades in the hippocampus during and after fear conditioning, as discussed earlier, might be related to formation of such explicit memories, and how regulation of these signals depends on the integrity of the amygdala and its neuromodulators. Interestingly, a recent study has shown that stimulation of the basal nucleus of the amygdala can modulate the persistence of LTP in the hippocampus (Frey et al., 2001), which provides a potential mechanism whereby the amygdala can modulate hippocampal-dependent memories.

NPY the BLC, CeA, and fear conditioning

Pavlovian fear conditioning is the neural process whereby an aversive unconditioned stimulus (US; footshock) becomes associated with a non-aversive signal (such as context, light, or tone) to produce a conditioned response (CS; freezing) to the previously non-aversive stimulus. Fear conditioning is necessary for survival yet when this process goes awry, there is inappropriate learning and retention of fear memories which can lead to a number of anxiety-related disorders including PTSD. PTSD is defined as the exaggerated and unrelenting unconditioned responses to stimuli (e.g., crowds, flashes of light, smells, sounds) which are associated with trauma (e.g., death, injury; Mahan & Ressler, 2012). These intense memories underlie some of the pathological conditioned fear responses that are associated with the generation of PTSD (Goode & Maren, 2019). Therefore, conditioned fear has clinical relevance as a model to study the normal responses as well as the maladaptive processes leading to PTSD (Paredes & Morilak, 2019).

Conditioned fear is generated by a well-defined neural circuitry: sensory information from the thalamus, auditory cortex or hippocampus for contextual fear conditioning, is processed in the La and Ba where projection neurons either directly or indirectly (via intercalated cell masses) innervate the CeL and CeM, respectively. Neurons from the CeM project to the periaqueductal gray (PAG) and BST. Additional projections from the infra- and prelimbic cortices modify amygdala activity and responses (reviewed in Perusini & Fanselow, 2015; also see Morozov, Chapter 6). The fear acquisition is dependent upon glutamatergic NMDA (N-methyl-d aspartate)-dependent signaling whereas fear extinction involves long-term depression (LTD)-like mechanisms that are in part dependent upon calcineurin in the amygdala (Lin, Lee, & Gean, 2003).

That NPY immunoreactivity and receptors are located within key sites associated with fear conditioning have implicated NPY signaling as a potential mechanism in modulating fear responses. In NPY KO animals, fear acquisition is accelerated whereas fear extinction is impaired (Verma, Tasan, Herzog, & Sperk, 2012). These responses are dependent upon the appropriate expression of both the Y1 or Y2 receptors, and in general, these studies in knockout mice implicate NPY and the Y1 and Y2 receptors in shaping fear acquisition and extinction. Intracerebroventricular injection of either NPY or [Leu31Pro34]NPY (Table 1) inhibits freezing during fear acquisition in a contextual fear paradigm; these responses were prevented by a Y1 antagonist (Lach & de Lima, 2013). Similarly, NPY inhibits freezing during the extinction trial, although this is not Y1R sensitive. The doses of NPY used in these studies did not alter locomotion or anxiety behaviors which could have influenced behavioral readouts for fear. Administration of NPY icv inhibits fear-potentiated startle through activation of an Y1- but not Y2-receptor-mediated mechanism (Broqua et al., 1995). While important in indicating a role for NPY, and predominantly Y1 receptors, in modulating fear responses, the use of global knockout animals and icv peptide administration do not identify a site of action.

Table 1. NPY Y1, Y2 and Y5 receptor pharmacology.

