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1521-0081/66/4/1002–1032$25.00 http://dx.doi.org/10.1124/pr.114.009126PHARMACOLOGICAL REVIEWS Pharmacol Rev 66:1002–1032, October 2014Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: LESLIE A. MORROW

Targeting the Modulation of Neural Circuitry for theTreatment of Anxiety Disorders

David H. Farb and Marcia H. Ratner

Laboratory of Molecular Neurobiology, Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine,Boston, Massachusetts

Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1003II. Types of Anxiety Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1004III. Brain-Imaging Studies: Assessment of Brain Activation in Patients with Anxiety Disorders . . 1005

A. Functional Magnetic Resonance Imaging as a Method for Biomarker Identification. . . . . . . 1006B. Positron Emission Tomography: Advances and Caveats. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1007C. Single Photon Emission Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008D. Magnetic Resonance Spectroscopy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009E. Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1010

IV. Neural Systems and the Neurochemical Basis of Anxiety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011A. Neural Systems Underlying Anxiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1011

1. A Connectome Hypothesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10112. Optogenetics and Electrophysiology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1012

B. Type A GABA Receptor and Serotonin Receptor Structure/Roles in Anxiety . . . . . . . . . . . . . . 10131. GABA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10142. Serotonin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1016

V. Neuropsychopharmacology of Anxiety Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018A. Positive Allosteric Modulation of GABAergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1018

1. Benzodiazepines and Related Modulators.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10182. Natural Product Modulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10193. Increasing GABA Levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020

B. Modulators of Glutamatergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10201. Inhibitors of Glutamatergic Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10202. Potentiators of Glutamatergic Activity.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10213. Neurosteroids as Modulators of Glutamatergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . 1021

Abstract——Anxiety disorders are a major publichealth concern. Here, we examine the familiar area ofanxiolysis in the context of a systems-level understandingthat will hopefully lead to revealing an underlyingpharmacological connectome. The introduction ofbenzodiazepines nearly half a century ago markedlyimproved the treatment of anxiety disorders. Theseagents reduce anxiety rapidly by allosterically enhancingthe postsynaptic actions of GABA at inhibitory type AGABA receptors but side effects limit their use in chronicanxiety disorders. Selective serotonin reuptake inhibitorsand serotonin/norepinephrine reuptake inhibitors haveemerged as an effective first-line alternative treatment ofsuch anxiety disorders. However, many individuals arenot responsive and side effects can be limiting. Research

into a relatively newclass of agents knownasneurosteroidshas revealed novel modulatory sites and mechanisms ofaction that are providing insights into the pathophysiologyof certain anxiety disorders, potentially bridging the gapbetween the GABAergic and serotonergic circuitsunderlyinganxiety.However, translating thepharmacologicalactivity of compounds targeted to specific receptorsubtypes in rodent models of anxiety to effectivetherapeutics in human anxiety has not been entirelysuccessful. Since modulating any one of several broadclasses of receptor targets can produce anxiolysis, weposit that a systems-level discovery platform combinedwith an individualized medicine approach based onnoninvasive brain imaging would substantially advancethe development of more effective therapeutics.

Address correspondence to: Dr. David H. Farb, Laboratory of Molecular Neurobiology, Department of Pharmacology and ExperimentalTherapeutics, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118. E-mail: [email protected]

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C. Dopaminergic Transmission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021D. Epigenetics of Anxiolysis: Histone Deacetylase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1021E. Modulators of Serotonergic and Noradrenergic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022F. Neuroactive Steroids as Neuromodulators of Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022

VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026

I. Introduction

Anxiety is a recognized symptom of many psychiatricdisorders, including generalized anxiety disorder (GAD),social anxiety disorder (SAD), obsessive-compulsivedisorder (OCD), and post-traumatic stress disorder(PTSD). Although anxiety is a commonly reported clinicalmanifestation, it can be severely debilitating if leftuntreated. According to the Epidemiologic CatchmentArea Study, the prevalence of anxiety disorders is about7% in the adult population of the United States (Regieret al., 1990, 1998). Studies looking at the incidence ofanxiety disorders indicate that about 18% of adults in theUnited States may seek treatment for anxiety in anygiven year (Kessler et al., 2005b). Women are more likelythan men to be diagnosed with an anxiety disorder buthow much of this sex difference is due to socioeconomicfactors has not been established. That said, the need fora unified theory that will aid the development of targetedanxiolytics could not be more urgent with the emergenceof PTSD as a major global public health challenge facedafter more than a decade of war.The importance of an improved approach to treating

anxiety is highlighted by the inconsistent results seenwith the class of drugs considered to be the contempo-rary first-line treatment: selective serotonin reuptakeinhibitors (SSRIs) and serotonin and norepinephrinereuptake inhibitors (SNRIs). Although these agentshave been shown to be beneficial for the treatment ofcertain anxiety disorders, not all patients achieve anadequate clinical therapeutic response. The existenceof “nonresponders” indicates that SSRIs and SNRIs arenot the fail-safe solution to treating anxiety clinicianshave been looking for. In addition, SSRIs and SNRIsare associated with complications that can limit their

use in some patients, including delays in producingthe desired clinical reduction in anxiety or even po-tential worsening of the anxiety (Ravindran and Stein,2010; Sertraline, 2012; Venlafaxine, 2012; Fluoxetine,2013), not to mention the risk for the SSRI discontin-uation syndrome in noncompliant patient populations.Furthermore, some SSRIs (e.g., citalopram) are notsuitable for patients with heart problems (Wang andPae, 2013).

Another class of drugs, benzodiazepine (BZD) anxi-olytics, has played a central role in the pharmacologicmanagement of anxiety disorders for about 50 years.Although not as widely prescribed as in the past, thesecompounds nevertheless remain an effective alterna-tive to SSRIs. That said, the nonselective BZDs are notdevoid of side effects, which can include drowsiness,ataxia, impairment of cognition, as well as the risk ofdependence and withdrawal symptoms, including in-creased anxiety, tremors, and seizures (Noyes et al.,1988; Bennett et al., 1998; Yamawaki, 1999; Allgulanderet al., 2003). The identification of subtype-selectiveGABA receptor–positive allosteric modulators, such aszolpidem, which have proven to be effective in thetreatment of patients with insomnia while producingfewer untoward side effects than nonselective BZDs,suggested that similar drugs could be developed for anx-iety disorders. Unfortunately, although subunit-preferringcompounds are effective for insomnia, they are not yetproven to be any more beneficial for the managementof anxiety disorders than the classic BZDs, such asdiazepam (Farkas et al., 2013).

Another alternative to enhancing inhibitory neuro-transmission mediated by GABA is modulating excit-atory glutamatergic neurotransmission mediated by

ABBREVIATIONS: 5-HT, serotonin; BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; BOLD, blood oxygen level–dependent; BZD,benzodiazepine; BZDR, benzodiazepine receptor; CBT, cognitive behavioral therapy; CL 218872, 3-methyl-6-[-3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine; CNS, central nervous system; Co 3-0593, 3b-ethenyl-3a-hydroxy-5a-pregnan-20-one; DSM,Diagnostic and StatisticalManual ofMental Disorders;fMRI, functional magnetic resonance imaging; GABAAR, type AGABA receptor; GAD, generalized anxiety disorder; GPCR, G protein–coupled receptor; HPA,hypothalamic-pituitary-adrenocortical; K36, 5,7,29-trihydroxy-6,8-dimethoxyflavone; L655,708, ethyl(13aS)-7-methoxy-9-oxo-11,12,13,13a-tetrahydro-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate; MRK-409, 7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,6-difluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; MRS, magnetic resonance spectroscopy; NMDA,N-methyl-D-aspartate; OCD, obsessive-compulsive disorder; PET, positron emissiontomography; PKA, protein kinase A; PTSD, post-traumatic stress disorder; rCBF, regional cerebral blood flow; Ro 15-4513, ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1,4-benzodiazepine-3-carboxylate; Ro 16-6028, (13aS)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylic acid 1,1-dimethylethyl ester; Ro 19-8022, (R)-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)carbonyl]-2-pyrrolidine-methanol; RU 28362, 11b,17b-dihydroxy-6,21-dimethyl-17a-pregna-4,6-trien-20yn-3-one; RY80, ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate,[ethyl-3H]; SAD, social anxiety disorder; SERT, serotonin transporter; SL651498, 6-fluoro-9-methyl-2-phenyl-4-(pyrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one; SNRI, serotonin/norepinephrine reuptake inhibitor; SPECT, single photonemission tomography; SSRI, selective serotonin reuptake inhibitor; TPA023B, 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine; VPA, valproic acid; VRE, virtual reality exposure.

