Serotonin in fear conditioning processes.

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Serotonin in fear conditioning processes Elizabeth P. Bauer ∗ Barnard College, Biology Department, 3009 Broadway, New York, NY 10027, United States

h i g h l i g h t s • • • •

5-HT2A agonists are anxiogenic and enhance fear learning. 5-HT1A agonists are anxiolytic and impair cued and contextual fear learning. Variation in 5-HT genes can influence fear conditioning and extinction. Roles of the amygdala, hippocampus and BNST in mediating acute SSRI effects.

a r t i c l e

i n f o

Article history: Received 9 May 2014 Received in revised form 18 July 2014 Accepted 21 July 2014 Available online xxx Keywords: Serotonin Fear conditioning Amygdala

a b s t r a c t This review describes the latest developments in our understanding of how the serotonergic system modulates Pavlovian fear conditioning, fear expression and fear extinction. These different phases of classical fear conditioning involve coordinated interactions between the extended amygdala, hippocampus and prefrontal cortices. Here, I first define the different stages of learning involved in cued and context fear conditioning and describe the neural circuits underlying these processes. The serotonergic system can be manipulated by administering serotonin receptor agonists and antagonists, as well as selective serotonin reuptake inhibitors (SSRIs), and these can have significant effects on emotional learning and memory. Moreover, variations in serotonergic genes can influence fear conditioning and extinction processes, and can underlie differential responses to pharmacological manipulations. This research has considerable translational significance as imbalances in the serotonergic system have been linked to anxiety and depression, while abnormalities in the mechanisms of conditioned fear contribute to anxiety disorders. © 2014 Published by Elsevier B.V.

1. Introduction One hallmark of several anxiety disorders is an abnormality in acquiring or extinguishing conditioned fear memories [1,2]. Manipulations of the serotonin (5-HT) system are widely used to treat a variety of anxiety disorders such as panic disorder, social phobia, generalized anxiety disorder and obsessive-compulsive disorder [3–7]. Thus, an understanding of how the serotonergic system modulates fear learning processes has been of considerable interest for decades. The advantage of studying classical Pavlovian fear conditioning is that it is a model of emotional learning for which the underlying neural circuitry has been described in detail. The structures involved in fear learning and expression, including the amygdala, hippocampus and prefrontal cortices, contain dense concentrations of 5-HT receptors [8–10]. Moreover, 5-HT levels in the amygdala increase during both cued and context fear conditioning

∗ Corresponding author. Tel.: +1 212 854 2349; fax: +1 212 854 1950. E-mail address: [email protected]

[11–13]. Here, recent advances in our understanding of the neural circuits involved in fear learning, expression and extinction are described. I then review the literature describing how manipulations of the serotonergic system affect each of these behavioral processes, attempt to reconcile seemingly contradictory findings and offer recommendations for future research. 2. The neural circuitry underlying classical fear conditioning Pavlovian fear conditioning is one of the most comprehensively studied behavioral paradigms. In classical fear conditioning, an initially neutral stimulus, such as a tone or light (conditioned stimulus; CS) is paired with an aversive stimulus, such as a brief electrical footshock (unconditioned stimulus; US). As a result of this pairing, the CS acquires aversive properties. Afterwards, when presented alone, the CS elicits responses in the animal that are characteristic of fear, including autonomic changes such as increased heart rate and blood pressure, as well as behavioral reactions such as the cessation of movement (freezing) and/or fear-potentiated startle

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Please cite this article in press as: Bauer EP. Serotonin in fear conditioning processes. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.07.028