A survey of NPY expression in the brain after fear conditioning demonstrates that NPY-like immunoreactivity is increased in the BLC but not the CeA or other neighboring nuclei (Krysiak et al., 1999; Leitermann et al., 2016). As the antibodies used in these studies detected pre-proNPY as well as the mature NPY, an additional antibody for the releasable, amidated-NPY (Grouzmann et al., 1992) was used to assess the releasable pool of NPY shortly after exposure to the conditioning chamber just prior to extinction. While pre-proNPY levels were increased at this time, the expression of amidated-NPY immunoreactivity was decreased in conditioned, but not control, rats approximately 20 min after exposure to conditioning chamber; this time course suggested the release of NPY upon exposure to the context prior to extinction testing (Leitermann et al., 2016). This mechanism was more directly tested by Gutman et al. (2008) who showed that intra-BLA injections of the Y1R antagonist, BIBO3304 (Table 1), do not alter fear learning but severely impact retention of extinction. In general, these studies demonstrate that exogenous NPY can modify fear acquisition and extinction, however, the important finding is the endogenous release of NPY which is important for appropriate fear extinction. This may occur through fear-induced changes in the biosynthetic capacity and activity of AStr NPY neurons projecting to the BLC. Others have demonstrated similar effects of NPY on conditioned fear and fear-potentiated startle, however, they were unable to implicate the Y1R in these processes whereas other NPY receptor subtypes have been implicated (Fendt et al., 2009).

The CeA also contributes significantly to fear responses participating as an output relay for the Ba and La (Perusini & Fanselow, 2015). As discussed, NPY, Y1, and Y2 receptors have a prominent expression within this nucleus and to date, the Y2R has been the most studied with respect to fear conditioning. Overexpression of the Y2 receptor-ligand NPY3-36 in the CeM reduces freezing during fear acquisition and recall. Freezing is also reduced during extinction which was more sensitive to cue-induced rather than contextual fear extinction (Verma et al., 2012). While the CeA acts as a transition between cortical processes and downstream regulation of freezing and sympathetic activity, NPY is positioned to regulate a number of responses associated with behavioral and physiological aspects of fear (Gray, 1993; Tovote et al., 2004).

In sum, NPY receptor activation alters the expression of conditioned fear through primarily Y1 and Y2 receptor-mediated mechanisms in the BLC and CeA, respectively. While many of these studies used exogenous administration of the peptide, a key finding of Gutman et al. (2008) demonstrates the active release of NPY within the BLC during fear extinction. This is an important finding from a clinical aspect that, if this mechanism is conserved from rat to humans, patients may receive added benefit from additional NPY treatment or therapies that interfere with the metabolism of NPY in vivo (Canneva et al., 2015; Zoellner, Roy-Byrne, Mavissakalian, & Feeny, 2019).

6.2 Memory extinction versus consolidation

In Pavlovian fear conditioning, a conditioned stimulus (CS; e.g., a context) is paired with an unconditioned stimulus (US; e.g., footshock). When the animal is placed back in the conditioned context, it displays conditioned fear responses such as freezing, in the absence of the US. Experimentally, cued recall typically involves re-exposing subjects to the CS without the US. This reminder initiates not only reconsolidation but also extinction in which the CS comes to predict no US and loses its ability to evoke a conditioned response (Baum, 1988; Bouton, 1993; Myers & Davis, 2002; Pavlov, 1927). Memory extinction is thought to reflect new learning of a CS–no US association that inhibits the conditioned response (Konorski, 1967; Rescorla & Heth, 1975; Rescorla, 2001; Robbins, 1990). Most important, similar to the molecular signatures of memory consolidation, extinction of fear memory was shown to be consolidated through new gene expression (Mamiya et al., 2009; Quirk et al., 2000; Santini et al., 2004).

Extinction is thought to depend on the medial prefrontal cortex (mPFC; Morgan & LeDoux, 1995, 1999; Morgan et al., 1993; Morrow et al., 1999; Quirk et al., 2000; Teich et al., 1989) and amygdala (Herry et al., 2008; Myers & Davis, 2002; Quirk et al., 2000). The amygdala plays significant roles in the acquisition of tone fear extinction and consolidation of contextual fear extinction, whereas the mPFC, especially the prelimbic cortex, is implicated in its consolidation (Mamiya et al., 2009; Quirk et al., 2000; Santini et al., 2004). Interestingly, a recent study identified a population of “extinction” neurons in the basolateral amygdala, whose activation decreases high fear behavior (Herry et al., 2008).