Targeted Modulation of Neural Circuits Implicated in Anxiolysis 1003

N-methyl-D-aspartate (NMDA) receptors. AlthoughNMDAreceptors offer a promising therapeutic target fortreating anxiety disorders, the efficacy of NMDA re-ceptor modulators developed to date also remains limited(Swanson et al., 2005; Barkus et al., 2010).Collectively, these findings reveal a clear need for an

improved understanding of the neural systems underly-ing anxiety as well as a more effective approach to de-veloping anxiolytic agents. We believe that three recentdevelopments—the use of noninvasive imaging in humansubjects to identify biomarkers implicated in neurologicdisorders, and a convergence of pharmacologic effects inhuman populations of drugs that augment GABAergictone or serotonergic tone, combined with the develop-ment of systems-based approaches to drug discovery—offer a promising path forward. Systems-based drugdiscovery differs from traditional target-based discoveryin that it utilizes an understanding of complex systems tosearch for potential therapeutic agents, as opposed toidentifying agents based solely on interactions witha single target (Desbiens and Farb, 2012).Research into neuroactive steroids recently revealed

an important role for these agents in the pathophys-iology of anxiety, both influencing and being influencedby the GABAergic, serotonergic, and other neural sys-tems associated with anxiety and demonstrates boththe complexity of the problem and why focusing ona specific target without regard for the entire neuralnetwork may actually be counterproductive. This re-view aims to consider the basic molecular mechanismsby which neurotransmission can be modulated acrossvarious neural systems implicated in anxiety disor-ders, as well as to parse out how best to approach thetreatment of specific anxiety disorders based on theneural networks involved in each, with the ultimategoal of developing a systems-based approach to thediscovery of novel anxiolytics.Our understanding of anxiolytics starts with the

recognition that at least in some cases, therapeuticsappear to effectively interact with specific receptorsto modulate apparently simple and conserved neuralsystems implicated in hypervigilance and fear in bothhumans and animals. A closer look at the problem ofanxiety in humans reveals that complex cognitiveprocesses such as executive function mediated by thefrontal lobes also play a role in how subjects respond toenvironmental stimuli and that a single drug targetmay not be the most effective approach in the treat-ment of all types of anxiety. A growing body of researchis now also shedding light on the multiple genetic,environmental, and social factors that interact in thedevelopment of anxiety disorders (Lépine, 2002; Steinet al., 2002, 2014; Pigott, 2003; Gelernter et al., 2004;Hettema et al., 2005; Porcelli et al., 2012). In addition,it is increasingly evident that a diverse array ofneurotransmitters and their respective receptors andtransporters, including those for GABA, serotonin

(5-HT), and NMDA, play critical roles in the develop-ment of anxiety disorders (Swanson et al., 2005; Barkuset al., 2010; Graeff and Zangrossi, 2010). An increasedunderstanding of the neural systems underlying a path-ologic response to environmental stimuli that do notinduce anxiety in healthy subjects therefore begins withthe development of “connectivity maps” that in turn maylead to more individualized approaches to the pharma-cological management of various anxiety disorders.

II. Types of Anxiety Disorders

A comprehensive review of anxiety disorders must beginwith a definition of the problem. Anxiety per se is generallyconsidered to be synonymous with acute nervousness,fretfulness, and apprehension. Although the words fear,anxiety, and worry can sometimes be used interchange-ably, these terms generally represent distinct emotionalresponses that can be distinguished clinically by intensityand duration. Among healthy individuals, the magnitudeof this type of emotional response is proportional to thespecificity and intensity of the stimulus. The specificity ofthe stimulus is determined by its relevance to a particularindividual based on his or her current circumstancecombined with past life experience with that particularstimulus (i.e., individual sensitivity). For example, expo-sure to a stimulus that has previously been paired with anadverse life event will produce a different response thanwill exposure to a novel stimulus. Likewise, fear, fright,flight, and panic are more intense and acute reactions thantrepidation, apprehension, and worry and normally occurin close chronological and physical relation to an exposureto the appropriate stimuli. For example, an individual mayfeel slightly nervous and fretful while waiting to meeta blind date, or may be apprehensive about his or herability to perform a novel task in public, but fearful ofencountering an armed robber while walking down a darkalley alone at night. All of these responses are typicallybrief and resolve spontaneously with termination of ex-posure to the respective stimuli.

Fear, an intense fight-or-flight response that occursin reaction to a real or perceived threat, is a survivalresponse that is in contrast with panic disorder, whichis a fight-or-flight response in the absence of any identifiabledanger, therefore appearing to be a pathologic condition.Because these symptoms occur spontaneously and withoutwarning, persons with panic disorder will typically avoidsituations that their past experience suggests can triggerthe onset of an attack. Patients who have difficultyassigning a specific trigger for their attacks may becomesocially withdrawn.

Worry is synonymous with a more persistent concernor preoccupation with the unknown outcome of futurelife events. Worrying can be controlled so as to permitnormal functioning in social and occupational settings.By contrast, worrying such as that which occurs amongpatients with GAD is associated with persistent concern

1004 Farb and Ratner

about routine life experiences, such as job performance orpersonal relationships, and is severe enough to interferewith the afflicted individual’s ability to concentrate andperform activities of daily living.The clinical anxiety disorders recognized in the fifth

edition of the Diagnostic and Statistical Manual ofMental Disorders (DSM-V), include the following: GAD,OCD, panic disorder, acute and chronic PTSD, and thevarious phobias, including agoraphobia, social phobia,and specific phobia (e.g., fear of flying) (American Psychi-atric Association, 2013). Each of these recognized clinicalanxiety disorders is unique with respect to the intensityand duration of the anxiety experienced as well as the typeof stimuli that can induce it.A systems-level approach to the development of

anxiolytic agents starts with a recognition of the rangeof anxiety disorders from which patients can suffer,including GAD, SAD, panic disorder, various phobias(e.g., fear of flying), and medication-induced anxietydisorder (DSM-V; American Psychiatric Association,2013). In addition, disorders that fall under their owncategory in the DSM-V, such as acute and chronic PTSD,include clinical manifestations, such as hypervigilance,exaggerated startle response, and fear as a component oftheir diagnostic criteria. Likewise, OCD includes fear ofsome dreaded event or situation among its diagnosticcriteria. Each of these disorders is unique with respect tothe intensity and duration of the symptoms experiencedby the patient as well as the type of stimuli that caninduce these. For example, persons with panic disorderexperience recurrent, discrete, and typically brief periodsof intense anxiety referred to as “uncued panic attacks”in the absence of any identifiable source of danger. Thisanxiety is associated with somatic symptoms, such asshortness of breath, chest pain, lightheadedness, andsweating. Persons with panic disorder often have per-sistent concerns about the causes or consequences ofhaving a panic attack. By contrast, “cued” anxiety attacksand phobias are defined as those in which the suffererexperiences debilitating symptoms in response to aclearly defined object or situation. In the cases ofphobias, exposure to the phobic stimulus produces animmediate onset of increased anxiety that is severeenough to interfere with the individual’s ability to functionnormally in social or occupational settings even when theperson recognizes that his or her own anxiety is excessiveor unreasonable.The onset of an anxiety disorder may be insidious or

may follow an identifiable life event. For example,acute stress disorder is characterized by symptomsthat occur during the first month after exposure to anextremely stressful event that evokes intense fear and/ora sense of helplessness, such as being in an automobileaccident or witnessing a murder. Symptoms of acutestress disorder include increased arousal, avoidance ofplaces and events that remind one of the traumaticevent, and recurrent intrusive memories or dreams

about the event. Although severe enough to interferewith activities of daily living, these symptoms typicallyresolve spontaneously within about 4 weeks after theevent. By contrast, chronic PTSD is characterized bysimilar symptoms that persist for more than 1 monthand, in many cases, can last for years after exposure toa traumatic event. These types of anxiety disordersrespond well to treatments that combine virtual realityexposure (VRE) therapy with pharmacologic interven-tions (Difede et al., 2014).

Unlike incident-evoked anxiety disorders, GAD ischaracterized by at least 6 months of persistent andexcessive apprehension and worry in response to aplethora of different stimuli, including such routine lifeevents as job performance, health of family members,or simply being on time for appointments. The onset ofGAD is typically insidious and is often not associatedwith a readily identifiable precipitating event. Individ-uals with GAD can be differentiated from those withnonpathological anxiety by two main criteria: 1) theyfind it difficult to control their worrying, and 2) thisperseverative thought process interferes with theirability to function in social and occupational settings.

The clinical manifestations of OCD are unique amongthe disorders that include fear and anxiety as a clinicalmanifestation in that the fear is about what will happenif the individual does not perform recurrent thoughts,impulses, or compulsions (e.g., repetitive hand washing)that the individual recognizes to be unreasonable andunrelated to real-life problems but nevertheless findsextremely difficult to stop. These symptoms cause distress,and thus, the affected individual may avoid situationsthat promote the obsession. For example, a person whoobsesses about personal contamination may not leavethe house to prevent placing himself in a situationwhere he might have to shake hands.

III. Brain-Imaging Studies: Assessment of BrainActivation in Patients with Anxiety Disorders

Although a range of clinical diagnostic criteria andassessment tools (e.g., Hamilton Anxiety Rating Scalesand the Minnesota Multiphasic Personality Inventory)have historically been used in the differential diagnosisof anxiety disorders, recent research has also shown thatvarious neuroimaging technologies can be employed toidentify common and unique brain regions associatedwith the different disorders. Neuroimaging studies canbe used to measure differences in regional cerebral bloodflow (rCBF) and for identifying significant changes inbinding to specific receptor subtypes and changes inuptake by transporters implicated in these disorders.Although it is premature to say that “silver bullets”targeting specific type A GABA receptor (GABAAR)subtypes or serotonin transporters (SERTs) withinparticular brain regions are on the horizon, it seemslikely that critical advances in brain imaging and


subunit subtype analysis are improving the chancesfor optimizing therapeutic approaches to patientswith anxiety disorders.