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[14,15]. While cued fear conditioning involves the association of the US with a discrete cued CS, animals are also capable of associating a collection of context cues with the US, producing context fear conditioning [16–18]. If an animal is exposed to repeated presentations of the CS in the absence of the US, a gradual reduction in the ability of the CS to elicit fear responses takes place [19]. This phenomenon, fear extinction, involves learning that the new CS no longer predicts the US. Rather than destroying the original memory, fear extinction is a new learning process that inhibits the original fear memory [20,21]. The gradual reduction in fear during CS presentations is referred to as within-session extinction. This new memory is consolidated and recalled at a later point in time, which is referred to as between-session extinction or extinction retention/recall. To appreciate how the serotonergic system modulates fear learning it is important to keep in mind that fear conditioning involves different stages of memory. Initial learning of the CSUS association during training is referred to as fear acquisition. These memories are thought to consolidate into stable, enduring protein-synthesis dependent memories during the 24 h after fear acquisition [22,23]. When the CS is later presented alone, the fear memory is retrieved, leading to expression of the conditioned fear response. Thus if a manipulation (such as the administration of a drug) occurs immediately before training and impairs both shortterm (STM) and long-term memory (LTM), it is said to disrupt the acquisition of a fear memory. However, a drug administered pretraining which does not affect STM but does impair LTM prevents memory consolidation. Similarly, a drug is given immediately after training which impairs the formation of LTM, also disrupts memory consolidation. Alternatively, if learning has already taken place and a drug is given immediately prior to testing, it can affect expression or recall of the fear memory. In this way, the timing of drug administration determines the stage of learning that is affected. It is important to keep in mind when studying fear conditioning and fear processes that the terms “fear” and “anxiety” can refer to conscious feelings and also to behavioral and physiological responses (see [24–26] for discussions of this idea). All animals, including humans, can detect threats and respond defensively. Studies that measure freezing behavior, or avoidance, or blood pressure are measuring behavioral or autonomic reactions to perceived threats. The conscious perception of fear and anxiety, however, can only be experienced by organisms capable of such conscious experience. In humans, emotions, including panic, can be consciously perceived even when the amygdala or insula is damaged [27,28], even though animals or humans with amygdala damage are not capable of Pavlovian fear conditioning [29,30]. Nevertheless, because the terms “fear conditioning” and “fear expression” are so widely used, this review will continue to use these terms. The robustness of the Pavlovian fear conditioning paradigm has led to a detailed understanding of the key neuronal circuits, neurochemicals and molecular mechanisms underlying fear learning, fear extinction and associative plasticity in general (for recent reviews, see [31–34]). Lesion, inactivation and unit recording studies originally identified the lateral nucleus of the amygdala (LA) as the main input station of the amygdala where CS and US sensory inputs from both cortical and subcortical areas synapse on individual LA neurons [29,35–39]. Activation of individual LA neurons by CS inputs is enhanced by US-mediated depolarization during fear learning [38,40]. As a result of contingent CS-US pairings, subsequent presentations of the CS alone evoke larger responses in LA neurons [37,38,41]. Several recent reviews explore the complex molecular mechanisms underlying the formation of the CS-US association [31,33]. In the original model of fear conditioning, the central nucleus (CE) functioned as the main output of the amygdala. Neurons in

the medial division (CeM) project to several fear effector systems in the hypothalamus and brain stem including the periaqueductal gray (PAG) which controls freezing behavior [15,42,43]. Disinhibition of the CeM allows for the expression of a range of defensive behaviors including freezing [44]. Newer research suggests that neurons in the lateral division of the Ce (CeL) contribute to fear acquisition. Pretraining inactivation of the entire CE, or CeL specifically, interferes with the acquisition of auditory fear conditioning [44,45]. Blockade of NMDA receptors or disruption of protein synthesis within the CE also impairs fear learning [45,46]. Neurons within the CeL receive input from the BLA and project to the CeM [44,47]. Plasticity of these glutamatergic inputs to CeL is induced by fear conditioning such that activation of these CeL neurons is necessary for fear memory recall [48]. Therefore, during CS presentations, subsets of LA and CeL neurons become active, disinhibiting CeM output neurons [44,48]. The bed nucleus of the stria terminalis (BNST), considered part of the extended amygdala, receives a strong glutamatergic input from the basolateral nucleus of the amygdala (BLA; which is composed of the LA and basal nuclei) and is reciprocally connected with the CE [49,50]. The CE and the BNST share a similar pattern of efferent targets including brainstem areas involved in fear and anxiety [49,51]. Unlike the BLA and CE, lesions of the BNST do not disrupt auditory fear conditioning or expression [15]. However, lesions or reversible inactivation of the BNST do attenuate the expression of context fear conditioning [52,53]. The BNST also contributes to an animal’s response to unpredictable stressful events and anxiety [54–57]. These data have led to the hypothesis that there is a distinction between fear (imminent threat or phasic fear) and anxiety (potential threat or sustained fear), which are mediated by the amygdala and BNST, respectively [56,58]. Animals are not only capable of associating discrete cues with an aversive stimulus, but can also associate a collection of contextual cues with a shock. A role for the hippocampus in this process is implicated by studies demonstrating that electrolytic lesions of the dorsal hippocampus before or after training impair context conditioning [16,17,59,60]. More recent experiments using optogenetic techniques reveal that during fear conditioning, subsets of granule cells in the dentate gyrus encode the memory of the context [61,62]. This memory of the context can be recalled when those specific subpopulations of neurons are activated [62,63]. Lesions or functional inactivation of the ventral hippocampus, however, produce inconsistent effects on context fear. Some studies show that the ventral hippocampus is required for the formation of a context representation [64–67], while others fail to replicate these findings [68]. Rather, the dentate gyrus of the ventral hippocampus seems to mediate anxiety [61]. Importantly, only the ventral hippocampus projects directly to the amygdala, thus damage to the ventral portion interferes with the transfer of information from the dorsal hippocampus to the amygdala [69,70]. Moreover, the ventral hippocampus modulates activity of BLA neurons [71]. The ventral hippocampus also sends a strong projection to the prelimbic cortex, which in turn forms a reciprocal connection with the BLA [72,73]. Finally, the BNST also receives input from both the ventral hippocampus and ventral subiculum and BLA [49]. However, lesions of the BNST selectively disrupt the expression of context but not cued fear conditioning [53,74]. After fear learning has taken place, it can be expressed via disinhibition of CeM neurons [44]. The increased responsiveness of CeM output neurons to the CS depends on disinhibition of CeL neurons as well as excitation from glutamatergic neurons in the BLA [44,75,76]. Indeed, the activity of BLA neurons correlates with high or low fear behavior (freezing) [71]. The prelimbic cortex (PL) integrates information from the BLA and ventral hippocampus, with increased activity in this area of the prefrontal cortex also correlating with increased fear expression [77,78].




