Serotonin in panic and anxiety disorders

C H A P T E R

36 Serotonin in panic and anxiety disorders He´lio Zangrossi, Jr. 1,*, Cristina Marta Del Ben2, Frederico Guilherme Graeff3,4, Francisco Silveira Guimara˜es1 1

Department of Pharmacology, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; Department of Neuroscience and Behavioral Sciences, School of Medicine of Ribeira˜o Preto, University of Sa˜o Paulo, Ribeira˜o Preto, SP, Brazil; 3Neurobiology of Emotion Research Centre (NAP-NuPNE), University of Sa˜o Paulo, Sao Paulo, SP, Brazil; 4Institute of Neuroscience and BehavioreINeC, Ribeira˜o Preto, SP, Brazil *Corresponding author.

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I. INTRODUCTION For the last 25 years serotonin (5-HT) has been the main neurotransmitter associated with anxiety (Fig. 36.1). The first suggestions of this association have been based on experimental evidence obtained in laboratory animals. Among the early animal models of anxiety, the so-called punishment or conflict test had shown the highest predictive value for the clinical response in anxious patients for the drugs available in the 1960s and 1970s, mainly barbiturates and benzodiazepines. Typically, drugs that relieved anxiety in humans were able to release behavior maintained by responsecontingent presentation of a reinforcer, such as food or water, which was also suppressed by the delivery of an aversive stimulus, such as electric shock, following the same response. In this way, an approach-avoidance conflict is generated, the avoidance tendency being decreased by anxiolytics. As a consequence, the observation that drugs that acted on 5-HT changed punished behavior has implicated this neurotransmitter in anxiety. Using the rat conflict test designed by Geller and Seifter (1960), Robichaud and Sledge (1969) have shown that decreasing brain 5-HT by injecting the selective inhibitor of 5-HT synthesis para-chlorophenylalanine (PCPA) releases bar pressing simultaneously rewarded by sweetened milk presentation and punished by foot shock. In the same direction, Graeff and Schoenfeld (1970) have reported that the nonselective 5-HT receptor antagonists, methysergide and bromolysergic acid (BOL), increase key-pecking rates maintained by food

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presentation, concurrently suppressed by responsecontingent electric shocks, whereas the receptor agonist a-methyltryptamine has the opposite effect. On the basis of these results, they have suggested that tryptaminergic mechanisms in the brain mediate the response suppression determined by punishment. Shortly thereafter, Wise, Berger, and Stein (1972) have shown that the benzodiazepine anxiolytic oxazepam reduces 5-HT turnover in the rat midbrain, at the same dose that released punished responding in the Gellere Seifter conflict test. Since it was already known that midbrain raphe nuclei contained the cell bodies of 5-HT neurons that project to the forebrain and brain stem, they have proposed that ascending 5-HT pathways would mediate the effects of punishment by acting on structures that suppress ongoing behavior localized in both the forebrain and in the periaqueductal gray matter (PAG) of the midbrain. Benzodiazepine anxiolytics would reduce anxiety by decreasing 5-HT release in these brain areas. Therefore, in the theoretical model designed by Wise et al. (1972), 5-HT is supposed to enhance anxiety by acting both in the forebrain and in the PAG (see Fig. 36.2). The first tenet of the above hypothesis has been supported by the results obtained by Tye, Everitt, and Iversen (1977) with the neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) that selectively damages 5-HT neurons. Their results have shown that microinjection of 5,7-DHT into the ventromedial tegmentum of the rat midbrain causes 70% depletion of cortical 5-HT, indicating a major lesion of ascending 5-HT pathways, and prevents the acquisition of

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FIGURE 36.1 Number of papers in the scientific literature associating serotonin (5-HT), GABA, noradrenaline (Na), glutamate (Glut), nitric oxide (NO), or endocannabinoid (eCB) with anxiety. Source: PubMed.

FIGURE 36.2 Classical view on the role of 5-HT in anxiety. 5-HT was proposed by Wise et al. (1972) to mediate the effects of punishment by acting on structures that suppress ongoing behavior localized in both the forebrain and in the periaqueductal gray matter (PAG) of the midbrain. Benzodiazepine anxiolytics would reduce anxiety by decreasing 5-HT release in these brain areas. þ: facilitation. Drawings based on the Paxinos, G., Watson, C. (2005). The rat brain in stereotaxic coordinates (5th ed.). Amsterdam: Elsevier.

response suppression induced by foot shock in a modified GellereSeifter procedure. In the same vein, Schoenfeld (1976) has reported that the hallucinogens lysergic acid diethylamide (LSD) and mescaline release

punished licking in the rat and suggested that this effect may be due to decreased activity of ascending serotonergic neurons. As complementary evidence, Graeff and Silveira Filho (1978) have found that electrical stimulation of the median raphe nucleus (MRN) inhibits ongoing lever-pressing behavior maintained by a variable-interval schedule of water presentation. In addition, such brain stimulation induces defecation, urination, piloerection, teeth clattering, and exophthalmos, mimicking the effect of a conditioned aversive stimulus in the conditioned suppression and conditioned emotional response procedures (Estes & Skinner, 1941; Millenson & Leslie, 1974). Moreover, pretreatment with PCPA has been shown to reduce the effect of MRN electrical stimulation, suggesting a 5-HT mediation of the response suppression. Since MRN 5-HT neurons project mainly to the dorsal hippocampus (Azmitia & Segal, 1978), the latter results agree with Gray’s (1982) proposal that anxiety is due to activation of the septohippocampal system. In addition to the septohippocampal system, 5-HT may also act in the amygdala to mediate the effects of punishment. For instance, microinjection of 5-HT antagonists into the basolateral amygdala (BLA) was been shown to release water licking suppressed by electric shock punishment (Petersen & Scheel-Kru¨ger, 1984). These results add to a large body of evidence implicating the amygdala in the learning and expression of conditioned fear and anxiety (e.g., Davis, 1992; LeDoux, 1993).

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III. HOW DO SSRIS AND BUSPIRONE WORK?

In summary, these initial studies clearly suggested that facilitation of 5-HT-mediated neurotransmission facilitates anxiety. As will be seen, a large body of clinical and animal studies, although clearly implicating 5-HT in the neurobiology of anxiety, indicates that this classical view cannot be sustained any longer.

II. 5-HT-RELATED DRUGS AND THE TREATMENT OF ANXIETY DISORDERS The clinical link between 5-HT and anxiety could be traced back to the key observation by Klein and Flink (1962) that chronic treatment with imipramine has beneficial effect in panic patients. Although it was subsequently recognized that imipramine is also effective in generalized anxiety disorder (GAD, Kahn et al., 1986), this initial finding was critical for the recognition of panic as a distinct anxiety disorder. Imipramine, however, is able to block the reuptake of both noradrenaline and 5-HT, and the involvement of the latter neurotransmitter in its anxiolytic effects was not immediately recognized. The link between 5-HT and clinical anxiolytic effects became clear during the late 1970s and 1980s, with the clinical introduction of buspirone, a partial 5-HT1A receptor agonist effective in GAD patients (Goldberg & Finnerty, 1979), and the selective 5-HT reuptake inhibitors (SSRIs). The latter group, being usually well tolerated and having a broad spectrum anxiolytic efficacy, is nowadays considered, along with selective 5-HT norepinephrine reuptake inhibitors (SNRIs), as first-line pharmacological treatments for several anxiety disorders, including GAD, panic (PD), social anxiety (SAD), and post-traumatic stress (PTSD) disorders (Baldwin et al., 2014; Bandelow, Michaelis, & Wedekind, 2017). Although other 5-HT-related compounds have also been tested in patients with anxiety disorders, results are far from encouraging. For example, buspirone has not been efficacious on PD (Sheehan, Raj, Sheehan, & Soto, 1988) and SAD patients (Blanco, Bragdon, & Schneier, 2013) and even its efficacy on GAD symptoms has not been replicated in all studies (Bandelow et al., 2017). Similarly, whereas ritanserin, a 5-HT2A/2C receptor antagonist, seems to be effective in GAD patients (Ceulemans, Hoppenbrowers, Gelders, & Reyntjens, 1985), it has no effect or even aggravates PD (den Boer & Westenberg, 1990). Mixed results have also been observed with 5-HT3 receptor antagonists (Hewlett, Schmid, & Salomon, 2003; Lecrubier, Pucch, Azeona, Bailey, & Lataste, 1999; Romach, Kaplan, Busto, Somer, & Sellers, 1998) and 5-HT1A agonists (van Vliet, Westenberg, & den Boer, 1996). Open studies have also suggested that d-fenfluramine, a 5-HT releaser, could improve panic patients resistant to traditional

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treatments (Hetem, 1996; Solyom, 1994). This drug, however, was withdrawn from the market due to side effects.