Amygdala

Acquisition of Pavlovian fear conditioning depends on cellular and molecular processes in the basolateral complex of the amygdala (BLA), which includes the basal and lateral amygdalar nuclei. After pairing a tone CS with a footshock unconditioned stimulus (US), for example, neurons in the lateral amygdala (LA) demonstrate increased firing in response to the CS. This increased activity reflects N-methyl-d-aspartate (NMDA) receptor- and protein synthesis-dependent synaptic plasticity. Lesions in or inactivation of the amygdala prevent the acquisition of conditioned fear as well as the expression of previously acquired fear memories. Thus, the amygdala is considered an essential site for the acquisition, consolidation, and retrieval of conditioned fear memories.

Given the critical role for the BLA in fear learning, it is not surprising that it is also involved in the inhibitory learning underlying extinction. Accumulating evidence suggests that the BLA mediates the acquisition of fear extinction, similar to its role in the acquisition of fear conditioning. Extinction of conditioned fear is similar to fear acquisition in that it requires the activation of glutamate receptors in the BLA. Within-session extinction is prevented by intra-BLA blockade of NMDA or metabotropic glutamate receptors. Extinction training engages several intracellular signaling cascades in the BLA, as indicated by increased levels of phosphorylated mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI)-3 kinase. Disruption of MAPK or PI-3 kinase activity in the BLA shortly after extinction training prevents the subsequent retrieval of extinction memory. These signaling pathways lead to the activation of immediate early genes such as c-fos and zif; failure to activate these genes is correlated with poor extinction learning. Within-session extinction of appetitive instrumental responses is blocked by inactivation of the BLA, suggesting that the BLA is similarly involved in the acquisition of extinction for other forms of affective learning.

Consolidation of extinction is also reliant on synaptic plasticity in the BLA. Protein synthesis inhibition in the BLA prevents extinction recall in conditioned fear and taste aversion paradigms. Shortly after extinction training, increased levels of mRNA for the neurotropic factor brain-derived neurotropic factor (BDNF) are observed in the BLA, indicating that structural changes help stabilize the extinction memory. Interestingly, some synaptic changes underlying extinction appear to directly oppose some of the plastic changes that underlie conditioning. For example, fear conditioning decreases levels of the γ-aminobutyric acid (GABA)-clustering protein gephyrin in the BLA, whereas extinction training is correlated with an upregulation of gephyrin and an increase in the surface expression of GABA receptors. Thus, extinction memories may be represented as increases in inhibitory neurotransmission within the amygdala. Similarly, extinction can reverse conditioning-induced phosphorylation of the transcription factor cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB). Extinction training increases the activity of the phosphatase calcineurin, which dephosphorylates CREB. This reversal suggests that, to a certain extent, extinction might cause an erasure of the conditioning memory stored in the BLA.

Neurocircuitry Models of PTSD

Studies of the neurocircuitry of Pavlovian fear conditioning and extinction in rodents and in healthy humans have elucidated the functional neural circuits that form the basis of a fear-conditioning neurocircuitry model of PTSD. Earlier rodent literatures have used the terms “infralimbic” and “prelimbic” when describing brain regions responsible in extinction learning and fear learning. However, in order to emphasize the differences between structures based on cytoarchitecture, Vogt and Paxinos (2014) have proposed using the terms “25” and “32” instead of “infralimbic” and “prelimbic,” respectively.

In rodents, area 25 is involved in the inhibition of fear responses; neurons in area 25 fire during the recall of extinguished fear, and stimulating this structure during CS presentation decreases freezing to subsequent presentations of that CS (Milad and Quirk, 2002). The human functional analogue includes area 25 of the ventromedial prefrontal cortex (vmPFC) (Vogt and Paxinos, 2014) and is part of ACC. Animal studies have shown that these structures directly and indirectly send excitatory projections onto the GABAergic intercalated neurons of the amygdala (Berretta et al., 2005), which in turn inhibit central amygdala output (Amano et al., 2010). Additionally, area 25 in rodents and the functional analogue in humans is necessary for the expression of fear to a conditioned context (as opposed to a single discrete cue), likely due to its connectivity with hippocampus and amygdala (reviewed in Maren et al., 2013).