A. Functional Magnetic Resonance Imaging asa Method for Biomarker Identification

It is natural to wonder whether serotonergic neuronsin the dorsal and median raphe nuclei that project tocorticolimbic circuits may be controlled by GABAergic,serotonergic, and neurosteroid modulators. A noninva-sive approach for assessing the elements of a potentialanxiety connectome is to evaluate the pharmacologic ef-fects of anxiolytics on specific brain regions by determiningchanges in rCBF associated with anxiety-inducingstimuli—these change in response to different pharmaco-logic interventions. Blood oxygen level–dependent (BOLD)functional magnetic resonance imaging (fMRI) can alsobe used to differentiate between types of anxiety, suchas between anxious apprehension (which involves verbalrumination and worry) and anxious arousal (which in-volves intense fear or panic or both) (Engels et al., 2007).An fMRI study of healthy subjects with no history

of psychiatric illness or treatment revealed that thosewho also tested positive for a regulatory variant(5-HTTLPR) in the SERT gene (SLC6A4), which waspreviously associated with reduced transcription of thetransporter, had greater amygdaloid reactivity in re-sponse to presentation of fearful and angry facesindependent of patient sex. These findings suggestthat the amygdala can be biased for reactivity basedon genetics and that this may represent a susceptibilityfactor for a pathologic response to stressful life ex-periences (Hariri et al., 2005). The observation thatSSRIs promote downregulation of SERTs (Mirza et al.,2007) also implicates dysregulation of serotoninergicneurotransmission in etiology of certain anxiety disor-ders. This polymorphism has also been associated withreduced responsiveness to SSRI treatment (Zanardiet al., 2001).The potential for an anxiolytic effect of 5-HT via

attenuation of amygdala activation was shown in astudy of healthy subjects treated with the SSRI citalopramfor 7 days. Compared with control subjects, the fMRIs ofindividuals treated with citalopram showed a bilateralreduction in activation of the amygdala, hippocampus,andmedial prefrontal cortex in response to presentation ofthreatening stimuli (Harmer et al., 2006).Evaluation of fMRI responses to the presentation of

pleasant or unpleasant words while monitoring leftversus right hemisphere activity indicates that the leftside (inferior frontal gyrus) is more active in patientswith anxious apprehension, consistent with the factthat this type of anxiety is more verbally mediated.These findings may seem to be discordant with datasupporting a left-sided bias in response to pleasantemotion, but positive emotional content preferentiallyactivates different regions of the left hemisphere: the

left frontal region (dorsolateral prefrontal cortex) (Engelset al., 2007).

Results from fMRI imaging studies support the clinicalimpression that GAD patients overreact to both pathology-specific and nonspecific cues and that treatment-inducedreduction of anxiety attenuates the response to both typesof cues (Hoehn-Saric et al., 2004). In a small cohort of sixpatients, subjects were initially studied using fMRIwhile each listened to verbal descriptions of a personalworry or a neutral statement before treatment withcitalopram and again after 7 weeks of treatment. ReducedBOLD responses to worry statements were observedin prefrontal regions, the striatum, and insula andparalimbic regions. In addition, contrasts before and aftertreatment revealed reductions in the differential responsethat existed between worry and neutral statements.Overall reduction of the BOLD response was mostprominent during neutral statements, particularly inthe left hemisphere. These findings are consistent withthe suggestion that frontolimbic structural connectivityunderlies the preservative thoughts as well as theemotional disturbances seen in patients with GAD(Tromp et al., 2012).

An fMRI study of 21 patients (8 males and 13 females)with generalized social phobia, characterized by anexaggerated and pervasive fear and avoidance ofscrutiny by others, suggested that activation of theamygdala in response to presentation of angry andfearful faces was attenuated relative to baseline after12 weeks of treatment with the SSRI sertraline (Phanet al., 2013). All patients received a stable dose (100–150mg/day) at the time of testing and had been titratedbased on their clinical response to treatment. At testing,14 of 21 patients were deemed to be responsive tosertraline treatment, whereas 7 were considered non-responders. All treated patients (clinical responders andnonresponders) were included in the study but bloodlevels of sertraline were not measured. A post hocanalysis looking at differences between responders andnonresponders indicated that the attenuation frompretreatment to post-treatment in amygdala reactivityto fearful faces was significant in responders (P = 0.01)but not in nonresponders (P = 0.22), as was attenuationin ventromedial prefrontal cortex reactivity to angryfaces (responders, P = 0.02; nonresponders, P = 0.29).Activation of the amygdala in all of the patients beforeand after treatment was also compared with that of19 healthy controls (10 males and 9 females). Consistentwith reports from other studies, activation of the amygdalain response to fearful faces before treatment was found tobe greater in the anxiety patients than in control patients;importantly, this difference was abolished by treatmentwith sertraline. By contrast, activation of the ventromedialprefrontal cortex in response to angry faces beforetreatment was found to be significantly lower in anxietypatients than in control patients, and this differencewas also abolished by sertraline treatment.


The effects of chronic paroxetine treatment (20 mg/day)versus placebo have also been measured in patients withSAD using fMRI, along with performance of several tasksincluding a public-speaking task, public exposition of arecorded performance task, and an emotional face-processing task (Giménez et al., 2014). Paroxetinetreatment was associated with reduced activation inthe insula, thalamus, and anterior cingulate cortex atrest and during performance of the public expositionof a recorded performance task. By contrast, duringthe emotional face-processing task, paroxetine treat-ment was associated with an increase in activation ofthe right amygdala and the insula bilaterally.Collectively, these findings point to a role for corticolimbic

circuits, including the amygdala and medial prefrontalcortex in SAD, and suggest that reductions in the level ofactivation in these brain regions may prove to be a usefulbiologic marker for the efficacy of novel anxiolytics.

B. Positron Emission Tomography: Advancesand Caveats

rCBF can also be measured using positron emissiontomography (PET) scans. Studies of rCBF using PETsuggest that paralimbic structures together with theright inferior frontal cortex and subcortical nuclei mediatesymptoms across three different anxiety disorders: OCD,simple phobia, and PTSD (Rauch et al., 1997). Oxygen15–labeled carbon dioxide was used as a tracer to measurerCBF in 23 right-handed adult subjects (8 men and 15women) meeting criteria for one of these three disordersand relative rCBF measured in the context of subjects’specific symptom provocation paradigms (e.g., exposure ofa patient with OCD to a “contaminated” object). Overall,analysis of pooled imaging data revealed activation in theright inferior frontal cortex and right posterior medialorbitofrontal cortex, and bilateral activation in the insula,lentiform nuclei, and brainstem during symptomaticversus control conditions.The limitations of measuring changes in rCBF as a

measure of SSRI efficacy in the treatment of anxietydisorders is hampered by a deficiency of knowledgeabout the underlying neural circuitry, or functionalconnectome, that gives rise to feelings of anxiety andpharmacologically induced anxiolysis. For example, whenPET techniques were used to assess the effects of SSRItreatment in patients with SAD, the SSRI-treated subjectsshowed a selective deactivation of the left lateralamygdala not associated with a reduction in anxietystatus. By contrast, the left basomedial/basolateraland right ventrolateral regions of the amygdala weredeactivated in both SSRI-treated and placebo patientswho showed improvement in anxiety symptoms (Fariaet al., 2012) (Fig. 1). This latter deactivation was notobserved in nonresponders (i.e., subjects who showed noreduction in anxiety scores) irrespective of treatmentgroup, suggesting that this change was mediated by areduction in anxiety unrelated to SSRI treatment. Thus,

SSRI treatment was no more effective than placebo fortreating SAD, perhaps due to the difficulty of separatinganxiolysis from a placebo effect in conjunction witha small sample size. Unfortunately, the subjects werenot tested for the 5-HTTLPR polymorphism in the 5-HTtransporter gene (associated with reduced responsive-ness to SSRI treatment; Zanardi et al., 2001), furtherconfounding interpretation of the results.

An advantage of PET versus fMRI as a clinical re-search tool can be exemplified by a study that observeddecreased flumazenil binding in specific brain regions inmedication-free persons diagnosed with panic disorder(Malizia et al., 1998). Global reduction in BZD site bindingthroughout the brain were found in patients with panicdisorder (n = 7) compared with controls (n = 8) (Fig. 2A).In addition, several loci thought to be essential in theregulation of anxiety (right orbitofrontal cortex and rightinsula) were found to have the largest regional decreasein binding. These results are consistent with previousclinical psychopharmacologic evidence of involvementof the GABAAR (Hasler et al., 2008) using PET with[11C]flumazenil in patients with panic disorder comparedwith healthy controls, which showed reduced bindingpotential across regions of the frontal, temporal, andparietal cortices, particularly the dorsal anterolateralprefrontal cortex.