Several lines of evidence implicate the amygdala in extinction learning. Reversible inactivation of the BLA with the GABA agonist muscimol or intra-amygdala infusions of NMDA receptor antagonists block the acquisition of extinction [71,79–82]. At the same time, CeM fear output neurons are inhibited during fear extinction through potentiation of BLA neurons to intercalated cell masses, a process that is dependent on the infralimbic prefrontal cortex (IL) [83]. The IL itself is a critical site of plasticity after extinction is learned and is involved in the retrieval of extinction memories [19]. Indeed, pairing of IL stimulation with a CS is sufficient to simulate extinction memory [84]. Recent data suggests that there exist distinct populations of neurons within the BLA which can be characterized by whether they project to IL or PL. Activation of PL-projecting BLA neurons produces fear-like behavior, while ILprojecting BLA neurons are recruited and exhibit plasticity during fear extinction learning [85].

3. Serotonergic modulation of fear learning and expression Fear learning and extinction are thus mediated by interactions between several nuclei of the amygdala, the hippocampus, the mPFC and the BNST. All of these structures contain dense concentrations of 5-HT receptors [8–10]. Moreover, there is an increase in the concentration of amygdalar 5-HT both during and after behavioral arousal and stress and in response to US presentations during fear conditioning [12,13,86–88]. After context fear conditioning, there are increases in extracellular 5-HT in both the BLA and mPFC [11,12,89]. However, 5-HT modulates activity in these structures in a complex manner. In general, 5-HT can have differential effects on behavior depending on the receptor subtypes present and the behavioral tests used. The 5-HT system is quite complex with multiple 5-HT receptor subtypes which have been classified into seven distinct families (5-HT1–7 ). The 5-HT3 receptor is the only ionotropic 5HT receptor and is permeable to cations [90]. 5-HT2 receptors, G-protein coupled receptors, are positively coupled to phospholipase C; their activation causes an increase in intracellular calcium [90]. Activation of 5-HT1A leads to hyperpolarization via opening of potassium channels [90]. Unlike the 5-HT2 receptors, the 5-HT1A receptor is expressed as both an autoreceptor in raphe nuclei and a heteroreceptor in forebrain regions. As an autoreceptor, it limits the release of 5-HT from raphe neurons, thus affecting overall serotonergic tone [91]. As a heteroreceptor in the amygdala, BNST and hippocampus, it mediates local responses to 5-HT. 5-HT modulation of neural activity is further complicated by the fact that one receptor subtype can exist on both glutamatergic and GABAergic cell types [92] and more than one 5-HT receptor subtype can be present on a neuron [93–95]. Moreover, 5-HT can have differential effects on behavior depending on the brain region that is targeted [96] or the behavioral paradigm used [97–99]. Thus 5-HT modulates the circuits and neurons mediating fear learning in a highly complex manner. 5-HT cell bodies are localized in the mid-brain raphe nuclei, comprised of the dorsal and median raphe nuclei, which send projections throughout the brain. The amygdala receives 5-HT innervation from the dorsal raphe nucleus [100], with the BLA receiving dense projections and the CE receiving weak input. In the BLA, 5-HT produces a primarily inhibitory response by depolarizing GABAergic interneurons [10]. This increase in GABAergic transmission results in feed-forward inhibition of the principal excitatory neurons of the BLA [10,101]. 5-HT2 receptors are found predominantly in the BLA on both the principal excitatory neurons as well as parvalbumin-positive GABAergic interneurons [92]. 5-HT1A receptors are concentrated in the CE, while 5-HT3 receptors are found in both the BLA and CE and are nearly exclusively