In rodents, neurons in area 32 activate to fear-conditioned stimuli during fear responses (i.e., freezing), including activation to conditioned stimuli that had undergone unsuccessful extinction and still elicit a freezing response (Burgos-Robles et al., 2009). Area 32 sends excitatory projections to the basolateral nuclei of the amygdala, which supports the binding together of CS–US associations and in turn projects to the central nucleus, the amygdala's “output.” In most of the human fear and PTSD literature, the functional analogue of rodent area 32 is referred to as the dorsal anterior cingulate cortex or dACC. However, according to more recent anatomic work discussed elsewhere in this volume (see Chapter 1 by Brent A. Vogt), this region in humans would perhaps more accurately be referred to as aMCC (e.g., Shin and Handwerger, 2009; VanElzakker et al., 2014). The human aMCC has been shown to activate to otherwise-neutral conditioned stimuli that have been fear conditioned, with a corresponding fear response of increased perspiration measured using skin conductance response (SCR) instead of freezing behavior (Milad et al., 2007). Likewise, in PTSD subjects the aMCC activated to conditioned stimuli that had undergone unsuccessful extinction and elicited increased SCR (Milad et al., 2009).

In a relatively simplistic model of PTSD, previously neutral stimuli are associated with traumatic event(s) and become CSs that can elicit a fear response. Fear responses may also generalize to other stimuli that resemble the original CSs. Failure to extinguish this fear response is considered a driving factor in sustaining PTSD symptoms. This extinction failure could be due to impaired extinction neurocircuitry and/or due to avoidance of triggering CSs, preventing the opportunity for extinction learning. Regarding PTSD neurocircuitry, this model would predict that the aMCC, amygdala, and insular cortex are hyperactivated, reflecting greater fear expression while the ACC is hypoactivated and fails to inhibit the amygdala, reflecting the observed extinction deficits in PTSD (refer to Fig. 20.1 for the anatomic regions of interest in the neurocircuitry of PTSD).

While a neurocircuitry model based on Pavlovian fear conditioning cannot explain all PTSD symptoms (e.g., ongoing anxiety in the absence of a reminder cue, endocrine and cognitive problems), it has offered a simplified way to understand some core symptoms better, and it is a useful model for exposure-based therapies used to treat PTSD.

Of course, other frameworks can be used to understand functional brain abnormalities in PTSD and they may not be mutually exclusive with Pavlovian fear conditioning-based neurocircuitry models (Patel et al., 2012). For example, the triple network model of psychopathology shifts the focus from the function of specific brain regions to that of large interconnected networks that underlie basic brain function: (1) the salience network, (2) the default mode network, and (3) the central executive network (Greicius et al., 2003; Seeley et al., 2007; Andrews-Hanna et al., 2010; Menon, 2011). Dysfunction in any (or all) of these networks can be associated with psychopathology. The salience network, which includes the amygdala, aMCC, and insular cortex, involves detecting and attending to salient stimuli and may help to switch between the other two networks in the model. The default mode network, which includes the vmPFC (including the ACC), hippocampus, PCC, and precuneus, mediates internally focused, self-referential thought and is measured during the absence of a specific task, at a resting baseline. Nodes in this network are typically deactivated during tasks. Finally, the central executive network, which includes the dorsal aMCC, dorsolateral prefrontal, and parietal cortices, mediates higher-level cognitive function like working memory, decision making, and problem solving. In PTSD, the triple network model would predict increased connectivity within the salience network (mediating hypervigilance symptoms) and between nodes in the salience network and those in the other networks (which could interfere with problem solving and self-referential thought). The model would also predict decreased connectivity within the default mode network (Koch et al., 2016).

The results of meta-analyses of functional neuroimaging studies are consistent with both the fear-conditioning neurocircuitry and triple network models (Hayes et al., 2012; Koch et al., 2016), and these models may be helpful in understanding related brain abnormalities. The text that follows uses these models to review the results of neuroimaging studies of PTSD, with a specific focus on the cingulate and its three subdivisions: the ACC, aMCC, and PCC.