Dynamic three-dimensional PET scans show that theBZD-GABAAR may play a role in the pathophysiologyof PTSD, a finding that is consistent with previousanimal and clinical pharmacological studies (Geuze et al.,2008). In this study, radiolabeled [11C]flumazenil was

Fig. 1. PET scans of subjects with SAD who were treated with either anSSRI or placebo for a minimum of 6 weeks does not identify a brain regionmodulated by drug and associated with anxiolysis. Scans were madewhile subjects performed a public-speaking task. Subjects were dividedinto two groups based on clinical response to treatment (respondersversus nonresponders). An attenuation of rCBF that was independent oftreatment was observed in the left basomedial/basolateral and rightventrolateral amygdala of responders. Red indicates the site of reducedactivation irrespective of treatment (placebo versus SSRI) seen in responders;blue indicates a purely pharmacodynamic nonaxiolytic effect on rCBF thatwas seen in both responders and nonresponders treated with an SSRI;and yellow indicates an effect related to repeated testing. Reprinted withpermission from Faria et al. (2012). L, left; R, right.


used as a tracer in nine drug-naïve male veterans withdeployment-related PTSD and sevenmale veterans withoutPTSD. After a scan of 60 minutes, reduced [11C]flumazenilbinding was observed throughout the cortex, hippocampus,and thalamus of the PTSD patients (Fig. 2B).PET techniques have also proven valuable in evaluating

variations in the distribution of specific subtypes of theGABAAR within the human brain and the roles of thesesubunits in mediating different functions. Competitionstudies in vivo in the rat revealed that [11C]Ro 15-4513(ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-1,4-benzodiazepine-3-carboxylate) uptake was reduced tononspecific levels only by drugs that have affinity forthe a5 subtype, such as flunitrazepam, RY80 (ethyl-8-acetylene-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate,[ethyl-3H]), Ro 15-4513,and L655,708 [ethyl(13aS)-7-methoxy-9-oxo-11,12,13,13a-tetrahydro-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylate], but not by the a1-selective agonist zolpidem.[11C]Ro 15-4513 uptake was relatively greater in limbicareas compared with [11C]flumazenil, but lower in theoccipital cortex and cerebellum (Lingford-Hughes et al.,2002). The authors concluded that [11C]Ro 15-4513 PETlabels the GABAAR containing the a5 subunit in vivo inlimbic structures and can be used to further explore thefunctional role of this subunit in humans. This techniquewas used to characterize BZD recognition site occupancyin the human brain associated with administrationof several a2-selective compounds, such as TPA023B[7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine] andMRK-409 [7-cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2,6-difluorophenyl)-1,2,4-triazolo[4,

3-b]pyridazine] (Fig. 3) (Atack et al., 2006b, 2011a,b; VanLaere et al., 2008).

C. Single Photon Emission Computed Tomography

A single photon emission computed tomography (SPECT)study using 123I-labeled 2b-carbomethoxy-3b-(4-iodophenyl)-N-(3-fluoropropyl)-nortropane revealed an increase indopamine transporter binding in the striatum of SADsubjects treated with escitalopram for 12 weeks(Warwick et al., 2012). These subjects also showed adecrease in anxiety scale scores associated with treat-ment, but the scores were not correlated with changesin dopamine transporter binding. The scores remainedrelatively high after therapy, suggesting that treatmentmay not have been completely successful for all par-ticipants. These subjects were also not stratified asresponders versus nonresponders, thus limiting theinterpretation of the data.

As revealed by SPECT scans using [123I]iomazenil,patients with panic disorder exhibit decreased benzo-diazepine receptor (BZDR) binding in the left hippo-campus and precuneus relative to controls (Bremneret al., 2000). Further comparisons of patients withpanic disorder who either did or did not have a panicattack at the time of the scan revealed a decrease inBZDR binding in the prefrontal cortex among patientswho had a panic attack. These findings suggest thatBZDR function in the prefrontal cortex appears to beinvolved in changes in state-related panic anxiety.

These findings also suggest a role for brain regionsimplicated in episodic memory function, such as theprecuneus. This in turn may be amenable to cognitivebehavioral therapy (CBT) and targeting by pharmacologic

Fig. 2. The BZD binding site on the GABAAR plays a role in the pathophysiology of PTSD. (A) Results of PET studies using radiolabeled[11C]flumazenil to study BZDR kinetics in vivo. Axial images of middle brain (12 mm above the anterior commissure–posterior commissure plane)showing median volume of distribution map for controls (left) and patients with panic disorder (right). Note the generalized decrease in volume ofdistribution affecting the thalami and the heads of the caudate nucleus among patients with panic disorder. Reprinted with permission from Maliziaet al. (1998). (B) PET scan images highlighting those areas that showed decreased binding of [11C]flumazenil in drug-naïve subjects with PTSD (n = 9)compared with controls (n = 7). Reduced binding was observed in the cortex, hippocampus, and thalamus. Reprinted with permission from Geuze et al.(2008). CN, caudate nucleus; FC, frontal cortex; Ins, insula; L, left; OC, occipital cortex; R, right; TC, temporal cortex; Th, thalamus.


interventions that can enhance extinction of salientmemories that trigger panic attacks. The results indicatethat the neuronal circuits implicated in panic attacks(i.e., the precuneus) are distinctly different from thoseimplicated in GAD (i.e., more involvement of the frontallobe).In another study of depressed patients with or without

panic disorder (N = 18), radiolabeled iomazenil SPECTscans demonstrated that those patients with panic disorderhad a significant decrease (P, 0.05) in the regional activityindex in the lateral inferior temporal lobes (right and left),medial inferior temporal lobes (left), and inferior frontallobes (right and left) after 2 hours (Kaschka et al., 1995).The findings may be due to either regional blood flowdifferences or BZDR effects, but the former hypothesis isconfirmed to some extent by similar findings in the scansacquired after 10 minutes. Only hypoactivity in the leftlateral temporal region seemed to be independent of thereduced blood flow seen in panic disorders.A SPECT study in patients with social phobia found

that citalopram therapy decreased activity in the leftcingulum, the anterior and lateral part of the left temporalcortex, and the anterior, lateral, and posterior part of theleft midfrontal cortex (Van der Linden et al., 2000).This broad engagement of cortical areas in anxiety

disorders and anxiolysis may be the consequence of aprimary abnormality or may be a cause of the disorders.

In some ways, similar to the phenomenon of “kineticambiguity” common to the study of enzyme mechanismsand bio-organic chemistry, “systems ambiguity” presentsa conundrum for the pharmacologist seeking to translatetarget-based pharmacology into pharmacotherapeuticeffects based on a mechanistic understanding of ananxiolysis connectome.

D. Magnetic Resonance Spectroscopy

Magnetic resonance spectroscopy (MRS) allows forthe noninvasive monitoring of levels of GABA as wellas other neurochemicals in the human brain (Goddardet al., 2001; Strawn et al., 2013; Zwanzger et al., 2013).MRS has been used to measure GABA levels in pa-tients with PTSD, social anxiety, and panic disorder(Ham et al., 2007; Pollack et al., 2008; Rosso et al.,2014). A study of 14 unmedicated patients with panicdisorder and 14 healthy control subjects matched forage and sex revealed a 22% mean reduction of GABA inthe occipital lobe of 12 of 14 subjects compared withcontrols. There were, however, no significant correla-tions between GABA levels in the occipital lobe andany measures of illness or state anxiety (Goddard et al.,2004). In a study of subjects with PTSD, MRS mea-surement of GABA levels in the insula revealed thatpatients had 30% less GABA than healthy controls. Thisfinding could not be accounted for by age or sex. This

Fig. 3. PET studies comparing TPA023B identify BZD binding sites in the human brain. (A) Comparison of the structures of TPA023B (Atack et al.,2011a), TPA023 (Atack et al., 2006b), and MRK-409 (Atack et al., 2011b). (B) Pseudocolor images on the left show the inhibition of [11C]flumazenilbinding produced by a single 1.5-mg oral dose of TPA023B in PET scans performed 1 hour before and 5 and 24 hours after dosing. TPA023B hasprolonged occupancy at the BZD binding site of GABAARs in the human brain. The right graph shows occupancy plotted as a function of thecorresponding plasma TPA023B concentrations. Modified with permission from Van Laere et al. (2008).


difference could also not be accounted for by the differencein the percent gray matter versus white matter propor-tions within the voxel of interest. In addition there was asignificant negative correlation between State-TraitAnxiety Inventory scores and GABA levels (Rosso et al.,2014) (Fig. 4).MRS can also be used to study acute drug-induced

changes in brain glutamate levels. Experimental pharma-cologic induction of panic has been used to elucidate theneurobiological correlates of panic attacks. Cholecystokinin-tetrapeptide-4–induced panic attack was studied usingMRS and was found to be associated with an increase inglutamate plus glutamine/creatine ratios in the anteriorcingulate cortex (Zwanzger et al., 2013). This suggests thata chemically induced (i.e., by cholecystokinin-tetrapeptide-4)panic attack alters the glutamatergic system in the anteriorcingulate cortex and may be involved in the pathogenesisof panic attack. Given the activation of precuneus in salientepisodic memory–induced panic attack, it is tempting tospeculate that a targetable downstream network involvingthe anterior cingulate cortex susceptible to glutamatergicpharmacotherapy could be an effective approach towardtreating panic attack.By contrast, in patients with PTSD, positive modulators

of glutamatergic transmission (e.g., D-cycloserine and

acamprosate; see section V.B) have been used in combi-nation with behavioral therapy to extinguish salientmemories associated with PTSD.