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localized to GABAergic neurons [9,10,102]. The BNST also receives serotonergic input from the dorsal raphe nucleus [103]. Although the response of BNST neurons to 5-HT depends on which specific receptors are activated, the primary response is inhibitory [95]. The prefrontal cortex receives input from both the dorsal and median raphe nuclei [104]. Within the prefrontal cortex the primary response to 5-HT is inhibitory through increased excitability of interneurons and decreased excitability of pyramidal neurons [105]. Similarly, 5-HT in the hippocampus, which receives innervation from the median raphe nucleus [106], depolarizes inhibitory interneurons and reduces pyramidal neuron excitability [107,108]. As a result of the complex nature of the serotonergic system in these structures, an appreciation for how 5-HT affects fear conditioning and fear extinction can be gained by understanding the contribution of specific 5-HT receptor subtypes to these learning processes. Most of these experiments have been done on rodents. 5-HT2A receptors, which are highly enriched in the amygdala, prefrontal cortex and hippocampus [8] modulate learning and memory in a variety of paradigms. Systemic injections of 5-HT2A agonists enhance conditioned avoidance memory [109], enhance eyeblink conditioning [110], and strengthen the consolidation of object memory [111] in animals. Conversely, 5-HT2A antagonists impair conditioned avoidance memory and eyeblink conditioning [110,112]. In humans, reduced binding capacity of 5-HT2A receptors has been observed in normal aging subjects as well as Alzheimer’s patients [113,114]. Moreover, humans who carry a polymorphism in the 5-HT2A receptor gene show impaired consolidation of explicit memory [115]. If the 5-HT2A agonist TCB-2 is administered immediately following fear learning, it enhances both cued and context fear conditioning in mice, suggesting an enhancement of the consolidation of fear memories by this receptor [111]. Similarly, a 5-HT2A agonist injected prior to extinction learning also facilitates extinction learning in both delay and trace fear conditioned mice [111]. Because this study used systemic injections of 5-HT2A agonists, it is unclear which structures mediate the effects of these drugs on fear conditioning and extinction. Considering the dense concentrations of 5-HT2A receptors in the amygdala, hippocampus and cortex, it is possible that agonists of this receptor facilitate their coordinated activity. In the cortex, 5-HT2A receptors influence low-frequency oscillations in the frontal cortex [116] and increase spinogenesis [117]. As described above, the 5-HT2A receptor is positively coupled to phospholipase C and its activation leads to an increase in internal calcium [90]. These receptors are present in the dendrites and dendritic spines of neurons [118] where they directly interact with the PSD-95 and regulate receptor trafficking and signal transduction [119]. Additionally, activation of 5-HT2A receptors enhances cortical presynaptic glutamate release [120,121] and NMDA receptor sensitivity [122]. 5-HT2A receptors facilitate NMDA receptor activity in the cortex [122] and BLA [123]. It should be noted, however, that 5-HT2A knockout mice do not show changes in fear conditioning, although they do exhibit reduced anxiety [124]. Both the 5-HT2A and 5-HT2C receptors in the amygdala are involved in the expression of anxiety-like behaviors. Fear can be characterized by arousal and fight-or-flight responses to imminent threats, while anxiety is characterized more by apprehension, tension, and worry, where the potential threat might occur in the distant future [125]. Infusions of 5-HT2A/C agonists into the BLA, as well as over-expression of 5-HT2C receptors in the BLA produce anxiety-like behaviors in animals as measured by the elevated plus maze and open field test [126–128]. Conversely, both 5-HT2A and 5-HT2C knockout mice show decreases in anxiety-like behavior [124,129]. There have been few direct studies on the contribution of 5-HT2C receptors to fear learning or extinction. However, one study examined the effects of the 5-HT2C receptor antagonist ritanserin on fear conditioning in humans [130]. Skin conductance responses