III. HOW DO SSRIS AND BUSPIRONE WORK? Buspirone is a partial 5-HT1A receptor agonist, whereas SSRIs acutely inhibit the neuronal reuptake of 5-HT (Artigas, Bertolozzi & Celada, 2018). Why, then, do the beneficial effects of these compounds need at least twoethree weeks to become evident in patients with anxiety disorders? Changes in receptors (both pre- and postsynaptic), signaling pathways, and neuroplastic mechanisms have been associated with this therapeutic delayed response (Artigas et al., 2017). The putative increase in extracellular 5-HT induced by acute administration of SSRIs is reduced by 5-HT activation of autosomic (5-HT1A) and presynaptic (5HT1B/1D) receptors. Postsynaptic 5-HT receptor activation can also modify 5-HT release. In the prefrontal cortex 5-HT1A and 5-HT3 receptors located in pyramidal and GABA interneurons, respectively, indirectly decrease raphe nuclei activity. On the other hand, 5-HT2A or 5-HT4 postsynaptic receptors in this brain area positively modulate raphe activity (Artigas et al., 2017). Also, the net result of SSRIs on the 5-HT level has been proposed to be dosedependent, with acute clinical doses decreasing (or not changing), and high doses increasing it (Artigas et al., 2017). Concomitant treatment with pindolol, a 5-TH1A receptor antagonist that shows a preference for the pre-over the postsynaptic receptor, accelerated in some studies the antidepressant effect of SSRIs. DU 125530, a 5-HT1A antagonist that does not discriminate these two sites, did not produce this effect (Artigas et al., 2017). Repeated administration of SSRIs or buspirone, but not imipramine, attenuates the function of inhibitory 5-HT1A autosomic receptors (Blier & El Mansari, 2013). Chronic administration of these drugs seems to differently impact on 5-HT1A postsynaptic function. For instance, whereas both fluoxetine and imipramine sensitize 5-HT1A postsynaptic receptors located in the dorsal PAG (dPAG), a critical panic-modulating area (see below), in other defense-related regions such as the hippocampus, dorsomedial hypothalamus, and amygdala sensitization of 5-HT1A receptor, has been reported only after chronic administration of the latter drug (de Bortoli et al., 2013; Gordon & Hen, 2004; Graeff & Zangrossi, 2010). Stimulation of 5-HT1A receptors located postsynaptically in limbic areas has also been proposed to mediate at least part of the anxiolytic effects of buspirone (Gordon & Hen, 2004; Guimara˜es, Carobrez, & Graeff, 2008). Contrary to 5-HT1A, postsynaptic 5-HT2C receptors seem to exert a negative action over mood (Millan, 2005; 2006) and those located at the BLA, as discussed

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below, have critically been implicated in the anxiogenic effect caused by short-term administration of antidepressants (Vicente & Zangrossi, 2012). These receptors are also desensitized by long-term treatment with several classes of antidepressants (Millan, 2005), facilitating their anxiolytic effects (Millan, 2005, 2006; Vicente & Zangrossi, 2014). Since 5-HT2C receptors may be constitutively active, whereas stimulation of postsynaptic 5-HT1A receptors requires the spontaneous release of 5-HT, a general decrease in 5-HT-mediated neurotransmission would shift the balance toward 5-HT2Cmediated anxiogenic effects (Millan, 2005; 2006). In addition to 5-HT2C, downregulation of 5-HT2Amediated neurotransmission could also be involved in the anxiolytic effects of repeated treatment with buspirone or antidepressants (Millan, 2003; Weisstaub et al., 2006). Corroborating the involvement of 5-HT in antidepressant effects, the therapeutic effect of SSRIs in PD and SAD is reverted by 5-HT reduction using dietary tryptophan depletion (Argyropoulos et al., 2004; Bell et al., 2002). The 5-HT precursor l-5-hydroxytryptophan (5-HTP), on the other hand, attenuated panic symptoms induced by CCK4 or CO2 in PD patients (Maron, Toru, Vasar, & Shlik, 2004; Schruers, van Diest, Overbeek, & Griez, 2002). Conversely, tryptophan depletion and metergoline, a nonselective 5-HT antagonist, facilitated these symptoms (Ben-Zion, Meiri, Greenberg, Murphy, & Benjamin, 1999; Miller, Deakin, & Anderson, 2000). Humans with PTSD and other stress-related disorders present larger amygdala and reduced hippocampal and prefrontal cortex volumes. These changes are similar to those reported in rodents after exposure to severe stress (Musazzi, Tornese, Sala, & Popoli, 2017). They are associated with altered density of dendrites and synaptic spines and, in the case of the hippocampus, reduced adult neurogenesis (Dean & Keshavan, 2017; Musazzi et al., 2017). The pathways that lead to these stress-induced neuroplastic changes have been nicely reviewed recently by several authors (Duman, Aghajanian, Sanacora, & Krystal, 2016; Harmer, Duman, & Cowen, 2017; Musazzi et al., 2017). They include hormonal (glucocorticoids, sex steroids), neurotrophins (reduced brain-derived neurotrophic factor, BDNF) and immunological (inflammatory cytokines, monocytes, and microglia) changes, together with genetic vulnerability and gastrointestinal and adipose tissue alterations (microbiome, ghrelin, leptin, Duman et al., 2016; Dean & Keshavan, 2017). Even a single, severe, stress exposure can induce long-term anxiogenic and neuroplastic effects that are reversed by repeated SSRI treatment (Campos, Ferreira, da Silva, & Guimara˜es, 2013). Repeated SSRIs and other antidepressant treatments, such as electroconvulsotherapy and noradrenaline reuptake blockers increase BDNF expression.

Although there are conflicting results (Greenwood et al., 2007), the behavioral effects of chronically administered SSRIs were dependent on BDNF signaling in some studies (Saarelainen et al., 2003). Moreover, the increase in hippocampal neurogenesis induced by fluoxetine was prevented in 5-HT1A knockout mice (Santarelli et al., 2003). More recent evidence indicated that 5-HT4 receptors also facilitate neurogenesis in this region (Segi-Nishida, 2017). Taken together, these results suggest that neuroplastic changes caused by chronic administration of SSRIs play a significant role in their therapeutic effect. However, the reappearance of anxiety symptoms in SSRI-treated patients induced by acute tryptophan depletion (Argyropoulos et al., 2004; Bell et al., 2002) indicates that caution must be exerted when considering these neuroplastic proposals as unitary explanations of the long-term effects of these drugs. In addition to these mechanisms, interference with psychological processes has also been proposed to underlie the therapeutic effects of antidepressants. Acute SSRI administration decreases amygdala response to negative affective faces in healthy subjects and patients with depression (Harmer et al., 2006, 2017; Murphy, Norbury, O’Sullivan, Cowen, & Harmer, 2009). This early effect on affective processing could facilitate symptom improvement over time (Harmer et al., 2017). Finally, it has also been suggested that antidepressants can interact directly with the glutamatergic system, reducing, for example, the release of this neurotransmitter (Bonanno et al., 2005). Additionally, fluoxetine has been shown to induce antiinflammatory, antiapoptotic, and antioxidant effects that could also help to explain its beneficial therapeutic effects (Caiaffo, Oliveira, de Sa´, & Eveˆncio Neto, 2016)

IV. ARE ANXIETY DISORDERS CAUSED BY DISTURBANCES IN 5-HT-MEDIATED NEUROTRANSMISSION? Several clinical studies have suggested that anxiety disorders are associated with disturbances of 5HT-mediated neurotransmission. For example, reinforcing a possible involvement of the serotoninergic neurotransmission in PD, a decrease in echogenicity was described in the brainstem raphe region of patients  ´n with this disorder using transcranial sonography (Silha et al., 2015). Moreover, using PET, increased 5-HT synthesis, and 5-HT transporter density was reported in patients with social anxiety (Frick et al., 2015). Alterations of 5-HT receptors have also been related to anxiety in humans. For example, changes in 5-HT2A and 5-HT1A receptors number or function in the medial frontal cortex during threat-associated emotional face

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IV. ARE ANXIETY DISORDERS CAUSED BY DISTURBANCES IN 5-HT-MEDIATED NEUROTRANSMISSION?

processing have been reported (for review, see Raab, Kirsch, & Mier, 2016). In PD, an impairment of 5-HT1A-mediated neurotransmission has been reported by several neuroendocrine studies (Broocks et al., 2000; Lesch et al., 1992; Mortimore & Anderson, 2000). These studies also suggest an increased 5-HT2C sensitivity in PD and and PTSD, but a decreased sensitivity in OCD. Since blunted cortisol responses to the 5-HT releaser d-fenfluramine was found in patients with PD and OCD, the increased sensitivity of 5-HT2C receptors in these disorders may be reflecting a compensatory up-regulation of 5-HT2C receptors due to a decrease in 5-HT neurotransmission (Guimara˜es et al., 2008). Gene variants of the 5-HT system may contribute to anxiety susceptibility, particularly in PD (for review see Maron & Shlik, 2006). Neuroimaging studies not only have supported this proposal but also have been helpful to elucidate genetic influences on these disorders (Hariri et al., 2005; Iidaka et al., 2005). An inverse correlation has been found between 5-HT1A binding and anxiety in healthy subjects and in patients with other neuropsychiatric disorders (Savic et al., 2004; Tauscher et al., 2001; Yasuno et al., 2004). However, there are negative studies (Iscan et al., 2017) or reports of higher 5-HT1A binding in the forebrain and brainstem of PTSD patients (Sullivan et al., 2013). There is also a report of association between a single polymorphism in the regulatory region of the 5-HT3 receptor coding gene and greater reactivity in the amygdala and in the dorsal and medial PFC (Iidaka et al., 2005). In patients with PD, less 5-HT1A receptors in the forebrain and raphe nuclei have been reported (Nash et al., 2008; Neumeister et al., 2004). A reduction of these receptors in limbic areas, particularly the amygdala, has also been shown in patients with SAD (Lanzenberger et al., 2007), but not in patients with PSTD or GAD (Bonne et al., 2005). In these patients, successful treatment with SSRIs antagonized activity changes during symptom provocation in the insula, and in prefrontal and inferior frontal cortices, suggesting the involvement of 5-HT (Fernandez et al., 2001; Hoehn-Saric, Schlund, & Wong, 2004). This same treatment has been shown to attenuate the increased activation of the anterolateral orbitofrontal cortex, caudate nucleus, thalamus, and temporal regions in OCD patients (Carey et al., 2004). In addition to 5-HT receptors, changes in the 5-HT transporter (5-HTT) have been described. They include an increased in 5-HTT binding in patients with GAD (van der Wee et al., 2008), although an inverse correlation between 5HTT binding and anxiety has also been reported (Maron et al., 2004). The presence of one or two copies of the short allele of the 5-HTT promoter predicts greater amygdala neuronal activity in response to fearful and angry facial