E. Overview

Taken as a whole, brain-imaging studies implicatethe amygdala, prefrontal, insular, limbic, paralimbic,and occipital cortices as well as the basal ganglia moreoften than other regions in pathologic anxiety. Giventhe role that the insula plays in integrating sensory,emotional, and cognitive information, it is certainly notsurprising to find changes in the level of activation inthis brain region. Given that the insula spans from theprefrontal cortex to the posterior parietotemporal lobesand thereby creates extensive connections between thelateral prefrontal cortex, ventromedial prefrontal cortex,orbitofrontal cortex, cingulate, amygdala, bed nucleusof the stria terminalis (BNST), and ventral striatum, itseems reasonable to think of the insula as a node in theanxiety connectome.

These neuroimaging findings also reveal some evi-dence of lateralization and localization based on how theanxiety-inducing stimulus is presented (e.g., verballyversus visually), suggesting a path forward for differen-tiating among types of anxiety and tailoring treatment

Fig. 4. MRS studies showing that brain GABA levels in the right insula are lower in patients with PTSD. (A) T1-weighted images depicting voxelplacement as well as difference edited and 68-millisecond spectra from the ACC (A and B) and insula (C and D) of a representative subject. (B)Associations of GABA/Cr in right anterior insula with state anxiety (A) and trait anxiety (B) in the whole sample. Circles correspond to PTSD patients(asterisk indicates the single medicated patient) and triangles are control subjects. All fitted metabolite areas were normalized to total creatine(creatine and phosphocreatine combined). ACC, anterior cingulate cortex; Cho, choline; Cr, creatine; NAA, N-acetylaspartate. Reprinted withpermission from Rosso et al. (2014).


appropriately based on systems-level analysis of data.For example, a meta-analysis of neuroimaging studiesrelated to emotional processing in SAD, PTSD, andspecific phobia suggests that common brain regions(hyperactivation of the amygdala and insula) play a rolein these anxiety disorders that can be triggered byspecific stimuli. In addition, the results revealed thathypoactivation in the dorsal and rostral anterior cingulatecortices and the ventromedial prefrontal cortex may play arole in the dysregulation of emotions associated withPTSD (Etkin and Wager, 2007).Human imaging studies in which both PET and

fMRI are used to study biomarkers in the same cohortmay be the most suitable for identifying relationshipsbetween individual variability in receptor binding andbrain region activation in response to treatments (althoughstudies employing one technology are also useful). Thisdual-technology approach was used by Fisher et al. (2006)to study 5-HT1A autoreceptor binding in relation toamygdala activation and by Rhodes et al. (2007) tostudy the association between 5-HT transporter bindingand amygdala activation. Kobiella et al. (2011) took thisapproach one step further and studied 5-HT transporterpolymorphisms in relation to amygdala region activation,further demonstrating the unique power of multimodalneuroimaging studies.Nonhuman animal imaging using experimental mod-

els of anxiety are also helping to parse out inferencesconcerning the possible differences and similaritiesunderlying various anxiety disorders. One MRS studyusing a chronic stress–induced depression model revealeda reduction in brain levels of N-acetyl-aspartate, totalcreatine, and choline-containing compounds in tree shrewsexposed to psychosocial stress. In this study, the voxelof interest was the forebrain including parasagittal neo-cortex, subjacent white matter, and portions of subcorticalforebrain structures, including striatum (Czéh et al.,2001). Postmortem analysis showed reduced cell pro-liferation in the dentate gyrus and a decreased GluR2expression in the prefrontal cortex. Concomitant admin-istration of a novel compound combining partial dopamineD2 receptor agonism with SSRI activity prevented thesechanges, suggesting that combining dopamine D2 receptoragonism with SSRI activity may serve as a novel treat-ment of those anxiety disorders presenting with comorbiddepression (Michael-Titus et al., 2008). In another studyusing fluorodeoxyglucose-PET, three specific character-istics of the anxious phenotype identified in rhesusmonkey (hypothalamic-pituitary-adrenal activity, freezingbehavior, and expressive vocalizations) were associatedwith a common elevation of activity in the central nucleusof the amygdala and the anterior hippocampus (Shackmanet al., 2013).However, can these types of imaging data produce

useful information for real-life therapy in humanpatients with a range of anxiety disorders? In patientswith SAD, fMRI was used to measure brain activation

prior to intervention with CBT and showed that changesin regional activation in response to angry versus neutralfaces or emotional versus neutral scenes are an effectivepredictor of response to CBT (Doehrmann et al., 2013).

IV. Neural Systems and the Neurochemical Basisof Anxiety

In the following sections, we explore the neurochemicalcomponents of the brain, placing these findings in thecontext of a systems-level approach to therapeutics basedon the real-time differences in rCBF demonstrated byfunctional neuroimaging studies and changes in brainlevels of GABA and glutamate revealed by MRS.

A. Neural Systems Underlying Anxiety

1. A Connectome Hypothesis. Anxiety is a complexemotional response that involves multiple neurotrans-mitters acting at various receptors within the limbicsystem (i.e., hippocampus, parahippocampal gyrus, cin-gulate gyrus, hypothalamus, and amygdala) and withinthe paralimbic regions of the cerebral cortex such as theorbital and medial prefrontal cortex (Rauch et al., 1997;Simpson et al., 2000; Pape and Stork, 2003; Phillipset al., 2003; Sah et al., 2003; Yilmazer-Hanke et al., 2003;Zwanzger et al., 2003; Kang-Park et al., 2004; Phan et al.,2004). From a neural systems perspective, fear andanxiety seem to be controlled by two major circuits: thoseinvolved in our most primitive innate responses to simpleand overt threatening stimuli, and those involved inmoderating responses to more complex situations. Theprefrontal cortex appears to be the brain region mostoften implicated in the latter.

The prefrontal cortex in particular is implicated inattenuation of responses to anxiety-inducing stimuli.Lesions to the ventrolateral prefrontal cortex havebeen associated with increased anxiety in nonhumanprimates (Agustín-Pavón et al., 2012). It has beenproposed that the amygdala is hyper-responsive tothreatening stimuli, whereas the response of theventromedial prefrontal cortex is a blunted in patientswith anxiety disorders, resulting in inadequate regu-lation of their responses to anxiety-inducing stimuli(Kent and Rauch, 2003; Rauch et al., 2003). Thishypothesis is supported in part by functional neuro-imaging studies showing that individuals with GADhave an attenuated discriminatory response pattern inthe ventromedial prefrontal cortex when presented withthreatening versus nonthreatening stimuli (Greenberget al., 2013).

The amygdala, which receives afferent inputs fromthe sensory cortex and sends its efferent projections tothe hypothalamus, periaqueductal gray matter, locusceruleus, raphe nuclei, and ventral tegmental area(Sah et al., 2003), appears to be primarily involved inthe encoding and consolidation of emotionally chargedmemories (Isenberg et al., 1999; Roozendaal et al.,


2001; Sah et al., 2003; Pelletier and Paré, 2004). Thebasolateral amygdala (BLA), which is composed of thelateral, basomedial, and basolateral nuclei, plays acentral role in this process and sends projections to themedial temporal lobe and nucleus accumbens andseptal nuclei via the stria terminalis (Roozendaalet al., 2001; Pelletier and Paré, 2004). Lesions of theBLA or its major efferent pathway, the stria terminalis,interfere with the encoding and consolidation of mem-ories induced by emotional arousal (McGaugh, 2000;Roozendaal et al., 2001). In addition, the effects ofglucocorticoids such as RU 28362 (11b,17b-dihydroxy-6,21-dimethyl-17a-pregna-4,6-trien-20yn-3-one) on memoryconsolidation are dependent on an intact BLA nucleus andits efferent fibers that run through the stria terminalis(Roozendaal et al., 2001).The subunit composition and pharmacological prop-

erties of GABAARs vary widely between and withinbrain regions. For example, receptors containing the a2

and a5 subunits, which show pharmacologic profilessimilar to the classic BZD II or type 2 receptors, arefound in higher concentrations in the amygdala andhippocampus, whereas receptors containing a6 subunits,which are insensitive to BZD anxiolytics, are localizedto the cerebellum (Wieland et al., 1992; McKernan andWhiting, 1996; Whiting et al., 1999).The neuroanatomic distribution of 5-HT receptors is

complex, as would be expected based on the pharma-cological effects of receptor agonists, antagonists, andSSRIs. The limbic system is critical for the generationof motivational and affective states, with the limbicforebrain containing high levels of 5-HT (an evolutionarilyancient neurotransmitter that has functional orthologreceptors even in the Drosophila fruit fly) (Nichols et al.,2002). The 5-HT fibers that project into the forebrainoriginate for the most part from the median raphe nucleusand the dorsal raphe nucleus. These projections areheterogeneous in terms of morphology and electro-physiology, representing yet another level of intricacythat could influence treatment outcomes (Hensler, 2006).The hippocampus is well known to be involved in

the formation of new memories. Neurogenesis in thehippocampal dentate gyrus has been implicated in theformation of both spatial memory and anxiety disor-ders, as well as in the action of SSRIs. The temporalbehavior of dentate gyrus granule cells with respect totheta rhythm activity is different between rats withnormal and impaired levels of neurogenesis, suggest-ing that immature neurons could distinctly affect thetemporal dynamics of hippocampal encoding (Rangelet al., 2013). Serotonergic antidepressants can reversethe established state of neuronal maturation in adulthippocampal cells, and upregulation of 5-HT4 receptor–mediated signaling may play a role in this process(Kobayashi et al., 2010). The role of dentate granulecell hypoactivity in anxiety and in the mechanism ofaction of SSRIs was recently suggested based on the

observation that challenge-induced hypoactivation ofdentate gyrus neurons in mice with high trait anxietyis normalized by fluoxetine treatment (Sah et al., 2012).