to a CS tone were enhanced after the tone was paired with an aversive stimulus, but this effect was blocked by ritanserin, suggesting that activation of 5-HT2C receptors contributes to fear conditioning. Moreover, as described below, co-administration of a 5-HT2C antagonist, but not a 5-HT3 receptor antagonist, blocks the acute effects of SSRI treatment on conditioned fear expression [131]. Together, these data suggest that activation of 5-HT2A/C receptors both increases anxiety and enhances fear learning and extinction. In contrast, 5-HT1A receptor agonists, such as the widely used 8OH-DPAT, produce anxiolytic effects in both humans and animals [132,133], and 5-HT1A knockout mice show increases in anxietylike and depressive-like behavior [134–136]. Several studies have revealed that 5-HT1A receptor agonists decrease context fear when given systemically [137,138]. However, as discussed above, the 5HT1A receptor is expressed as both an autoreceptor in raphe nuclei and a heteroreceptor in forebrain regions. To disambiguate the contribution of these two receptor subtypes to fear conditioning, several studies in animals have used local infusions into the raphe nuclei, the amygdala or the hippocampus. When the 5-HT1A agonist 8-OH-DPAT is infused directly into the median raphe nucleus to stimulate 5-HT1A autoreceptors, it impairs both the acquisition and expression of context fear memories [139]. Testing the contribution of post-synaptic 5-HT1A receptors, pretraining intra-hippocampal infusions of 8-OH-DPAT impair context fear conditioning [140]. Pre-testing infusions of 8-OH-DPAT impair both the expression of context conditioned freezing as well as fear-potentiated startle [141]. In contrast, pre-testing infusions of this agonist into the median raphe nucleus decreases context fear without affecting fear-potentiated startle, which suggests that postsynaptic 5-HT1A receptors play a greater role in the modulation of context fear memories [141]. Similarly, when 5-HT1A agonists are infused into the BLA or the BNST they impair the expression of context fear conditioning [142,143]. Using a conditioned defeat paradigm, it was found that activation of 5-HT1A receptors in the BLA decreases both acquisition and expression [144]. In sum, 5-HT1A agonists, which have been shown to be anxiolytic, decrease both acquisition and expression of fear conditioning. These inhibitory effects may be explained by the fact that activation of 5-HT1A receptors inhibits neuronal activity in both the hippocampus and the BNST [145,146]. A recent study examined the role of 5-HT1A receptors in the BLA, PL and dorsal PAG in low or high-anxious animals on unconditioned and conditioned fear [147]. The examination of individual differences in anxiety and fear conditioning is useful in developing models of psychopathology that affect a relatively small proportion of the population [148,149]. In low-anxious rats, activation of 5-HT1A receptors in either the BLA or dorsal PAG reduced fear potentiated startle. In contrast, in high-anxious rats, 5-HT1A receptor activation in the prelimbic area reduced fear potentiated startle. This type of behavioral study highlights the importance of examining the serotonergic system in a heterogeneous population. The 5-HT3 receptor is the only ionotropic 5-HT receptor and is permeable to cations. Mice in which this receptor is overexpressed demonstrate enhanced context but not cued conditioning, increased exploratory behavior but decreased anxiety-like behavior as measured in the elevated plus maze [150]. Conversely, 5-HT3 antagonists impair the expression of context fear conditioning [89,151]. 5-HT3 antagonists do not block the effects of SSRIs on the expression of cued fear conditioning [131]. Together, these data suggest that 5-HT3 receptors modulate context but not cued fear conditioning. The contribution of 5-HT3 receptors to extinction learning is not clear. 5-HT3 receptor knockout mice show reduced fear extinction learning to both cues and context [152]. However, systemic injections of 5-HT3 receptor antagonists prior to extinction training enhance cued and context fear extinction [153]. This discrepancy is difficult to resolve given the different behavioral

paradigms, species and methods of disrupting 5-HT3 receptor function.