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expressions, compared with individuals homozygous for the long allele, suggesting a bias toward increased reactivity to stressful life experiences (Hariri et al., 2005). However, studies comparing the presence of the short allele in anxiety disorders, such as OCD or GAD, have produced mainly negative results (Hesse, Barthel, Schwarz, Sabri, & Mu¨ller, 2004; Maron et al., 2004). Nevertheless, SAD patients with one or two copies of the short allele in the promoter region of the 5-HTT gene show increased anxiety-related traits, state anxiety, and enhanced right amygdala responding to anxiety provocation (Furmark et al., 2004). In PD patients, there are also negative (Blaya, Salum, Lima, Leistner-Segal, & Manfro, 2007; Strug et al., 2008) and positive (Bauer, 2015; Strug et al., 2008) results regarding an association between a polymorphism in the 5-HTT coding gene and the disorder. Subjects with the short allele present smaller hippocampi and amygdala, functional changes in the amygdalaecingulate circuit and increased fear conditioning (for review see Bauer, 2015). Also, patients with PTSD present reduced amygdala 5-HT transporter binding (Murrough et al., 2011). Although several results support the presence of an association between changes in 5-HTT expression and emotional processing in the amygdala, the functional consequences of these changes on 5-HT-mediated neurotransmission are still not clear. A decrease in the expression of 5-HTT would, theoretically, increase synaptic 5-HT. There is evidence, however, that this polymorphism is associated with blunted 5-HT function (Reist, Mazzanti, Vu, Tran, & Goldman, 2001). Moreover, it is probable that this genetic trait interferes with neuronal development, causing long-term effects that go far beyond a simple change in 5-HT. Some studies have also focused on the brain-specific tryptophan hydroxylase-2 (TPH2) gene, the ratelimiting enzyme in 5-HT synthesis. Although no association between polymorphisms in this gene and PD was found (Maron & Shlik, 2006), they can modulate amygdala responsiveness to emotional arousal. There is also an additive effect on emotional processing in individuals carrying the combination of the T variant of TPH2 with the short variant (l/l) of the 5-HT transporter gene (Canli, Congdon, Todd Constable, & Lesch, 2008). In agreement with the reports of TPH2 changes in anxiety disorder, tryptophan depletion has also been shown to facilitate a negative bias, i.e., increase the responses to fearful relative to happy faces, and altering prefrontale amygdala connectivity (Robinson et al., 2013). Taken together, these studies support the association between anxiety disorders with disturbances of 5-HT mediated neurotransmission. How these disturbances bring about the complex and sometimes distinct symptomatology found in these disorders is still unclear.

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V. THE DUAL ROLE OF 5-HT IN ANXIETY. THE DEAKIN AND GRAEFF THEORY The complexity of the 5-HT system greatly contributes to the difficulty in understanding its role in anxiety. 5-HT can act on more than 14 receptor subtypes, located either pre- or postsynaptically and, in some cases, in both locations. Moreover, their activation could produce opposite effects (Hoyer, Hannon, & Martin, 2002). In addition to this intricate neurochemistry, the system has also a complex neuroanatomy. The widespread distribution of 5-HT fibers in the forebrain arrives from two distinct raphe nuclei, the MRN and the dorsal raphe nucleus (DRN). These nuclei innervate partially distinct structures (Barnes & Sharp, 1999; Hensler, 2006; Pazos & Palacios, 1985) and may present a high degree of heterogeneity (Clark, McDevitt, & Neumaier, 2006). For example, it has been shown that the projections to cortical and limbic structures arise from neurons located in a specific part of the caudal DRN that is particularly sensitive to stressful stimuli (for review see Lowry, Johnson, Hay-Schmidt, Mikkelsen, & Shekhar, 2005). Studies with animal models of anxiety can potentially overcome some limitations of clinical investigations and have been extensively used to investigate the role of 5-HT in anxiety (for comprehensive reviews see Millan, 2003; Bourin & Hascoet, 2003; Sanchez, 2003; Prut & Belzung, 2003; Olivier et al., 2003; Blanchard, Griebel, & Blanchard, 2003; Griebel, 1995; Zangrossi & Graeff, 2014). Although the evidence reviewed so far using these models supports the hypothesis that 5-HT enhances anxiety by acting upon forebrain limbic structures, contrary results have been obtained in the PAG. Electrical stimulation of the dPAG in laboratory animals induces defensive reactions, such as vigorous flight or defensive aggression (Hunsperger, 1956). These defense strategies are expressed in natural conditions when a predator is very close to or in direct contact with the prey (Blanchard, Hynd, Minke, Minemoto, & Blanchard, 2001). Testifying to the aversive character of such stimulation, laboratory animals easily learn to switch off electrical stimulation of the dPAG (Delgado, Roberts, & Miller, 1954; Hunsperger, 1956). According to the above theoretical model, 5-HT is expected to facilitate such escape behavior. However, reported results with a procedure in which rats are trained to lever press in order to decrease the intensity of dPAG electrical stimulation suggest the opposite action. Indeed, using this decremental escape procedure, Kiser and coworkers have been shown that PCPA markedly increased the rate of bar pressing (Kiser, Lebovitz, & German, 1978b; Kiser & Lebovitz, 1975). Further results have shown that the precursor of 5-HT synthesis 5-hydroxytryptophan

(5-HTP) reduces the same bar pressing, and that the SSRI clomipramine has the same effect (Kiser, German, & Lebovitz, 1978a). Finally, electrical stimulation of the DRN, which sends 5-HT nerve fibers to the PAG, has also been reported to depress responding (Kiser, Brown, Sanghera, & German, 1980). Altogether, these results show that manipulations that increase 5-HT activity in the dPAG have an antiaversive effect, whereas 5-HT depletion results in facilitation of escape from dPAG electrical stimulation. Further supporting an inhibitory function of 5-HT on aversion generated in the dPAG, Schenberg and Graeff (1978) have shown that the 5-HT receptor antagonists cyproheptadine and methysergide decrease responding that switches-off electrical stimulation of the dPAG, whereas the nonsedative doses of the benzodiazepine chlordiazepoxide facilitate switch-off responding. Such opposite effects of a benzodiazepine anxiolytic and the 5-HT antagonist contrast with the similar antipunishment effects of the two classes of drugs verified in conflict tests. Furthermore, using electrical stimulation of the dPAG as a punishing stimulus, Morato de Carvalho et al. (1981) have shown that, as expected, the anxiolytics chlordiazepoxide and pentobarbital released responding maintained by water reinforcement, suppressed by the response-contingent delivery of electrical stimuli in the dPAG. In contrast, the 5-HT receptor antagonist cyproheptadine did not increase punished responding at doses that had been shown to markedly release behavior punished by foot shock (Graeff, 1974), and methysergide was also ineffective. To explore the function of 5-HT in the PAG more directly, rats bearing a chemitrode implanted into the dPAG allowing microinjection of drugs associated with electrical stimulation were placed inside a shuttle box, and the intensity of a sinusoidal current was gradually increased until the rat ran toward the opposite compartment of the shuttle box. This response was made to switch off the brain stimulation. The same procedure was repeated 3 or 10 times, according to the experimental protocol, to determine the basal aversive threshold. Soon after, a drug microinjection was made and the aversive threshold was determined again. The difference between the magnitude of the postdrug and that of the basal threshold measures the drug effect on aversion. In the first study with this method, conducted by Schu¨tz, de Aguiar, and Graeff (1985), microinjection of 5-HT as well as of the 5-HT receptor agonist 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) into the dPAG raised the aversive threshold dosedependently. Local pretreatment with either metergoline or ketanserin, which are 5-HT receptor antagonists, blocked the antiaversive effect of 5-HT. Since ketanserin is a relatively selective 5-HT2A-receptor antagonist, these