Differences in the intrinsic physiology and synapticinhibition between the mature and immature cell pop-ulations could separate the activity of differently agedneurons in a temporal coding regimen, influencingdownstream CA3 neuron integration of informationconveyed by young and mature granule cells. In linewith this hypothesis, fluoxetine treatment induces activesomatic membrane properties in a manner reflective ofimmature granule cells (Kobayashi et al., 2010). Thesechanges do not seem to be explained by an increase innewly minted immature neurons and may be character-ized as “dematuration” of mature granule cells. Theseobservations suggest that normalization of hippocampalhypoactivity may serve as a neurobiological marker ofanxiolytic effectiveness and successful remission.

2. Optogenetics andElectrophysiology. Optogenetic tech-niques have been used to further elucidate the role of theamygdala in anxiety. Tye et al. (2011) used viral trans-fections to study the role of BLA–centrolateral nucleuspathway in the expression of anxiety. Illumination ofchannel rhodopsins expressed in BLA terminals located atthe central nucleus of the amygdala was shown to induce anacute reversible anxiolytic effect. Selective illumination ofspecific BLA terminals produces a behavioral responseopposite that induced by activation of all glutamatergicBLA terminals suggesting that multiple subpopulationsor projections of BLA neurons can act in opposition (e.g.,direct excitation of centromedial nucleus along with feed-forward inhibition of centromedial nucleus) (Fig. 5).

Subsequent studies demonstrated that bilateraloptogenetic inhibition of BLA axon terminals in theventral hippocampus reduces anxiety-related behaviors inmice, suggesting that BLA inputs to the ventral hippo-campus may play a role in modulating basal levels ofanxiety (Felix-Ortiz et al., 2013). A reduction of hippo-campal theta activity has also been implicated in theanxiolysis induced by various classes of anxiolytics, in-cluding buspirone (McNaughton et al., 2007; Chee et al.,2014). A reduction of hippocampal theta activity has alsobeen implicated in the anxiolysis induced by variousclasses of anxiolytics, including buspirone (McNaughtonet al., 2007; Chee et al., 2014). McNaughton et al. (2013)proposed that conflict-specific, right frontal activationrecorded noninvasively with surface electrodes is a read-ily obtainable noninvasive human functional homologof rodent hippocampal-cortical activity that may alsobe useful as an objective biologic marker of anxiolyticefficacy.

An optogenetics approach has also been used toelucidate the role of the dentate gyrus in the processesof learning and memory, as well as the pathophysiol-ogy of anxiety (Kheirbek et al., 2013). Specifically,granule cells in the ventral dentate gyrus suppressinnate anxiety but do not affect contextual learning,


whereas those in the dorsal region control the encodingof contextual fear memories but not their retrieval. Asnoted, findings from this study suggest that distur-bances in pattern separation may underlie anxietydisorders in some patients, and therapies targeting theexcitability of the ventral dentate gyrus may offera beneficial approach in this population. These kinds oftechniques have also born fruit in animal models aswell, potentially allowing for translation of data fromanimal models to studies in humans. The connectionbetween hippocampal-mediated learning and anxiety

was further elucidated using optogenetic techniques,showing that hippocampal dentate gyrus neuronsactivated during fear learning in mice can be reac-tivated later using light stimulation, producing anincreased freezing response outside of the originalcontext (Liu et al., 2012).

Optogenetics combined with in vivo electrophysio-logical recordings may further elucidate activity inthe neural networks implicated in anxiety disorders.Optogenetic inhibition of the BNST in mice increasedexploration of open spaces in the elevated plus maze testand open field test, whereas stimulation of the BNSTwith the excitatory channelrhodopsin-2 was associatedwith increased anxiety in both tests. To elucidate theneural circuitry between the BLA and the anterodorsalBNST, microdrive electrode arrays containing stereotrodessurrounding a fiber-optic were implanted in the anterodorsalBNST of mice expressing channelrhodopsin-2 in theBLA. Excitation of the glutamatergic BLA terminalswas associated with increased activity of anterodorsalBNST neurons. Conversely, optically stimulating in-puts to the oval nucleus of the BNST induced a netinhibition of neural activity in the BLA, suggestingthat the oval nucleus and anterodorsal BNST playantagonistic roles in modulating anxiety (Kim et al.,2013). These findings are consistent with observationsthat the extended amygdala plays an important role inthe neural circuitry implicated in anxiety.

The BNST occupies a central position in the neuralcircuitry regulating the HPA axis response to stressand thus plays an important role in regulating theneuroendocrine and behavioral responses to stress (Henke,1984; Dunn, 1987; Fernandes et al., 2002; Walker et al.,2009). The release of neuroactive steroids in this brainregion may represent an endogenous homeostatic mech-anism for modulating anxiety and restoring normalGABAergic neurotransmission and HPA axis activityafter exposure to stress-inducing stimuli (Patchev et al.,1994; Barbaccia et al., 1996; Fernandes et al., 2002).Stressors that activate the BNST also activate seroto-nergic systems, and changes in 5-HT receptor expressionin this region were suggested to play a role in pathologicresponses to stress (Guo et al., 2009; Hazra et al., 2012).Thus, modulation of the activity of specific 5-HT receptorsubtypes in this region may be a novel approach totreating anxiety disorders.

B. Type A GABA Receptor and Serotonin ReceptorStructure/Roles in Anxiety

A disturbance of the nuanced integration of excit-atory and inhibitory transmission can occur on a localnetwork level that ultimately affects interconnecteddomains of larger-order structures linking to the outsideworld. Unraveling this will undoubtedly involve theaction of networks serving as low-pass as well as high-passfilters, all of which are based on the integrated action ofmultiple transmitters at their respective receptors.

Fig. 5. Projection-specific excitation induced by illumination of BLA terminalsin the CeA induces acute reversible anxiolysis in mice. (A) Illustrations showingwhere illumination was directed in the channelrhodopsin-2 (ChR2):BLA-CeA,eYFP:BLA-CeA, and ChR2:BLA (somata) mice. The mice received 5-millisecondlight pulses at 20 Hz for all light-on conditions. (B) Representative pathsin the elevated plus maze during light-on and light-off conditions. (C)Histograms showing increased probability of open arm entries during lighton phase in ChR2:BLA-CeAmice. (D and E) Open field test data demonstratingthat ChR2:BLA-CeA mice spend more time in center with light on than doeYFP:BLA-CeA and ChR2:BLA (somata) controls (P , 0.00001). CeA, centralamygdala; CeL, centrolateral nucleus; CeM, centromedial nucleus; eYFP,enhanced yellow fluorescent protein. Reprinted from Tye et al. (2011).


1. GABA. There is general agreement that GABA-mediated inhibitory neurotransmission plays a partic-ularly important role in the modulation of emotionalresponses to fear-inducing stimuli (Davis and Myers,2002; Stork et al., 2002, 2003; Walker et al., 2003), andthe concept of positive and negative allosteric modulationof fast postsynaptic GABAergic inhibitory neurotrans-mission by BZDs was central to anxiolytic pharmacologyfor decades (Choi et al., 1977; Macdonald and Barker,1978; Chan and Farb, 1985). GABAARs are locatedprimarily in the postsynaptic membrane at synaptic andextrasynaptic sites and mediate the majority of fastpostsynaptic inhibitory neurotransmission in the adultcentral nervous system (CNS) (Rabow et al., 1995;Barnard et al., 1998; Ben-Ari, 2002). The action ofGABA on GABAARs can modulate both the frequencyand duration of chloride channel openings to maintainthe membrane near its resting voltage (Macdonald et al.,1989; Twyman et al., 1989).The GABAAR is a member of the cys-loop superfamily

of receptors, which includes the nicotinic acetylcholinereceptors, the glycine receptors, and the 5-HT3 receptors.These receptors all have a characteristic loop formed bya disulfide bond between two cysteine residues (Sieghartand Sperk, 2002). The GABAAR is a pentameric complex,which can be composed from the members of eightrecognized families of glycoprotein subunits (a, b, g, d, «,p, u, and r) (Fig. 6). Several of these subunit families areknown to include multiple isoforms (e.g., a1–a6 andg1–g3), giving rise to at least 19 distinct subunits that,when combined, permit the assembly of a large numberof receptor subtypes with variable affinities for GABAARmodulators (Barnard et al., 1998; Smith et al., 2001).Each of these receptor subunits has an extracellulardomain, which serves as a ligand-binding site, and fourtransmembrane domains, which are involved in chlorideion channel structure (Fig. 7).The majority of GABAARs are composed of two a

subunits, two b subunits, and a single g subunit (Pritchettand Seeburg, 1991; Chang et al., 1996; Graham et al.,1996; Tretter et al., 1997; Wingrove et al., 2002), with theGABA binding site located on the a/b subunit (Casalotti

et al., 1986). Residues that contribute to the GABAbinding site are F64 on the a subunit and Y157, T160, andY205 located on the b subunit (Amin and Weiss, 1993;Amin et al., 1997; Baur and Sigel, 2003). Substitution ofany of these four amino acids decreases the sensitivityof the GABAAR to activation by GABA and muscimol.However, these same amino acid substitutions do not alterGABAAR activation by pentobarbital, indicating thatthese three compounds act at distinct binding sites (Aminand Weiss, 1993). A large fraction of GABAARs have asubunit composition of a1b2g2, and possess many of thepharmacologic properties associated with the classic BZDI or type 1 receptors.