4. Serotonin transporter gene variation Variations in serotonergic genes can influence fear conditioning and extinction processes. The most widely studied is a functional polymorphism in the promotor region of the 5-HT transporter gene (5-HTTPR). The 5-HT transporter 5-HTT regulates 5-HT signaling via reuptake of 5-HT from the extracellular space [154]. In humans, the short (s) allele of the 5-HTT promotor region, which is associated with decreased transcription of the gene compared to the long (l) allele, leads to reduced 5-HTT function and presumably increased concentrations of extracellular 5-HT [155]. At the behavioral level, the s allele is associated with abnormal levels of anxiety [155,156]. Humans with one or two copies of the s allele exhibit greater amygdala activity in response to fearful stimuli [157]. Carriers of the s-allele possess smaller hippocampal and amygdala volumes, as well as a functional uncoupling of the amygdala-cingulate circuit [158,159]. Human carriers of the s-allele show increased fear conditioning as well as stronger fear potentiated startle [160–162]. They also produce enhanced autonomic responses when watching another subject undergo fear conditioning, a process referred to as observational fear learning [163]. Conversely, mice in which 5-HTT is overexpressed throughout the brain show impaired fear learning and reduced theta oscillations in the amygdala [164]. However, 5-HTT knockout mice show intact fear conditioning and initial extinction learning but impaired extinction recall [165,166]. Thus, although variations in 5-HTT affect fear learning and extinction, there are several inconsistent findings. This might be due to different methodologies and subjects, but might also be due to variable correlation of the 5-HTTPR with other genetic alterations. For example, a recent study in humans examined the interaction between two polymorphisms: the 5-HTTPR and variation in the gene encoding the neuropeptide S receptor (NPSR1). Only carriers of both risk alleles exhibited higher fear potentiated startle [167]. Similarly, only human subjects carrying the risk allele of both the 5HTTLPR and that of a polymorphism of the corticotropin releasing hormone receptor 1 (CRHR1–rs878886) show increased fear potentiation of the eyeblink startle reflex [168]. There is a considerable amount of data showing that environment can influence anxiety-like behaviors and anxiety disorders [169–171]. Interestingly, variations in the 5HTTLPR can make individuals more or less susceptible to the effects of environment. For example, humans with one or two copies of the s-allele have an increased risk of developing depression if they experience at least three “stressful” life events during childhood [172]. After repeated social defeat, 5-HTT knockout mice showed higher levels of anxiety-like behavior than normal mice [173]. In addition, homozygous (5-HTT−/− ) and heterozygous (5-HTT+/− ) 5-HTT knockout mice show significantly higher freezing behavior during extinction and extinction recall compared to wild-type mice, and this difference is enhanced after social defeat [165]. Furthermore, socially defeated 5-HTT−/− mice show increased theta correlations between LA and mPFC during extinction learning and recall [165]. A second functional gene variation affecting 5-HTT is the STPP/rs3813034 serotonin transporter polyadenylation polymorphism. Polyadenylation is a posttranscriptional modification; the polymorphism influences the balance of two polyadenylation forms of 5-HTT [174]. The G-allele carriers have reduced 5-HTT levels, which presumably leads to an increase in extracellular 5-HT, and they exhibit increased risk for panic disorder [174]. Carriers of the risk allele also show impaired retention of fear extinction memory, heightened anxiety and depressive symptoms [175]. These




data suggest that 5-HTT down-regulation can increase fear learning and impair extinction retention, resulting in persistent fear memories. Future studies should genotype for both 5-HTTPR and STPP in order to clarify their relative phenotypic effects.

5. Acute SSRI administration and fear conditioning SSRIs, such as citalopram, escitalopram, fluoxetine, fluvoxamine and paroxetine are widely used for the treatment of anxiety disorders including panic disorder, generalized anxiety disorder, phobias and obsessive-compulsive disorder [4,6,176,177]. SSRIs act pharmacologically by inhibiting the serotonin transporter, leading to an increase in 5-HT availability. These increased levels of 5-HT produce an increase in anxiety that can be measured in animals using a variety of behavioral tests including the elevated plus maze [178–181], novelty suppressed feeding [182], social interaction [183,184] and the free-exploration test [185]. These behavioral results are consistent with clinical data suggesting that patients experience an initial increase in anxiety and an increased risk of suicide ideation when given SSRIs; these anxiogenic effects eventually subside after chronic treatment [186–192]. Several hypotheses have been suggested for how SSRIs mediate their long-term effects of anxiety including the disinhibition of autoreceptors in the raphe nuclei [193,194], and adaptive changes in glutamatergic neurotransmission [195–197]. Yet despite extensive research, it is still unclear how SSRIs mediate their long-term effects on anxiety. Acute SSRI treatment has been shown to increase extracellular 5-HT within the amygdala, hippocampus and frontal cortex [198–200]. We can therefore gain insight into the role of 5-HT in fear conditioning processes by examining how SSRIs affect fear learning, expression and extinction. Systemic injections of citalopram or fluoxetine prior to training enhance the acquisition of auditory fear conditioning in animals [201–203]. Acute systemic injections of these drugs prior to testing enhance the expression of auditory fear conditioning, an effect which is blocked by coadministration of 5-HT2C antagonists, but not by co-administration of 5-HT3 antagonists [131]. In humans, acute SSRI treatment enhances fear potentiated startle to a discrete cue [204]. Together, these results indicate that overall, acute SSRI treatment increases both fear acquisition and expression when animals or humans are fear conditioned to a discrete cue. Acute SSRI injections increase immediate-early gene expression within the amygdala and BNST [203,205–208]. However, when SSRIs are infused directly into the amygdala, they do not affect fear learning [203]. Rather, direct infusions of SSRIs into the BNST prior to training both enhance immediate early-gene expression in the amygdala and enhance fear conditioning [203]. Although the BNST is not normally required for the acquisition of cued fear memory [53,58], these data suggest that SSRI administration recruits the BNST into the fear circuit during conditioning. The neuronal interactions between the BNST and amygdala during SSRI administration remain to be explored. The effects of SSRIs on the acquisition of context fear conditioning are contradictory, with some finding that SSRI treatment decreases the acquisition of context fear [209,210] while others report an increase in acquisition [202,211]. However, when the SSRI is administered immediately before testing, there is general agreement that it results in a decrease in fear expression [137,202,209,211–214]. SSRIs decrease neuronal activity, immediate-early gene expression and plasticity in the hippocampus [203,215,216]. They may therefore be interfering with the recall of context information [65,217], and thus impairing the expression of context fear conditioning. The contradictory effects of SSRI administration on acquisition of context fear conditioning might be explained by different strategies animals use to form