3.24.5.4 Fear-Potentiated Acoustic Startle

Another important model system for analyzing the neural mechanisms of Pavlovian fear conditioning is the fear-potentiated acoustic startle paradigm (Davis, 1992, 2006; Davis and Whalen, 2001). Similar to conditioned freezing, lesions placed in the amygdala, including excitotoxic lesions in the basolateral complex or central nucleus, produce severe impairments in the acquisition and expression of fear-potentiated startle to a visual CS in rats (Hitchcock and Davis, 1986; Sananes and Davis, 1992; Kim and Davis, 1993; Campeau and Davis, 1995b; Lee et al., 1996). Similarly, pharmacological inactivation of either the central nucleus or basolateral complex of the amygdala prevents the acquisition and expression of fear-potentiated startle (Kim et al., 1993b; Walker and Davis, 1997a; Walker et al., 2005). Both thalamic and cortical afferents of the amygdala transmit sensory information to the amygdala for the acquisition and expression of potentiated startle (Rosen et al., 1992; Campeau and Davis, 1995a; Shi and Davis, 1999, 2001). Extensive pharmacological evidence indicates that synaptic plasticity in the amygdala contributes to both the acquisition and extinction of fear-potentiated startle (Miserendino et al., 1990; Falls et al., 1992; Gewirtz and Davis, 1997; Walker and Davis, 2000; Lu et al., 2001; Josselyn et al., 2001; Lin et al., 2001, 2003a,b; Walker et al., 2002; Chhatwal et al., 2006).

In addition to learning-induced potentiation of acoustic startle, ambient illumination (bright light) can lead to nonassociative increases in acoustic startle (Davis, 1998). Unconditioned increases in potentiated startle also involve the amygdala, but interestingly there is a double dissociation in the circuitry for conditioned and unconditioned potentiated startle. Inactivation of the central nucleus of the amygdala affects conditioned increases in startle without influencing unconditioned increases in startle, whereas inactivation of the bed nucleus of the stria terminalis (which receives input from the amygdala) produces the converse pattern of results (Walker and Davis, 1997a). Inactivation of the basolateral complex of the amygdala influences both conditioned and unconditioned potentiated startle.

Although fear-potentiated startle is a widely used measure of fear conditioning, it displays some properties that differentiate it from other indices of conditioned fear. Unlike many of the other measures of conditioned fear, the magnitude of fear-potentiated startle decreases with increases in US magnitude. This decrease in the amplitude of the acoustic startle response is not predicted by formal models of learning and appears to be due to competition with freezing behavior. That is, lesions of the periaqueductal gray that eliminate freezing behavior permit the expression of potentiated acoustic startle by CSs trained with high US intensities (Walker et al., 1997; Walker and Davis, 1997b). Another factor that differentiates the acoustic startle response from other measures of fear is the timing of the conditioned response relative to the CS. Whereas freezing or hypoalgesic responses are tonic and expressed for minutes after the delivery of a brief CS, acoustic startle is only potentiated within a narrow time window that envelopes the expected time of US delivery (Davis et al., 1989; Burman and Gewirtz, 2004). These factors prove valuable for the analysis of temporal relationships that modulate fear expression but also suggest that the neural network involved in the expression of fear-potentiated startle is quite different from that involved in the expression of other fear responses.

Pavlovian Fear Conditioning as a Paradigm for the Acquisition of Phobias

There is a long tradition of understanding phobias as learned through Pavlovian fear conditioning, in which a stimulus that is in itself relatively innocuous (the conditioned stimulus (CS)) comes to signal the occurrence of a threatening stimulus (the unconditioned stimulus (US)). By associating cue to consequence, organisms may prepare themselves for the consequences by recruiting defense behavior in anticipation of the US (e.g., an attacking predator). Thus, Pavlovian fear conditioning favors prey animals in the evolutionary arms race between predator and prey, making this learning process a ubiquitous phenomenon in animal life that emerged early in evolution.