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V. THE DUAL ROLE OF 5-HT IN ANXIETY. THE DEAKIN AND GRAEFF THEORY

results suggest that 5-HT inhibits aversion in the dPAG by stimulating this 5-HT receptor subtype. This view has been further supported by results showing that the 5-HT2A/2C agonist 2,5- DOI increases the aversive threshold (Nogueira & Graeff, 1995). The latter study has also shown that the 5-HT1A agonists, 8-OH-DPAT and ipsapirone, have a similar effect, while the nonselective 5-HT2C receptor agonist mCPP was ineffective. Using chemical, instead of electrical stimulation, Marsden and coworkers have shown that microinjection of 5-HT1A agonists into the PAG attenuated running behavior induced by microinjection of an excitatory amino acid into the dPAG; the 5-HT1A-receptor blocker WAY 100635 antagonized this effect, whereas mCPP facilitated running (Beckett, Lawrence, Marsden, & Marshal, 1992; Beckett & Marsden, 1997). Therefore stimulation of either the 5-HT1A or the 5-HT2A receptor in the dPAG seems to inhibit escape. It is worth remarking that in the results reported by Schu¨tz et al. (1985), microinjection of SSRI into the dPAG not only potentiated the antiaversive effect of the following administration of 5-HT, but also had an antiaversive effect of its own. This implies the existence of 5-HT nerve fibers in the dPAG that are physiologically regulating defense. This view has been strengthened by further experimental evidence showing that blockade of presynaptic 5-HT1B receptors that inhibit 5-HT release with isamoltane, microinjected into the dPAG, also has an antiaversive effect, which is blocked by local pretreatment with the 5-HT2-receptor blockers, ketanserin and ritanserin (Nogueira & Graeff, 1991). Interestingly, administration of 5-HT receptor antagonists alone into the dPAG has no effect on the aversive threshold (Nogueira & Graeff, 1991; Schu¨tz et al. 1985). This finding contrasts with the major aversive effects caused by compounds like bicuculline, which block GABAA receptors in the dPAG (Branda˜o, de Aguiar, & Graeff, 1982). It may be concluded that while GABAergic terminals tonically inhibit the neurons of the dPAG that control defensive behavior, serotonergic fibers seem to exert a phasic inhibition. As a consequence, it has been suggested that the modulatory action of 5-HT would only appear under conditions, such as stressful situations, that engage 5-HT systems (Deakin & Graeff, 1991). Overall, the results reviewed so far indicate that 5-HT facilitates inhibitory avoidance by acting on forebrain structures such as the septo-hippocampal system and the amygdala, whereas inhibiting escape in the dPAG. The amygdala and dPAG, plus the medial hypothalamus, constitute a set of interrelated structures, often referred to as the brain defense system (BDS), which controls defensive strategies and elaborates the accompanying emotional and motivational states. Although the three components of the BDS would work together to generate different kinds of defensive behavior, each structure is

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likely to exert specific functions. In this regard, Fanselow (1991) has suggested that the amygdala synthesizes the stimulus input from the environment and then signals to the PAG the degree of threat that they represent to the organism, whereas the PAG would be in charge of selecting, organizing and executing the appropriate behavioral and neurovegetative defensive reactions. On the basis of the systematic study of antipredator defense in wild rats, Blanchard and Blanchard (1988) have formulated the concept of defense levels. Three such levels have been identified as a function of the threat being potential (uncertain), distal or proximal, each evoking a specific defense reaction. Thus, when danger is uncertain, like in novel environments, rats perform cautious exploration aimed at risk assessment. When the predator is perceived at safe distance, tense and attentive immobility (freezing) ensues, and when the predator is near or in actual contact with the rat, the animal rapidly flees, whenever possible or otherwise threatens back or even attacks the predator defensively. Comparative studies led to the conclusion that these basic defense strategies can be found in other animals, including nonmammalian species (Blanchard et al., 2001). The defense levels named by the Blanchard’s “potential” and “proximal” correspond to the postencounter and circa-strike defense strategies, defined by Fanselow and Lester (1988) as a function of predatory eminence. Attempts to relate each level of defense with critical brain structures and to particular emotions (e.g., Graeff, 1994) led to the suggestion that the septohippocampal system and the amygdala would be the key structures for risk-assessment behavior and inhibitory avoidance, the related emotion being anxiety. The septo-hippocampal system would provide the cognitive component of anxiety, while the affective component would be integrated in the amygdala (Gray & McNaughton, 2000). At the other extreme, the vigorous undirected flight elicited by proximal danger would be related to panic, the critical structure being the dPAG. At the intermediate level of distal threat, well-directed escape related to fear would be elaborated by the medial hypothalamus. Concerning psychopathology, the first level of defense has been related to GAD and the third with PD, the second being implicated in specific phobias (Deakin & Graeff, 1991; Gray & McNaughton, 2000). This approach, relating animal and human defense strategies with fear and anxiety disorders, has recently been adopted by the Research Domain Criteria (RDoC) initiative of the US National Institute of Mental Health (NIMH), which is aimed to classify psychiatric disorders for research purposes on the basis of both behavioral and neurobiological data (Kozak & Cuthbert, 2016). Both the amygdala and the PAG receive serotonergic input mainly from the DRN. The axons that project onto the amygdala follow the DRNeforebrain tract, while

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36. SEROTONIN IN PANIC AND ANXIETY DISORDERS

those that go to the PAG run through the DRNe periventricular tract (Azmitia & Segal, 1978). Most of the nerve fibers that originate in the DRN are thin and have small varicosities that make preferential contact with 5-HT2 receptors (Mamounas et al., 1991). Given this background, a working hypothesis has been conceived to reconcile the experimental evidence on the role of 5-HT in defense, reviewed above (Graeff, 1991). According to this view, activation of the DRN results in facilitation of defense strategies that are mainly integrated at the amygdala and other forebrain areas, such as risk assessment and inhibitory avoidance. At the same time, proximal defense reactions that are chiefly organized in the dPAG, such as escape/flight, are inhibited. The adaptive function of this disposition would be to inhibit extreme defense patterns, like flight or fight, in situations where predatory threat is uncertain or far from the prey. In these circumstances, such reactions are inappropriate, because they enhance the probability of detection by the predator. Instead, more flexible, largely learned, responses are likely to lead to successful defense. This construct has been extended to psychopathology, relating inhibitory avoidance and risk assessment to GAD and proximal defense to PD (Deakin & Graeff, 1991, Fig. 36.3).

FIGURE 36.3 The dual role of 5-HT on defensive behaviors proposed by Deakin and Graeff (1991). By activating the dorsal raphe nucleus threatening stimuli would facilitate defense strategies that are mainly integrated at the amygdala and other forebrain areas, such as risk assessment and inhibitory avoidance. At the same time, proximal defense reactions that are chiefly organized in the dorsal periaqueductal gray (dPAG) such as escape would be inhibited. In humans these strategies have been associated, respectively to generalized anxiety (GAD) and to panic, phobias and social anxiety. þ: facilitation; : inhibition. Drawings based on the Paxinos, G., Watson, C. (2005). The rat brain in stereotaxic coordinates (5th ed.). Amsterdam: Elsevier.

VI. EXPERIMENTAL TESTS OF THE DEAKIN AND GRAEFF THEORY: CLINICAL STUDIES Two experimental models of anxiety in humans have been used to test this theoretical model: the simulated public speaking (SPS) and the skin conductance response (CSCR) tests (review in Graeff, Parente, Del-Ben, & Guimara˜es, 2003). The SPS test consists, basically, in the preparation and performance of a speech in front of a video camera. During the procedures the participant can see his/her own image on a TV screen, and anxiety and other subjective measures are taken before, during, and after the speech. Since fear of speaking is highly prevalent and generates anxiety in healthy persons irrespective of trait anxiety level (Palma, Guimara˜es, & Zuardi, 1994), it is supposed to be a species-specific fear. Pharmacological studies have shown that benzodiazepines decrease anxietye measured by the anxiety factor of the Visual Analog Mood Scale (VAMS)ebefore and after the speech, but do not attenuate the increase of VAMS anxiety during speech preparation or performance (speaking fear). In contrast, and in agreement with the predictions of Deakin and Graeff’s proposal, drugs that facilitate 5-HTmediated neurotransmission decrease, whereas drugs that impair 5-HT function increase speaking fear (Graeff et al., 2003). The CSCR test consists in the presentation, through earphones, of 10 neutral tones (habituation phase), followed by a neutral tone paired with a loud white noise (acquisition phase) and by the representation of 10 neutral tones (extinction phase). During the procedures, measures of skin conductance (level, SCL; response, SCR and spontaneous fluctuations, SF) are taken. Anxiolytic drugs, such as diazepam and the 5-HT1A partial agonist buspirone, impaired conditioning, while anxiogenic drugs, such as the 5-HT agonist methyl-chlorophenylpiperidine (mCPP) and the 5-HT releaser d-fenfluramine tend to facilitate conditioning (Graeff et al., 2003). The pharmacological profile of the above experimental human tests keeps some similarities with the pharmacological response observed in the clinical practice of patients with the diagnoses of anxiety disorders (reviews in Graeff & Del-Ben, 2008; Garcia-Leal, Graeff, & Del-Ben, 2014). Buspirone and ritanserin seem to be effective in the treatment of GAD, but not of PD. Both drugs had an anxiolytic effect in the CSCR, without modulating the fear induced by the speaking performance. Sumatriptan, a 5-HT1D/1B agonist largely used for the treatment of migraine, has been related to a worsening of panic/anxiety symptoms in panic patients and acutely increased the fear induced by the SPS test (de Rezende et al., 2013).