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representations of a context. Animals are capable of using the discrete cues that make up a context to successfully fear condition when hippocampal function is impaired [17,217]. If SSRI administration decreases hippocampal activity, animals might use this strategy to successfully fear condition. If this solution is not efficient due to a decrease in salience of the contextual cues used [218], then acute SSRI administration prior to training would impair the animal’s ability to form a representation of the context and would thus impair context fear conditioning. Chronic administration of SSRIs impairs fear extinction learning, while also down-regulating the NR2B subunit of the NMDA receptors in the BLA [219,220]. However no study has observed an effect of acute SSRIs on fear extinction recall, despite an increase in fear expression and within-session fear extinction [131,220]. 6. Serotonin depletion Lastly, reducing central 5-HT levels and examining the effects on fear conditioning can also provide insights into the normal role of 5-HT in this behavioral paradigm. 5-HT can be reduced throughout the brain in several ways. One method is acute tryptophan depletion, which deprives the subject of the precursor of 5-HT [221] and decreases the release of 5-HT [222,223]. Another is to interfere with the synthesis of 5-HT by systemic or local injection of p-chlorophenylalanine (PCPA) which inhibits tryptophan hydroxylase, the enzyme which synthesizes 5-HT from tryptophan [224]. A third method is to destroy serotonergic neurons via a neurotoxin (5,7-DHT) which is taken up into cells by 5-HTT and destroys them [225]. Tryptophan depletion in healthy humans does not affect fearpotentiated startle to short-duration cues, but does decrease fear-potentiated startle to long-duration cues [226]. Previous work has shown that fear potentiated startle to short-duration cues is mediated by the amygdala and can be interpreted as a phasic fear response to an explicit threat, whereas startle to long duration cues is mediated by the BNST and is a more sustained anxietylike response [56,125]. In line with this data, research on rodents found that tryptophan depletion impaired the formation of context fear memory but did not affect cued fear memory [227]. Confusingly, PCPA administration in rodents selectively enhances fear-potentiated startle to short duration cues [228], but does not affect the expression of context fear conditioning [229]. And others have found that depletion of tryptophan in humans leads to attenuated autonomic responses to the CS during fear conditioning [230]. Both tryptophan depletion and PCPA administration reduce 5-HT levels to a modest extent, and they do so globally. Unfortunately, this prevents researchers from drawing conclusions about region-specific effects. Moreover, tryptophan depletion can have differential effects on behavior that can be explained by genetic variation. In a recent study in humans, tryptophan depletion attenuated motivation, as measured by a cued-reinforcement reaction time task. This effect was only seen in subjects with two copies of the s-allele of 5-HTTPR, but not in subjects with the l-allelic variation [231]. 7. Conclusions A considerable body of literature has examined how modulation of the serotonergic system affects Pavlovian fear conditioning and extinction. Strategies for manipulating this system include the administration of agonists or antagonists directed against one of the 17 different 5-HT receptor subtypes, administration of SSRIs and serotonin depletion. Moreover, there is considerable genetic variation among individuals resulting in altered functionality of the 5-HT transporter. Given the complex, and occasionally contradictory