A fear-conditioning perspective on phobias proposes that a phobic stimulus acquires its fear-eliciting potency by association with other dangerous or traumatic events. For example, a dog phobia may have its origin in a childhood dog attack. However, there are several problems with this account. First, it does not explain the relative selectivity of phobias. Furthermore, it implies that phobias should primarily be seen as being to objects and situations that are threatening in a contemporary, rather than an evolutionary, perspective. Second, not all dog phobics recall being attacked by a dog, nor do members of their family. Therefore, direct Pavlovian conditioning may not be the only route to phobia. Other possibilities included observational learning (the dog phobic may have witnessed another person being attacked by a dog, in which case the fear evoked by witnessing this events may become conditioned to dogs) or verbal conditioning (e.g., from admonitions about the dangers of dogs from anxious parents).

The neural basis of fear conditioning is relatively well understood. Basically, the associative link between the CS and the US is formed in the lateral nucleus of the amygdala by N-methyl-d-aspartate (NMDA)-mediated potentiation of glutaminergic synaptic connections in a convergence zone of CS and US pathways from the thalamus. In this way, the CS may gain access to some of the US efference to elicit a plethora of fear-related changes mediated by the central nucleus of the amygdala, which is often seen in individuals diagnosed with phobia.

Conclusion

Extant research across species suggests that observational fear learning draws upon similar neural circuitry to that of Pavlovian fear conditioning, although some notable differences between these types of learning have been documented (see Bennet, Galef, & Durlach, 1993; Lancet & Orr, 1980; White & Galef, 1998). The research reviewed here and elsewhere (Olsson et al., 2007) suggests the amygdala and the surrounding brain structures that mediate empathically driven appraisals of another's aversive experience support the learning and expression of observational fear. Specifically, the aversive emotional response elicited from a conspecific's distress (US) is likely relayed to the LA through pathways that convey affective input related to the perceived aversive state of another. This input converges with cue-related sensory input (CS) that reaches the LA through sensory pathways to form a CS–US association. A CS that is later encountered could then excite the CE, leading to fear expression that is mediated by similar brain regions to those involved in Pavlovian fear learning and expression. Additional research, however, is necessary to develop a clearer understanding of the circuitry that is involved in this form of learning. Such investigation will further illustrate how learning in a social context can shape emotional functioning and offer insight into how socially acquired fear may contribute to clinical disorders marked by emotional dysfunction.

4 The medial prefrontal cortex encoding of fear states and fear learning

Fear is generally defined as distressing emotions caused by impending and certain danger. MPFC encoding of fear is commonly assessed using Pavlovian fear conditioning. This procedure applies an aversive unconditioned stimulus (US), such as a footshock, paired with a neutral conditioned stimulus (CS), such as a tone, that elicits a conditioned behavioral fear response (CR) such as freezing or darting (Gruene, Flick, Stefano, Shea, & Shansky, 2015; Johansen, Cain, Ostroff, & LeDoux, 2011). Thus, the main measure in these and similar paradigms is the strength of the association between the CS and US through the CR.

It is well documented that expression of freezing behavior in Pavlovian conditioning requires the PL-mPFC. Inactivation of the PL-mPFC after fear learning prevents conditioned freezing (Corcoran and Quirk, 2007). However, when this region is inactivated during the acquisition of fear learning and the subject is tested for fear expression later, the fear expression remains intact (Corcoran & Quirk, 2007). Furthermore, fear responses to predator odor and in a novel open field, where learned associations are not required, remain intact following inactivation of the PL-mPFC. Thus, mPFC circuitry is critical for expression of learned fear responses but not necessary for the acquisition of fear learning itself nor for innate fear expression. At the neural level, single unit recording from the PL-mPFC neurons of putative excitatory and inhibitory subtypes shows heterogeneous but time-locked changes in firing rate when fear associated stimuli are presented and during freezing (Baeg et al., 2001; Courtin et al., 2014). In addition, the excitatory PL-mPFC response to a CS is associated with higher levels of fear expression across all phases of fear learning (i.e., habituation, conditioning, and extinction; Burgos-Robles, Vidal-Gonzalez, & Quirk, 2009). IL-mPFC neuron inactivation, however, does not influence fear learning (Sierra-Mercado, Padilla-Coreano, & Quirk, 2011) and IL-mPFC neurons fail to show time-locked responses to CS during the acquisition phase of fear learning (Milad & Quirk, 2002). These findings suggest that for the development of associations between CS and US, the PL-mPFC shows high involvement, while the IL-mPFC does not.