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VII. EXPERIMENTAL TESTS OF THE DEAKIN AND GRAEFF THEORY IN LABORATORY ANIMALS: THE ELEVATED T-MAZE

Some antidepressants largely used for the treatment of anxiety disorders have also been tested in these models of human experimental anxiety. The acute administration of clomipramine (Guimara˜es, Zuardi, & Graeff, 1987) and nefazodone (Silva, Hetem, Guimara˜es, & Graeff, 2001) increased speaking fear in the SPS test, and this effect has been related to the worsening of anxiety symptoms frequently observed at the beginning of the treatment with antidepressants. However, a single dose of escitalopram, an SSRI, did not enhance the fear caused by the speaking performance itself, as observed with other antidepressants, but prolonged the fear induced by the SPS test (Garcia-Leal, Del-Ben, Leal, Graeff, & Guimara˜es, 2010), similar to that observed with a single dose of ritanserin, which allegedly decreases serotonergic function. It has been proposed that the fear-enhancing effect of a single dose of antidepressants in SPS is due to a lack of 5-HT inhibition of brain systems that generate panic attacks, likely to be localized in the dPAG. As discussed previously, it is possible that acute doses of antidepressants preferentially increase 5-HT concentration near the cell bodies of serotonergic neurons, reducing their firing rate due to the activation of somatodendritic 5-HT1A autoreceptors, decreasing 5-HT release and lowering the postsynaptic concentration of 5-HT. Taken together, the pharmacological data obtained so far with human experimental models are compatible with the hypothesis that 5-HT enhances anxiety, evaluated by the CSCR test, whereas inhibits fear, accessed by the SPS test. If the predictions derived from pharmacological tests discussed above are correct, it is expected that panic patients would perform in SPS differently from healthy volunteers, but not in CSCR, since the former test would engage the neural substrates that would be implicated in the neurobiology of PD, but the latter would not. It is important to highlight that the SPS is not considered as a model of panic attack or a probe aimed at provoking panic attacks in susceptible individuals. The rationale of the relationship between the model and the anxiety disorder is that the public speaking would engage the brain mechanisms involved in the process of innate fear, which would be impaired in PD. As expected, our studies have shown that the panic patients’ response to CSCR is similar to that of controls, except for the occurrence of more spontaneous fluctuations of skin conductance along the experimental session in panic patients, a finding that is consistent with their high baseline level of subjective anxiety. In the SPS test, panic patients showed higher levels of anxiety than normal controls along the whole session, but were less responsive to the speaking challenge (Del Ben et al., 2001; Garcia-Leal et al., 2005; Parente, Garcia-Leal, Del-Ben, Guimara˜es, & Graeff, 2005). The

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profile of panic patients during the SPS test resembles the effect of the nonselective 5-HT-receptor blocker metergoline in healthy volunteers, which increased anxiety before and after the speech, but not during the speech preparation or performance (Graeff, Zuardi, Giglio, Lima Filho, & Karniol, 1985). These results support the suggestion that 5-HT deficiency, and therefore less inhibition of PAG, may be present in PD. Interestingly, the above SPS task did not increase cortisol secretion (Garcia-Leal et al., 2005). Accordingly, neither panic attacks (reviewed in Graeff, Garcia-Leal, Del-Ben, & Guimara˜es, 2005) nor the electrical stimulation of the dPAG of the rat (Schenberg, Dos Reis, Ferreira Povoa, Tufik, & Silva, 2008) seem to activate the hypothalamice pituitaryeadrenal axis. Another evidence that panic patients process innate fear responses abnormally has come from a study carried out in our laboratory with patients with SAD submitted to the SPS test (MC Freitas, A Santos Filho, F Oso´rio, SR Loureiro, CM Del-Ben, AW Zuardi, FG Graeff, JAS Crippa, unpublished results). Although SAD and PD are different anxiety disorders, they keep some similarities, such as the response to the treatment with antidepressants that act on 5-HT function. Nevertheless, differently from panic patients, SAD patients have shown a greater increase of the fear induced by the SPS, in comparison to healthy controls. Therefore, the low sensitive to SPS test seems to be a specific feature of PD.

VII. EXPERIMENTAL TESTS OF THE DEAKIN AND GRAEFF THEORY IN LABORATORY ANIMALS: THE ELEVATED T-MAZE Since the initial findings showing that impairment of 5-HT-mediated neurotransmission causes anticonflict effects (Graeff & Schoenfeld, 1970; Robichaud & Sledge, 1969), a large number of studies aimed at investigating the role of 5-HT in anxiety has been performed. The results, however, were far from conclusive (Belzung, 2001; Blanchard et al., 2003; Bourin & Hascoet, 2003; Griebel, 1995; Millan, 2003; Olivier et al., 2003; Prut & Belzung, 2003; Sanchez, 2003). This fact is easily illustrated with results obtained in the elevated plus-maze (EPM, Handley, McBlane, Critchley, & Njung’e, 1993; Griebel, 1995, for review see Pinheiro, Zangrossi, Del-Ben, & Graeff, 2007). Trying to explain these contradictory results, the British psychologist Sheila Handley proposed that the EPM engages different defense strategies, which could be influenced in opposite directions by 5-HT (Handley et al., 1993). These strategies would include avoidance of the open arms, when the rat is in one of the enclosed arms, and escape from these same arms

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36. SEROTONIN IN PANIC AND ANXIETY DISORDERS

to enter a safer, enclosed arm. Based on this proposal, and trying to develop an experimental approach to test the predictions of the Deakin and Graeff (1991) theory, Graeff and coworkers have developed the elevated T-maze (ETM, Graeff, Viana, & Tomaz, 1993, 1998; Zangrossi & Graeff, 2014). The apparatus was built by closing the entrance of one of the enclosed arms of the elevated plus-maze. The test measures both inhibitory avoidance (the latency to withdraw from the enclosed arm) and one-way escape (the withdrawal latency from the open arm) in the same animal. Pharmacological results have shown that diazepam impairs inhibitory avoidance but does not affect one-way escape, whereas chronic treatment with imipramine, fluoxetine, and other antidepressants increased escape latency (for a review see Zangrossi & Graeff, 2014). The latter parameter was also impaired by panic-effective benzodiazepines such as alprazolam and clonazepam (Pobbe et al., 2014; Sant’Anna et al., 2016), while enhanced by the panicogenic agent cholecystokinin (Zanoveli, Netto, Guimara˜es, & Zangrossi, 2004). These results indicate that inhibitory avoidance and escape measurements in the ETM may be modeling two different anxiety disorders, respectively GAD and PD. In the first test of the dual role of 5-HT in defense hypothesis, it has been shown that systemic administration of d-fenfluramine, a drug that selectively releases 5-HT from the thin serotonergic fibers that come mainly from the DRN, impairs escape, whereas tending to increase inhibitory avoidance (Graeff, Viana, & Mora, 1997). These results not only fulfill the predictions from the above hypothesis, but also correlate with the results described above with the same compound administered to healthy subjects submitted to the SPS and CSCR models. Moreover, they agree with clinical reports on antipanic effects of this drug in PD patients (Hetem, 1996; Solyom, 1994). To test the dual 5-HT hypothesis more directly, a series of experiments has been conducted to explore the effects of intracerebral injection of 5-HT-acting compounds in rats submitted to the ETM (Table 36.1, Fig. 36.4). Results employing microinjection of the 5-HT1A receptor antagonist WAY100635 and the 5-HT1A agonist 8-OH-DPAT into the DRN have produced consistent results. Whereas the former drug, supposedly enhancing neuronal activity of 5-HT neurons, impaired escape and facilitated inhibitory avoidance, the latter did exactly the opposite (Pobbe & Zangrossi, 2005; Sena et al., 2003). Although not originally contemplated in Deakin and Graeff’s theory or in the studies just mentioned, a wealth of evidence gathered in the last years suggests that the DRN is not a homogenous structure, but an aggregate of distinctive populations of serotonergic and nonserotonergic neurons, which are supposed to play different

FIGURE 36.4 An alternative view of the proposal by Deakin and Graeff (1991). Instead of the rostro-caudal hierarchy originally suggested, 5-HT would play a dual role in two parallel, longitudinally organized systems (McNaughton & Corr, 2004). The approach defense system would be engaged mainly by approacheavoidance conflict. Its main neural representation would involve the septum-hippocampus and other forebrain structures. The avoidance defense system, on the other hand, would be activated when there is no tendency to approach the source of danger. In this case, the experienced emotion is fear, not anxiety. According to this proposal, fear or panic would be elicited depending on the defensive distance. Distal threat would activate forebrain structures and elicits fear while proximal threat would activate the PAG and elicit panic. The figure also illustrates the results obtained after intracerebral injections of 5-HT receptor agonists, more specifically 5-HT1A and 5-HT2C agonists (see table 1) into the dorsal periaqueductal gray (dPAG), medial hypothalamus (MHyp), hippocampus (Hipp), amygdaloid nuclei (Amyg), or medial prefrontal cortex (PFC) on inhibitory avoidance and escape latencies of rats submitted to the elevated T-maze (ETM). þ: facilitation; : inhibition; 0: no effect. ? indicates areas where the effects of these drugs have not yet been tested. Drawings based on the Paxinos, G., Watson, C. (2005). The rat brain in stereotaxic coordinates (5th ed.). Amsterdam: Elsevier.

roles in the pathophysiology of GAD and PD (Lowry et al., 2008; Paul & Lowry, 2013). For instance, using the ETM, Spiacci, Coimbra, and Zangrossi (2012) reported that whereas acquisition of inhibitory avoidance activates serotonergic neurons primarily within the

V. SEROTONIN IN PSYCHIATRIC DISORDERS

VII. EXPERIMENTAL TESTS OF THE DEAKIN AND GRAEFF THEORY IN LABORATORY ANIMALS: THE ELEVATED T-MAZE

TABLE 36.1

Effects in the elevated T-maze of 5-HT related drugs injected into brain areas involved in defensive responses.