results reviewed here, it is still possible to draw some general conclusions about the role of 5-HT in fear conditioning processes. Pharmacological manipulations which are anxiogenic generally facilitate cued and context fear conditioning. For example, systemic injections of 5-HT2A receptor agonists, which are anxiogenic when infused directly into the amygdala [126,127] facilitate cued and context fear conditioning as well as fear extinction [111]. Acute administration of SSRIs enhances both acquisition and expression of cued fear conditioning, although it impairs expression of context fear conditioning [131,202,203,212]. And carriers of the s-allele of the 5-HTTPR show both increased anxiety and enhanced fear conditioning [155,156,160–162]. Conversely, administration of 5-HT1A agonists, which are anxiolytic, impair the acquisition and expression of context fear conditioning [132,133,140,141]. Given our knowledge of the neuronal circuitry underlying cued and context fear conditioning, studies which use local infusions rather than systemic manipulations are particularly illuminating. For example, 5-HT1A receptor agonists infused into the hippocampus, BLA or BNST impair the expression of context fear conditioning [140,142,143]. Infusion of an SSRI into the BNST prior to fear conditioning is sufficient to increase fear acquisition and enhance expression of the immediate-early gene Arc in the CE, mirroring the results of acute systemic SSRI administration. While the neuronal interactions between the BNST and amygdala during SSRI administration remain to be explored, these types of experiments underscore the utility of using local drug infusions to identify relevant structures. A second general observation can be made based on the experiments reviewed here. There exist several functional polymorphisms which affect the 5-HT transporter and thus the concentration of 5-HT in the extracellular space. There might be considerable interaction between these genetic alterations, whereby only carriers of several risk alleles exhibit increased fear or anxiety [167,168]. Furthermore, a recent study suggests that human carriers of the s-allele of 5-HTTPR show downregulation of 5-HT1A receptors [232]. This raises the intriguing question of whether pharmacological manipulations targeting the 5-HT1A receptor, for example, show differential efficacy in these individuals. As these functional polymorphisms are discovered and characterized, researchers should perform more extensive genotyping in order to clarify relative phenotypic effects. Moreover, recent experiments suggest that variations in the 5HTTLPR can make both humans and animals more or less susceptible to the effects of environment [165,172]. Finally, in the past few years, new technologies have greatly expanded our ability to study the neural circuitry underlying behavior. Using optogenetic tools, for example, it has been recently discovered that two populations of neurons within the BA which can be characterized by whether they project to PL or IL. BA-PL neurons are activated by fear conditioning; optogenetically inhibiting these neurons promotes extinction. BA-IL neurons, in contrast, are activated by fear extinction and silencing them impairs extinction [85]. Within the CEL , experiments using optogenetic tools have revealed that there are distinct subpopulations which bidirectionally regulate conditioned fear [44,47]. Meanwhile, the effects of optogenetic manipulations can depend on the cell type targeted: stimulating the subpopulation of BLA neurons expressing Thy1 impairs fear learning but strengthens fear extinction [233]. Vectors can selectively target serotonergic neurons [234,235], as well as excitatory pyramidal neurons and astroglia [236]. These techniques have the potential to provide enormous insight into how the serotonergic system modulates the fear memory system. While the relationship between the serotonergic system and fear conditioning processes is not straightforward, there is continued interest in this topic. Modulations of the serotonergic system

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Human serotonin transporter availability predicts fear conditioning.

Interplay between serotonin and cannabinoid function in the amygdala in fear conditioning.

Brief fear preexposure facilitates subsequent fear conditioning.

Trace fear conditioning in mice.

Pharmacological depletion of serotonin in the basolateral amygdala complex reduces anxiety and disrupts fear conditioning.

Contextual fear conditioning in zebrafish.

Contextual fear conditioning modulates hippocampal AMPA-, GluN1- and serotonin receptor 5-HT1A-containing receptor complexes.

Stimulus compounding in classical fear conditioning.

Serotonin and conditioning: focus on Pavlovian psychostimulant drug conditioning.

Prefrontal neuronal circuits of contextual fear conditioning.

Contextual fear conditioning induces differential alternative splicing.

Contextual fear conditioning depresses infralimbic excitability.

Fear conditioning and extinction in anxious and nonanxious youth and adults: examining a novel developmentally appropriate fear-conditioning task.

Learning pain-related fear: neural mechanisms mediating rapid differential conditioning, extinction and reinstatement processes in human visceral pain.

Origins of fear of dogs in adults and children: the role of conditioning processes and prior familiarity with dogs.

Fear conditioning to subliminal fear relevant and non fear relevant stimuli.

Serotonin, Amygdala and Fear: Assembling the Puzzle.

Contextual fear conditioning in humans using feature-identical contexts.

Conditioning- and time-dependent increases in context fear and generalization.

Sound tuning of amygdala plasticity in auditory fear conditioning.

Value conditioning modulates visual working memory processes.

Increased stathmin expression strengthens fear conditioning in epileptic rats.

Enhanced extinction of contextual fear conditioning in ClockΔ19 mutant mice.

Fear conditioning induced by interpersonal conflicts in healthy individuals.