Compounds that affect GABAAR-mediated responsesfall into three categories: agonists, antagonists, andmodulators. Agonists, such as muscimol, directly andspecifically interact with the GABA recognition site onthe GABAAR to induce a response. Competitive antag-onists, such as bicuculline, or noncompetitive antago-nists, such as picrotoxin, bind to the receptor and blockGABA from activating a response via its recognitionsite. Allosteric modulators bind at modulatory sites andincrease, decrease, or null modulate the binding ofGABA to the GABAAR, thereby enhancing, inhibiting,or null modulating the ensuing response to GABA (Farbet al., 1984; Chan and Farb, 1985). Chlordiazepoxideand diazepam were the first demonstrated therapeuticsto act as positive allosteric modulators at the GABAAR(Braestrup and Squires, 1977; Choi et al., 1977; Möhlerand Okada, 1977; Macdonald and Barker, 1978; Gallagerand Tallman, 1983; Ferrero et al., 1984). These com-pounds appear to impart a conformational change in thetransmembrane receptor that alters its affinity for GABAand GABA agonists (Czajkowski and Farb, 1986; Lavoieand Twyman, 1996; Boileau and Czajkowski, 1999).Activation of GABAARs with the agonist muscimol alsoincreases the affinity for the receptor of positive allostericmodulators [e.g., diazepam and the triazolopyridine CL218872 (3-methyl-6-[-3-(trifluoromethyl)phenyl]-1,2,4-triazolo[4,3-b]pyridazine)], indicating that this rela-tionship is also reciprocal (Chiu and Rosenberg, 1979;Lippa et al., 1979; Villiger, 1984). Sensitivity to BZDs

Fig. 6. Schematic representation of GABAAR and the GABAergic synapse. (A) Top view of GABA and BZD binding sites. (B) Side view. Adapted fromBerezhnoy et al. (2007).


appears to be mediated primarily by the g2 and a1, a2,a3, and a5 subunits (Brooks-Kayal and Pritchett, 1993;Boileau and Czajkowski, 1999).Dissimilar compounds influence GABAARs differently.

For example, although BZDs and barbiturates both acton GABAARs, electrophysiological studies suggest thatthe actual mechanism of action is different. These studiesindicate that diazepam increases the duration of GABA-induced channel openings or burst by only 9.3%, butincreases the number of bursts by 102.5%. In contrast,phenobarbital increases burst duration by 81.9%, butincreases the number of bursts by only 6.3% (Twymanet al., 1989). Although the mechanism of receptormodulation has not been fully elucidated, BZDs appearto enhance single-channel burst frequencies by increas-ing the affinity of GABA for its binding site, whereasbarbiturates appear to slow receptor deactivation bystabilizing the interaction between GABA and theGABAAR (Mathers, 1985; Twyman et al., 1989; Rabowet al., 1995; Steinbach and Akk, 2001). The binding ofBZDs to the allosteric BZD site increases the affinity ofthe receptor for GABA and enhances GABAergic neuro-transmission by increasing the number of chloridechannels opened at a given synaptic concentration ofGABA, which in turn hyperpolarizes the cell membrane.

The a subunit appears to be intimately related toGABAAR sensitivity to the prototypical BZDs andparticularly to diazepam, playing an important rolein drug binding and concentration response relation-ships (Pritchett et al., 1989; Pritchett and Seeburg,1990; Wafford et al., 1993; Smith et al., 2001) viainteracting with the a/g interface (Buhr and Sigel,1997). The g subunit contributes to receptor affinity forspecific ligands as well as the single-channel character-istics (Herb et al., 1992; Günther et al., 1995; Benkeet al., 1996).

GABAAR subtypes that are modulated by BZDs canbe divided into those that are diazepam sensitive andthose that are not (Lo et al., 1982). BZDRs that recog-nize the classic 5-phenyl-1,4-BZDs (e.g., diazepam andflunitrazepam) are referred to as “diazepam-sensitive”receptors, whereas those that do not recognize theseligands are referred to as “diazepam-insensitive” recep-tors (Malminiemi and Korpi, 1989; Hadingham et al.,1996; Knoflach et al., 1996). It was originally thought thatthere were just two subtypes of BZD-sensitive GABAARs.The first group, type 1 receptors that are enriched in thecerebellum and globus pallidus, display a higher affinityfor the triazolopyridine CL 218872 and the b-carboline[3H]propyl-b-carboline-3-carboxylate. By contrast, type II

Fig. 7. Molecular docking and transmembrane structure of the GABAAR. (A and B) The GABAAR interface (A) and structures (B) of BZD binding siteligands used in the study. Panel A shows the homology model of a1 and g2 subunits of the GABAAR with BZD binding site loops highlighted in yellow;loop F is green. Dashed lines indicate limits of cell membrane. Figure courtesy of Dr. Cynthia Czajkowski. Also see Hanson and Czajkowski (2008).


receptors, which are found in the superficial layer of thesuperior colliculus, striatum, and the dentate gyrus ofthe hippocampus, show low affinity for CL 218872 and[3H]propyl-b-carboline-3-carboxylate (Braestrup andSquires, 1977, 1978; Braestrup and Nielsen, 1981;Young et al., 1981). It has since been shown that thereare multiple classes of BZDRs, reflecting differencesin GABAAR subunit compositions such as substitutionof the g3 and g2 subunits.Overall, it appears that sedation and amnesia associated

with classic BZD actions are mediated primarily via a1

subunit–containing GABAARs in rodents, whereas re-duction of vigilance is mediated primarily via the a2 anda3 subunits (Löw et al., 2000; McKernan et al., 2000; Diaset al., 2005), with activation of the g subunit also playing arole in BZD efficacy (Rudolph et al., 1999). The a2 subunitis largely expressed in limbic structures implicated inanxiety and fear, including the amygdala and hippocam-pus. Staining intensities of the a2 subunit are highest inthe accessory olfactory bulb, molecular layer of the dentate,hippocampal area CA3, central and lateral amygdaloidnuclei, septum, striatum, accumbens, and hypothalamus.The lateral septum has a high concentration of a2

subunits and has been shown to participate in theregulation various visceral and somatic functions aswell as in modulation of anxiety, aggression, and fearresponses in rodent models (Pesold and Treit, 1996;Gavioli et al., 1999). Neurons in this region are alsonot sensitive to zolpidem-induced enhancement ofGABAergic inhibition, which has been associated withactivation of receptors containing the a1 subunit (Duncanet al., 1995). Cat odor exposure induces Fos expres-sion in the posterior accessory olfactory bulb, whichhas also been shown to contain a high concentrationof the a2 subunit (McGregor et al., 2004). Cat odorinduction of Fos expression is also seen in the mainolfactory bulb and midazolam inhibited Fos expres-sion in this region. Midazolam also inhibited Fosexpression in key limbic regions involved in pheromonetransduction (medial amygdala and BNST) and de-fensive behavior (prelimbic cortex, lateral septum, lateraland medial preoptic areas, and dorsal premammillarynucleus).H101R mice in which the a2 subunit was rendered

insensitive to diazepam by a knock-in point mutation(His101→Arg) show reduced sensitivity to the anxio-lytic effects of diazepam. This mutation was notassociated with any overt phenotypic changes and theanimals expressed all subunits tested (a1, a2, a3, b2/3,and g2) at normal levels and distribution. After ad-ministration of diazepam, the a2 knock-in mice did notshow the increase in exploratory behavior (as mea-sured by the amount of time spent and the number ofentries into the open arms) on the elevated plus mazethat is typically seen in wild-type mice. This differencein behavior could not be attributed to motor impair-ment because the motor activity in the enclosed arms

was similar in a2 H101R and wild-type mice irrespectiveof the treatment (Löw et al., 2000).

A subsequent study looked at the behavior of H101Rmice in a conditioned emotional response task (Morriset al., 2006). In these experiments, lever pressing forfood on a variable interval schedule of reinforcementwas suppressed by delivery of a foot shock pairedwith a conditioned stimulus tone or light. During theconditioned emotional response test session, wild-typeand H101R mice were pretreated with diazepam(0, 0.5, 1, and 2 mg/kg) 30 minutes prior to testing.The wild-type mice showed an increase in lever pressingduring treatment with diazepam, whereas H101R micewere resistant to the anxiolytic effects of diazepam.