Region

Avoidance

Escape

Reference

8-OH-DPAT



þ

5,7 DHT (5-HT lesion)



þ

Sena et al., 2003; Pobbe and Zangrossi, 2005

WAY-100635

þ



Dorsal raphe (lateral wings)

8-OH-DPAT

0

þ

WAY-100635

0



Dorsal raphe (dorsomedial subnucleus)

8-OH-DPAT



þ

WAY100635

þ



dPAG

5-HT

þ



mCPP

þ



MK-212

þ

0

8-OH-DPAT





DOI

0



WAY-100635

0

0

Ketanserin

0

0

SDZ SER082

0

0

SB-242084

0

0

5HT



0

8-OH-DPAT



0

DOI



0

WAY-100635



0

Ketanserin

0

0

Dorsomedial hypothalamus

8-OH-DPAT

0



WAY-100635

0

0

Ventromedial hypothalamus

8-OH-DPAT

0

0

DOI



0

MK-212



0

5-HT

þ

0

MK-212

þ

0

SB-242084



0

8-OH-DPAT





8-OH-DPAT

þ

0

WAY-100635

0

0

5-HT



0

8-OH-DPAT



0

Hernandes PM, Ku¨mpel VD, de Andrade TGC, and Zangrossi Jr. H, personal communication

8-OH-DPAT

þ

0

Viana et al., 2008

5-HT

0

0

Dorsal raphe

vlPAG

BLAmyg

Dorsal hippocampus

Ventral hippocampus

Lateral septum

Drug

þ, facilitation, , inhibition, 0, no effect.

V. SEROTONIN IN PSYCHIATRIC DISORDERS

Spiacci et al., 2016

Spiacci et al., 2016

Zanoveli et al., 2003; de Paula Soares et al., 2004; Yamashita et al., 2011.

de Paula Soares et al., 2009

Nascimento et al., 2014

da Silva et al., 2011

Vicente and Zangrossi Jr., 2012; Strauss et al., 2013

dos Santos et al., 2008

621

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36. SEROTONIN IN PANIC AND ANXIETY DISORDERS

dorsomedial and caudal DRN subnuclei, escape expression activates nonserotonergic neurons within the lateral wings of the DRN (lwDR). Results such as these have led to the proposal that whereas the former nuclei are the critical source of serotonergic innervation to limbic forebrain areas implicated with anxiety processing, particularly the amygdala, the lwDR gives rise to the DRN-5-HT periventricular pathway implicated by Deakin and Graeff in escape/panic regulation (Lowry et al., 2008; Paul & Lowry, 2013). In support, nonselective chemical stimulation or disinhibition of lwDR neurons with kainic acid or bicuculline, respectively, evoked escape behavior in an open-field and facilitated escape expression in the ETM, without affecting inhibitory avoidance acquisition (Matthiesen, Spiacci, & Zangrossi, 2017; Spiacci, Pobbe, Matthiesen, & Zangrossi, 2016). This selective effect on the escape task in the ETM was also observed after local microinjection of WAY-100635 or 8-OH-DPAT in this subnucleus, but with these drugs causing opposed consequences for the animal’s response; whereas WAY-100635 facilitated, 8-OH-DPAT impaired it. On the other hand, kainic acid or WAY100635 administration into the dorsomedial DRN facilitated avoidance acquisition, as predicted, but unexpectedly also impaired escape performance. The opposed effect on these defensive tasks was observed after 8-OH-DPAT microinjection into this subnucleus (Spiacci et al., 2016). As shown in Table 36.1 and Fig. 36.4, results obtained with direct injections into projecting areas of 5-HT neurons have also produced results that are, in general, compatible with the proposal of a dual role of 5-HT (Deakin & Graeff, 1991). There are, however, some inconsistencies. Injection of the endogenous agonist 5-HT into the dPAG not only inhibited escape performance, as expected, but also caused an anxiogeniclike effect on inhibitory avoidance (Zanoveli, Nogueira, & Zangrossi, 2003). Other 5-HT receptor agonists, such as 8-OH-DPAT and DOI, similarly impaired escape, but with a differential effect on inhibitory avoidance; i.e., while the former compound was anxiolytic, the latter had no effect (Zanoveli et al., 2003). Intra-dPAG administration of either, the 5-HT1A receptor antagonist WAY-100635 or the 5-HT2A/2C receptor antagonists, ketanserin, SDZ SER082, and SB-242084, was ineffective, corroborating previous evidence indicating that in this brain region 5-HT exerts a phasic rather than tonic regulatory influence on defensive behaviors. Studies with these antagonists also revealed that whereas the anxiogenic effect of 5-HT in the dPAG is mediated by 5-HT2C receptors (Yamashita, de Bortoli, & Zangrossi, 2011), the antiescape effect is due to the recruitment of 5-HT1A or 5-HT2A receptors (de Paula Soares and Zangrossi Jr., 2004), as previously shown in the dPAG stimulation model (Nogueira & Graeff,

1995). Interestingly, a more recent study with the ETM shows that pharmacological activation of 5-HT1A receptors in the dorsomedial hypothalamus, a brain area also implicated in the pathophysiology of PD (Johnson et al., 2012), causes antiescape effect similarly as observed in the dPAG, but without affecting inhibitory avoidance acquisition (Nascimento, Kikuchi, de Bortoli, Zangrossi, & Viana, 2014). In the ventromedial hypothalamus, an area in the vicinity of the dorsomedial hypothalmus, the same procedure was ineffective on both ETM tasks (da Silva, Poltronieri, Nascimento, Zangrossi, & Viana, 2011) Similar to the results obtained in the dPAG, the role of different amygdaloid 5-HT receptor subtypes in the modulation of the two tasks generated by the elevated T-maze seems to be more complex than originally thought. Thus, stimulation of 5-HT1A receptors by 8-OH-DPAT in the BLA impaired, rather than facilitated, inhibitory avoidance acquisition, indicating an anxiolytic effect. Also unexpectedly, this agonist equally inhibited escape behavior (Strauss, Vicente, & Zangrossi, 2013). However, in agreement with Deakin and Graeff’s proposal, local injection of 5-HT itself or of the 5-HT2C receptor agonist MK 212 caused an anxiogenic effect upon inhibitory avoidance, without changing escape performance. On the other hand, microinjection of the 5-HT2C receptor antagonist SB 242084 had the opposite effect on inhibitory avoidance, also without interfering with escape (Vicente & Zangrossi, 2012). Since antidepressants have been shown to downregulate 5-HT2C receptors after repeated administration, this finding may be related to their therapeutic effect in GAD patients, as further discussed below. Altogether these results indicate that 5-HT, through the activation of BLA 5-HT2C receptors, plays a tonic and selective role in the mediation of inhibitory avoidance. Considering that the BLA has been proposed to assign emotional salience to both rewarding and aversive stimuli, the contradictory findings found after direct injections of 5-HT1A and 5-HT2C related compounds may involve a complex interference on both approach and avoidance behaviors (Lowry et al., 2005). Regarding the septo-hippocampal system, only studies assessing the involvement of 5-HT1A receptors have been published using the ETM, the results being compatible with the proposal that 5-HT is mainly implicated in the regulation of anxiety. Accordingly, 8-OHDPAT injection either into the lateral septum or dorsal hippocampus has an anxiogenic effect upon inhibitory avoidance, without affecting escape. In the lateral septum, the effect of 8-OH-DPAT on anxiety was opposed to that caused by the benzodiazepine midazolam. Microinjection of WAY-100635 into either the septum or the hippocampus had no effect on the two ETM tasks (dos Santos, de Andrade, & Zangrossi,

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VII. EXPERIMENTAL TESTS OF THE DEAKIN AND GRAEFF THEORY IN LABORATORY ANIMALS: THE ELEVATED T-MAZE

2008; Viana, Zangrossi, & Onusic, 2008). However, several conflicting results suggest that the role of 5-HT on defensive responses that involve the septohippocampal system is more complex than originally believed. Although effects on different 5-HT receptor subtypes could help to explain these results (Gordon & Hen, 2004), most studies so far have targeted the dorsal hippocampus, an area that receives 5-HT fibers from the median, rather than the DRN. The latter structure innervates mainly the ventral hippocampus, a region that has been particularly related to anxiety (Bannerman, Rawlins et al., 2004). Interestingly, a recent analysis revealed that administration of 5-HT or 8-OH-DPAT in the ventral portion of the hippocampus causes anxiolytic and not anxiogenic effect in the ETM as previously observed in the dorsal area. In both cases, no change on escape expression was observed (PM Hernandes, VD Ku¨mpel, TGC de Andrade, and H Zangrossi Jr., unpublished data). As a final remark, results obtained with the administration of 5-HT-acting drugs in the ventrolateral column of the PAG point to the selective involvement of this area in anxiety modulation. To this respect, it is noteworthy that whereas electrical or chemical stimulation of the dPAG evokes flight/escape responses accompanied by tachycardia and hypertension, as mentioned before, the same procedure in the ventrolateral PAG leads to behavioral inhibition (hyporeactive immobility), accompanied by bradycardia and hypotension (Carrive & Bandler, 1991). In agreement, intraventrolateral PAG injection of 5-HT, 8-OH-DPAT, and DOI has been shown to consistently impair inhibitory avoidance acquisition, without interfering with one-way escape. Curiously, while both ketanserin and WAY-100635 were also ineffective in changing escape performance, the latter drug impaired avoidance acquisition, an effect that has been attributed to the drug interference with nonserotonergic mechanisms, while ketanserin did not affect inhibitory avoidance (de Paula Soares and Zangrossi Jr., 2009). Studies in the ETM have also been employed to unveil the mechanism of action of therapeutic drugs. Several pieces of evidence suggest that whereas the antipanic effects of SSRIs and imipramine depends on facilitation of 5-HT1Aand/or 5-HT2A-mediated neurotransmission in the dPAG, the anxiolytic effect caused by these compounds involves a desensitization of 5-HT2C receptors and the recruitment of 5-HT1A receptor in the BLA (for a review see Graeff & Zangrossi, 2010). Regarding the antipanic effect of antidepressants, repeated treatment (21 days) with imipramine, sertraline or fluoxetine enhanced the inhibitory effect of intra-dPAG injection of 5-HT1A or 5-HT2A agonists on escape responses either induced by local electrical stimulation or measured in the ETM (de Bortoli et al., 2006; Zanoveli, Nogueira, & Zangrossi, 2005). The same effect