Fear conditioning paradigms can also assess behavioral flexibility by a process known as fear extinction. The extinction procedure involves extinguishing the CS-US association, after freezing in response to the CS is learned, by presenting the CS while omitting the US. Animals' ability to learn the change in contingencies is measured by a reduction (or extinction) in fear responses after the CS. While extinction of freezing is unchanged by lesions in the PL-mPFC, it is impaired by lesions of IL-mPFC (Quirk, Russo, Barron, & Lebron, 2000). Consistent with lesion studies, single unit recording from IL-mPFC and PL-mPFC neurons showed that IL-mPFC responses correlate with the extinguishing of fear behavior (Chang, Berke, & Maren, 2010). Moreover, animals that displayed a large IL-mPFC neural response to the CS showed deficits in an extinction retention test (Chang et al., 2010). This is intriguing because earlier studies showed that larger phasic responses of IL-mPFC neurons in response to the CS in extinction was associated with improved learning (Milad & Quirk, 2002). To resolve this, a recent study used optogenetics to selectively stimulate or inhibit IL-mPFC neurons at the time of CS delivery during extinction training. Optogenetic stimulation produced a dramatic effect nearly obliterating freezing during training and enhancing extinction learning in a later recall test in the absence of optogenetic stimulation. Surprisingly, inhibition of these neurons during extinction training had no effects on extinction per se, but produced deficits in extinction memory because freezing was elevated in the retrieval test the following day (Do-Monte, Manzano-Nieves, Quiñones-Laracuente, Ramos-Medina, & Quirk, 2015).

While fear conditioning paradigms lack explicit goal-directed actions, other procedures such as active avoidance can measure how the mPFC is involved in planning and encoding actions that are executed after cued footshock threat. In active avoidance paradigms, subjects learn to move to a safe location or execute an action (such as pressing a lever) with the goal of avoiding an imminent aversive outcome signaled by a cue. Thus the latency to escape the aversive stimulus is the main measure of how well subject learns this action-outcome association. A recent comprehensive study recording from PL-mPFC found that these neurons encode cues that signal avoidance, and that inhibiting the phasic response of mPFC neurons (through excitation) delays the latency to actively avoid the footshock (Diehl et al., 2018). Thus, PL-mPFC was interpreted to be necessary for facilitating actions toward the goal of avoiding an aversive outcome. This is similar to previous studies where IL-mPFC lesions before avoidance resulted in impaired active avoidance (Moscarello & LeDoux, 2013). While the results of the IL-mPFC study may be due to enhancement of freezing behavior seen after the lesion, facilitatory roles for PL and IL-mPFC in avoidance have been shown in more recent studies which required animals to discriminate active and inhibitory avoidance cues (Capuzzo & Floresco, 2020). However, when the active avoidance task was simple, i.e., required no discrimination, this study failed to show an effect of PL-mPFC lesion even though IL-mPFC lesions continued to perturb avoidance responses. This is interesting because the avoidance study outlined earlier (Diehl et al., 2018) also had a food reinforcement contingency in the task to coax animals away from the safety platform. Taken together these findings suggest that PL-mPFC may be involved in avoidance actions when multiple contingences are in play.

Collectively fear learning literature suggests that PL-mPFC activity subserves the expression of fear whereas the IL-mPFC activity exhibits greater involvement in promoting the extinction of fear. These regions, however, are also important for execution of actions motivated by the goal of avoiding stressors in active avoidance procedures. Thus while the computational and functional processes of neural activity in the mPFC in response to fear learning and avoidance remain to be fully determined, mPFC subregions are convincingly involved in encoding fear related information and behavior.