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is also reported after long-, but not short-term treatment with alprazolam, a high potency benzodiazepine receptor agonist successfully used in the treatment of PD. Curiously, chronic treatment with alprazolam facilitates of 5-HT1A- or 5-HT2A-mediated neurotransmission in the dPAG, without affecting the responsiveness of benzodiazepine receptors in the same brain area (de Bortoli et al., 2008). Differently from SSRIs, buspirone failed to enhance the inhibitory effects of 5-HT1A or 5-HT2A agonists on escape responses mediated by the dPAG (de Bortoli et al., 2006; Zanoveli et al., 2005), what could explain its lack of therapeutic efficacy in PD. A microdialysis study also revealed that chronic, but not acute, fluoxetine treatment, besides causing a functional sensitization of 5-HT1A receptors in the dPAG, also increases 5-HT release in this midbrain area. Such an effect is not observed after chronic systemic administration of buspirone (Zanoveli et al., 2010). Also of importance, injection of WAY-100635 into the dPAG antagonized the antiescape effect caused by chronic systemic injection of fluoxetine in rats tested in the ETM (Zanoveli et al., 2010). The ETM has also been employed to unveil the mechanisms through which antidepressants can affect anxiety; worsening it at the beginning of the treatment, but with beneficial therapeutic effects after continued administration. Vicente and Zangrossi (2012) reported that administration of the 5-HT2C antagonist SB-242084 into the BLA blocked the acute anxiogenic effect caused by a single systemic injection of imipramine. This anxiety-blocking effect of SB-242084 in the BLA was also observed in rats acutely injected with imipramine or fluoxetine and tested in another anxiety-associated model, the Vogel conflict test. These observations add to a series of evidence showing that the 5-HT2C receptor in the BLA mediates the anxiogenic effect generated by different conditions/situations such as exposure to stressful stimuli (Christianson et al., 2010) and discontinuation of chronic ethanol ingestion (Overstreet, Knapp, Angel, Navarro, & Breese, 2006). As previously mentioned, according to Millan (2003, 2005) the shift from anxiogenesis to anxiolysis caused by antidepressants over time involves both the desensitization of 5-HT2C and the sensitization of 5-HT1A receptors located in limbic areas. There is now evidence that strongly implicates the BLA as the fulcrum of this change. Vicente and Zangrossi (2014) reported that chronic, but not acute administration of imipramine or fluoxetine, abolished the anxiogenic effect caused by the injection of the 5-HT2C-receptor agonist MK-212 in the ETM and lightedark transition tests. Therefore, long-term treatment with these two prototypical antidepressants causes a desensitization of BLA 5-HT2C receptor-mediated increase in anxiety, and this phenomenon comes into play during the time these drugs cause

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anxiolysis. Furthermore, it was also shown that when 5-HT2C-mediated neurotransmission in the BLA is inhibited, 5-HT1A receptors in this nucleus remain operationally active. More specifically, the authors observed that intra-BLA microinjection of the 5-HT1A-receptor antagonist WAY-100635 counteracted the anxiolytic effect of chronic imipramine both in the ETM and the lightedark transition test. This is in line with evidence showing that 5-HT1A receptor activation in the BLA causes anxiolysis in the ETM (see above) and other animal models of anxiety (for a review see Strauss et al., 2013). Finally, Vicente and Zangrossi (2014) also showed that the blockade of 5-HT1A receptors in the BLA did not affect the antiescape effect caused by chronic imipramine in the ETM, supporting the view that the anxiolytic and panicolytic effects of chronic antidepressant administration are mediated by different mechanisms/neural substrates. Overall, the results obtained in the ETM, particularly those in the dPAG and BLA, agree with a proposed dual role of 5-HT in defensive responses. They question, however, the rostro-caudal hierarchy originally suggested by Deakin and Graeff (1991, Fig. 36.3). More recently, McNaughton and Corr (2004) proposed that defensive responses are organized in two parallel, longitudinally organized systems. In this view, the “approach defense system” would deal with anxiety and be engaged mainly by approache avoidance conflict. Its main neural representation involves the septum-hippocampus and other forebrain structures. The “avoidance defense system”, on the other hand, would be activated when there is no tendency to approach the source of danger. In this case, the experienced emotion is fear, not anxiety (Fig. 36.4). According to this proposal, fear or panic would be elicited depending on the defensive distance. Distal threat would activate forebrain structures and elicit fear while proximal threat would activate the dPAG and elicit panic. This proposal has received strong support from a study in healthy volunteers using functional magnetic resonance imaging (MRI, Mobbs et al., 2007), the results of which have shown that brain activity shifts from the prefrontal cortex (PFC) to the midbrain PAG as a virtual predator capable of inflicting pain grows nearer to the virtual prey. In addition, the activity of the DRN correlated with reported subjective degree of dread and decreased confidence of escape. In the same line, an fMRI study using audio descriptions of threatening scenarios taken from of a validated fear questionnaire (Shuhama, Del-Ben, Loureiro, & Graeff, 2008), and asking the participant to imagine him/herself in the given situation, showed that imagination of a situation of potential threat, e.g., hearing noises outside the house in the night, has activated the ventromedial prefrontal cortex. Whereas

imagining a proximal threat, e.g., being trapped inside an elevator by an attacking man, has activated the PAG (Shuhama et al., 2016).

VIII. GENETIC MANIPULATIONS OF THE 5-HT SYSTEM Using genetic manipulations to explore the biological basis of anxiety (Gottesman & Gould, 2003; Hariri & Holmes, 2006; Holmes et al., 2005; Leonardo & Hen, 2006; Lesch, 2005; Wrase, Reimold, Puls, Kienast, & Heinz, 2006), several studies have investigated the 5-HT system. They have initially focused on the deletion of genes coding for 5-HT receptors, 5-HT-related enzymes, and the 5-HT transporter. In the last decade, new studies using an optogenetic approach have also contributed to this area (Dolzani et al., 2016; Garcia-Garcia et al., 2017; Marcinkiewcz et al., 2016; Ohmura, Tanaka, Tsunematsu, Yamanaka, & Yoshioka, 2014; Spoida, Masseck, Deneris, & Herlitze, 2014). Most of these studies have shown that impairment of 5-HT1A neurotransmission increases anxiety (Heisler et al., 1998; Parks, Robinson, Sibille, Shenk, & Toth, 1998; Ramboz et al., 1998; Dai et al., 2008). This effect was proposed to depend on postsynaptic, rather than inhibitory autosomic receptors (Garcia-Garcia et al., 2017; Gross et al. 2002). Surprisingly, these animals perform normally in the Vogel conflict test, an animal model based on approacheavoidance conflict that is sensitive to anxiolytic benzodiazepines. Different from the open field, elevated zero-maze or EPM, the Vogel test involves discrete, a nonspatial threatening stimulus (Lemenhagen, Gordon, David, Hen, & Gross, 2006). Rather than being more “anxious”, it was suggested that 5-HT1A knockout mice might show a subtype of anxiety-related behavior characterized by excessive behavioral inhibition to ambiguous and complex partial cues (Klemenhagen et al., 2006). Data showing that 5-HT1A knockout mice exhibit enhanced fear conditioning to ambiguous conditioned stimuli are compatible with this proposal (Tsetsenis, Ma, Iacono, Beck, & Gross, 2007). Pharmacogenetic inhibition of the dentate gyrus blocked this latter effect, suggesting that the hippocampal formation plays a crucial role in the anxiety-related changes observed in these knockout animals (Klemenhagen et al., 2006). Neurodevelopmental factors could also influence anxious behavior in 5-HT1A knockout mice. The presence of forebrain 5-HT1A receptors between the 5th and the 21st postnatal day is necessary for the development of normal adult defensive behavior (Gross et al., 2002; Leonardo & Hen, 2006). However, using new genetic tools, Piszczek, L., Piszczek, A., Kuczmanska, Audero and Gross (2015) have recently shown that the

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IX. 5-HT-OPIOID INTERACTION

expression of 5-HT1A receptors in principal cortical neurons of adult mice is sufficient to decrease anxiety in mice. Agreeing with these findings, decreased 5-HT1A-receptor binding has been observed in PD and PTSD patients (Klemenhagen et al., 2006; van Praag, 2004). Moreover, genetically induced deficiency of 5-HT synthesis facilitated panic-like behavior in mice (Waider et al., 2017). Even if neurodevelopmental factors could help to explain part of the results observed with 5-HT1A genetic manipulations, they contrast with the original predictions from the DeakineGraeff theory (Fig. 36.3). However, studies investigating the impact of disturbance of forebrain 5-HT2 receptors on defensive-like behaviors mostly agree with their proposal. For example, disruption of 5-HT2A decreases anxiety in animal models (Cohen, 2005; Weisstaub et al., 2006), an effect that disappears after selective restoration of 5-HT2A signaling in the cortex (Weisstaub et al., 2006). Also, optogenetic stimulation of 5-HT projections from the DRN to 5-HT2C receptors in the extended amygdala (Marcinkiewcz et al., 2016) increased defensive-like behavior. It is possible, therefore, that a decrease in forebrain postsynaptic 5-HT1A-mediated neurotransmission moves the 5-HT balance toward 5-HT2C receptors, facilitating anxiety. Moreover, even if a recent study also using an optogenetic approach suggested that 5-HT neurons in the MRN, rather than those in the DRN, are associated with anxiety (Ohmura et al., 2014), interference with 5-HT2C receptors located in GABAergic interneurons in the DRN has also been shown to modulate anxiety in mice (Spoida et al., 2014). Compatible with the clinical reports in subjects carrying the short allele 5-HTT gene polymorphism, 5-HTT knockout mice present increased anxiety-like behavior (Holmes 2003a; 2003b) and a failure to cope with stress (Adamec, Burton, Blundell, Murphy, & Holmes, 2006; Hariri & Holmes, 2006). As discussed above, however, genetic changes in the 5-HT system produce neuroadaptive compensatory effects (Li, Wichems, Heils, Lesch, & Murphy, 2000, 2003, 1999; Bengel et al., 1998; Fabre et al., 2000; Gobbi, Murphy, Lesch, & Blier, 2001; Hariri & Holmes, 2006; Kim et al., 2005; Mathews et al., 2004; Mo¨ssner et al., 2004; Rioux et al., 1999). Corroborating these findings, a similar increase in anxiety-like behavior was observed in wild-type mice treated with fluoxetine between postnatal days 4 and 21 (Ansorge, Zhou, Lira, Hen, & Gingrich, 2004).

IX. 5-HT-OPIOID INTERACTION Besides the Deakin and Graeff theory, which proposes, as aforediscussed, that susceptibility to panic

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attack is due to deficient serotonergic mechanisms, another influential theory on the pathophysiology of PD, elaborated by Preter and Klein (2008), known as the amplified false suffocation alarm (FSA) theory, stresses the role of endogenous opioids. In the original FSA theory, Klein (1993) suggested that the spontaneous panic attack is due to a hypersensitive CO2 detector that triggers an inappropriate suffocation alarm, producing acute respiratory distress. Supporting this concept, Klein remarked that dyspnea, air hunger, and urge to flee are chief symptoms of panic attacks, and that PD patients present a high rate of respiratory abnormalities and diseases. The amplified FSA theory connects the regulatory function of endogenous opioids in both respiratory regulation and separation distress, which has also been associated with vulnerability to PD (Klein, 1995). As a consequence, Preter and Klein proposed that endogenous opioids prevent the behavioral and physiological manifestations of the panic attack in normal subjects. An opioid deficit would thus heighten suffocation sensitivity and separation anxiety in panic patients, resulting in abnormal vulnerability to panic attacks. To verify whether there is a connection between 5-HT and opioids in the regulation of panic-related proximal defense mechanisms, a series of experiments has been carried out in rats performing in the two already mentioned animal models of panic, the escape task in the ETM and the escape response elicited by electrical stimulation of the dPAG. In the first study, Roncon et al. (2012) showed that the antiescape (panicolytic) effect of chronic (21 days) treatment with fluoxetine was antagonized by pretreatment with the nonselective opioid receptor antagonist naloxone, both drugs given systemically. Because previously reported results indicate that the antipanic action of fluoxetine is enacted through 5-HT1A receptors localized in the dPAG (Zanoveli et al., 2010), subsequent experiments were performed using the intra-dPAG route of administration. It was thus verified that microinjection of naloxone blocked the antiescape effect of chronic, systemically administered fluoxetine. Therefore, endogenous opioids in the dPAG seem to participate in the panicolytic action of fluoxetine. Furthermore, the antiescape effect of 5-HT was blocked by previous microinjection of naloxone, indicating that 5-HT interacts with local opioids in the dPAG to dampen proximal defense and, supposedly, panic attacks. The second study by Roncon et al. (2013), undertaken with intra-dPAG microinjections only, showed that the nonselective opioid agonist morphine reduced the escape response in the ETM, an effect that was antagonized by naloxone pretreatment, as well as by previous administration of WAY-100,635. Conversely, pretreatment with naloxone attenuated the antiescape effect of

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8-OH-DPAT. In contrast, the antiescape effect of the 5-HT2A/2C receptor agonist DOI remained unchanged following naloxone. Moreover, combined administration of subeffective doses of 5-HT and morphine impaired escape. Altogether, these results point to the existence of a synergic interaction between 5-HT1A receptors and some opioid receptors, both localized postsynaptically. To determine the opioid receptor type, selective m-opioid receptor (MOR) ligands were used. The obtained results showed that the MOR antagonist CTOP blocked the antiescape effect of either CTOP or 8-OH-DPAT, and that the association of subeffective doses of the MOR agonist DAMGO and 8-OH-DPAT impaired the escape response in the ETM. Therefore, 5-HT1A receptors and MORs located in the dPAG seem to cooperate in the regulation of proximal defense/panic attacks. Similar results were obtained after electrical stimulation of the dPAG (Rangel, Zangrossi, Roncon, Graeff, & Audi, 2014). Again with the ETM, it was shown that pretreatment with CTOP antagonized the antiescape effect of chronic (21 days) fluoxetine, given intraperitoneally (i.p.), indicating MOR mediation of the panicolytic action of the antidepressant. In addition, the association of subeffective doses of DAMGO with subeffective doses of 21 day as well as 7 day i.p. injection of fluoxetine increased escape latencies, suggesting that MOR activation may both facilitate and accelerate the effects of fluoxetine (Roncon et al., 2015), a desirable goal for the pharmacotherapy of PD. The concept that 5-HT1A receptors and MORs act in cooperation is supported by electrophysiological evidence showing that the stimulation of presynaptic 5-HT1A receptors and MORs synergistically inhibited the release of GABA in the ventrolateral PAG, suggesting a cellular mechanism for analgesia (Kishimoto, Koyama, & Akaike, 2001). Using tagged 5-HT1A receptors and MORs in cell lines, and their corresponding antitag antibodies, Cussac, Rauly-Lestienne, and Heusler (2012) found that the immune-precipitated receptor complexes contained both coexpressed receptors, forming heterodimers. If occurring in dPAG neurons, this arrangement would provide a molecular basis for the hypothesis based on the presently discussed pharmacological results obtained in animal models of panic. The above-suggested interaction between 5-HT and endogenous opioids in the dPAG regulating proximal defense may lead to new strategies for the drug treatment of PD, as for example, the addition of MOR direct or indirect agonists to SSRIs in patients refractory to the latter, given alone. In addition, this concept might contribute to a better understanding of the pathophysiology of PD, since it

allows reconciliation between the opioid-deficiency and the 5-HT-deficiency hypotheses of vulnerability to panic attack. Furthermore, the influence of a likely 5-HT-opioid interaction in the brain may not be restricted to PD, since there are indications in the literature that it could apply to other psychiatric conditions, particularly depressive disorders (for discussion of these topics, see Graeff, 2012; 2017).

X. CONCLUSIONS In the last decade, studies combining the traditional behavioral, pharmacological, anatomical, and electrophysiological approaches with the new molecular genetic and functional neuroimaging techniques have helped us to further understand, particularly at the circuitry level, the complex role of 5-HT in normal and pathological anxiety. Converging evidence has shown that 5-HT: (1) modulates normal defensive responses; (2) is altered in anxiety disorders; and (3) is involved in the therapeutic effects of drugs, particularly SSRIs. However, it is clear now that, rather than playing a single and straightforward role, the modulatory effects of 5-HT on anxiety-related behavior depend on factors such as the nature of the threatening stimulus, the defensive strategies available, and the brain structures engaged. A consistent finding in this area, however, is that 5-HT inhibits defense reactions triggered by proximal threats. Facilitation of 5-HT-mediated neurotransmission in brain structures related to these responses, such as the dPAG, could help to explain the therapeutic effects of SSRIs in panic and other anxiety disorders that share some neurobiological features with panic, such as SAD (Johnson, Truitt, Fitz, Lowry, & Shekhar, 2008). Several studies have also been compatible with the Deakin and Graeff (1991) proposal that 5-HT facilitates anxiety while inhibiting panic. However, contradictory data show that the precise role of 5-HT in defensive responses associated with generalized anxiety is more complicated than initially thought. In this regard, most studies indicate that facilitation of 5-HT2C-mediated neurotransmission in different brain sites, including the BLA, increases anxiety. However, 5-HT1A receptor manipulations in the same areas suggest that these receptors inhibit, rather than facilitate, anxiety-like reactions. Despite the contradictory results, 5-HT remains the most investigated neurotransmitter in association with the anxiety (Fig. 36.1). However, even considering the great progress made in the last 55 years that followed the original proposal by Stein and coworkers (Wise et al., 1972), it is clear that much more work is needed to understand the complex full picture of its role in the neurobiology of defensive responses.

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