These observations naturally suggest that develop-ment of compounds that are selective for those receptorsubtypes implicated in anxiety may prove to be extremelyfruitful in the management of anxiety disorders. To date,efforts to fine-tune the binding affinity or functionalefficacy of compounds that display selectivity or prefer-ence for specific receptor subtypes remain promisingin rodent experimental animal systems, yet elusivein human clinical trials (Ballenger et al., 1991, 1992;Rudolph et al., 1999; Löw et al., 2000; Sandford et al.,2001; Atack, 2003; Lippa et al., 2005; Rudolph andMöhler, 2006; Atack et al., 2011a,b; Olivier et al., 2013).Additional consideration for targeting therapeutics,which take advantage of the rapid rates of GABAARturnover and the nonlysosomal pathway for degradation(Borden et al., 1984; Borden and Farb, 1988), may alsoyield greater selectivity and reduced adverse events.Further research directed along a neural-systems levelmay well resolve the drug discovery puzzle at hand ifthe receptor subtype and selective modulatory thera-peutic can be effectively aligned for the treatment ofspecific anxiety disorder subtypes.

2. Serotonin. The role of 5-HT in the pathophysiol-ogy of anxiety disorders has become increasingly wellestablished, and agents that inhibit the reuptake ofthis neurotransmitter now represent important com-ponents of current approaches to anxiolysis (Graeff andZangrossi, 2010; Sertraline, 2012; Venlafaxine, 2012;Fluoxetine, 2013). 5-HT is amonoamine whose postsynapticaction is terminated via 5-HT reuptake into the presynapticnerve terminal via a 5-HT transporter. The efficacy of SSRIsis thus based on augmenting the concentration of 5-HT inthe synaptic cleft by inhibiting its clearance (Ressler andNemeroff, 2000; Nichols and Nichols, 2008).

Effects of 5-HT are mediated by as many as 14 receptorsubtypes, including 13 G protein–coupled receptors(GPCRs) and a single ligand-gated ion channel. Thesereceptors are divided into seven distinct classes (5-HT1

to 5-HT7) (Fig. 8); however, postgenomic modificationscan produce at least 20 additional GPCRs, leaving morethan 30 5-HT receptors that signal through G proteins.In addition, activation may result in variable effects,depending on the brain region in which the receptors


are expressed and on individual differences in respon-siveness mediated by genetic polymorphisms (Raymondet al., 2001; Hoyer et al., 2002; Gray and Roth, 2007).Although it is tempting to conceptualize the actions

of 5-HT at its receptor sites as being either inhibitoryor excitatory, the complexity of the signal transductionpathways activated ultimately may confound suchan appealing target- and hypothesis-driven approach.That said, 5-HT receptors generally have the followingfunctions (Polter and Li, 2011): 1) 5-HT1 receptors inhibitadenyl cyclase, reduce cyclic AMP, and inhibit proteinkinase A (PKA); 5-HT1A receptors are the prototypical5-HT1 receptors; 2) 5-HT2 receptors increase inositoltrisphosphate, increase intracellular calcium levels, increase

diacylglycerol, and activate PKA; 5-HT2A receptors are theprototypical type 5-HT2 receptors; 3) 5-HT3 receptors areligand-gated cation channels; 4) 5-HT4 receptors activateadenyl cyclase, increase cyclic AMP, and activate PKA; 5)5-HT5 receptors inhibit adenyl cyclase, reduce cyclic AMP,and inhibit PKA; and 6) 5-HT6 and 5-HT7 receptors activateadenyl cyclase, increase cyclic AMP, and activate PKA.

The complex distribution and functionality of thesereceptor subtypes contributes to their unique ability tomodulate emotional responses and other bodily func-tions via the systems into which they feed forward.The 5-HT3 receptor is a ligand-gated ion channel. Theremaining subtypes are GPCRs with the latter alsobeing defined as a type A family, rhodopsin-like receptors.Evidence suggests that most, if not all, of the 5-HT GPCRsundergo dimerization, and that dimerization may benecessary for proper functioning of the receptor (Nicholsand Nichols, 2008). For example, when inactive 5-HT2C

receptors are coexpressed with a wild-type 5-HT2C re-ceptor, the inactive receptor is able to inhibit theactivity of the wild-type receptor through the formationof a nonfunctional heterodimer, suggesting that di-merization is essential for receptor function (Herrick-Davis et al., 2005). Studies to date suggest that the5-HT receptors primarily related to anxiety and moodare 5-HT1A, 5-HT2A, and 5-HT2C (Toth, 2003; Van Oekelenet al., 2003; Mato et al., 2010).

Interestingly, the roles of these receptor subtypesmay vary depending on the form of anxiety involved, asoutlined in a review by Graeff and Zangrossi (2010).The authors suggest that SSRIs improve symptomsin patients with panic disorder by enhancing theresponsiveness of 5-HT1A and 5-HT2A receptors in thedorsal periaqueductal gray matter in the midbrain, butimprove symptoms in patients with generalized anxi-ety through the desensitization of 5-HT2C receptors and,perhaps, through stimulation of 5-HT1A receptors in theforebrain.

Understanding the effects of 5-HT in anxiety disordersrequires attention to variations in transporter reuptakesubtypes that may potentially influence the clinicalresponse to SSRIs. Genetic polymorphisms of the 5-HTtransporter gene promoter (5-HTTLPR) have beenassociated with efficacy of antidepressants (Porcelliet al., 2012), as well as with anxiety. For example,a functional polymorphism of the 59-flanking regionof the 5-HT transporter gene (SLC6A4) is associatedwith differences in anxiety-related personality traits(Heils et al., 1996; Lesch et al., 1996). A 43-bp deletion/insertion in 5-HTTLPR is associated with long “L”(16 repeats) and short “S” (14 repeats) alleles (Heilset al., 1996). There appear to be ethnic differences in theprevalence of these polymorphisms as well, with Cau-casians having about 22% S/LG alleles and Asians havingabout 60% S/LG alleles (Hu et al., 2006). Gene/environmentinteractions may therefore account for susceptibilityto anxiety disorders and for patient response to

Fig. 8. Model of a serotonergic neuron and synapse. 5-HT1A receptors canfunction both as autoreceptors on the serotonergic cell body and as postsynapticreceptors. Most 5-HT receptors are GPCRs and are located postsynaptically toserotonergic neurons. The 5-HT3 receptor is the exception because it is a ligand-gated ion channel. AC, adenylyl cyclase; DAG, diacylglycerol; GIRK, G protein–coupled inwardly rectifying potassium channel; IP3, inositol trisphosphate;PLC, phospholipase C; TPH, tryptophan hydroxylase. Adapted from Gray andRoth (2007).


pharmacological treatment with SSRIs and SNRIs (Leschet al., 1996; Nakamura et al., 2000; Hu et al., 2006;Klumpers et al., 2012). The L(A) allele is overtransmittedby 2-fold to patients with OCD (Hu et al., 2006), whereassubjects with at least one short allele exhibit greaterfear-potentiated startle than do subjects with the longallele (Klumpers et al., 2012).Another genetic factor implicated in the clinical re-

sponse to SSRIs is a polymorphism in the gene that codesfor RGS2, a protein involved in neuronal GPCR responsesto neurotransmitters including 5-HT. Stein et al. (2014)found that a mutation that reduces expression of RGS2is associated with a poor clinical response to sertralinetreatment.

V. Neuropsychopharmacology ofAnxiety Disorders

Different combinations of subunits comprise theGABAA, NMDA, and 5-HT receptors, providing tre-mendous variability in affinity for endogenous andexogenous ligands and thus for the ability of the receptorto modulate neuronal function (Smith et al., 2001;Nichols and Nichols, 2008). Below, we review a rangeof established and investigational anxiolytic agents,outlining the impact of selective modulation of receptorsubtypes where data are available. The pharmacology ofdrug action is inherently the result of the net outcome ofsystems-level actions of the neuroactive agent. Therefore,it must be considered that, although subtype-preferringcompounds exist, highly selective therapeutics have notyet been developed.

A. Positive Allosteric Modulation ofGABAergic Transmission

1. Benzodiazepines and Related Modulators.Although the actions of BZDs on GABAARs can reduceanxiety, this therapeutic effect is not without severaladverse side effects, including drowsiness and ataxiaas well as the risk of dependence and withdrawal reactions(Noyes et al., 1988; Bennett et al., 1998; Yamawaki, 1999;Allgulander et al., 2003). Because of these untoward effects,the use of BZDs for long-term treatment of chronic anxietydisorders such as GAD is undesirable (Allgulander et al.,2003). As indicated previously, side effects and efficacy ofanxiolytic effects may be mediated through differenta subunits (Löw et al., 2000; McKernan et al., 2000;Dias et al., 2005), providing one impetus for researchinto the development of more selective compounds.Partial positive allosteric modulators have a lower

intrinsic ability to induce a modulatory effect at givenfractional occupancy as compared with full allostericpositive modulators and represent an approach todeveloping anxiolytics with fewer side effects and toxicity(Atack, 2003). The relationship between fractional re-ceptor occupancy and fractional effect can be assessed bydetermining the percentage of inhibition of flumazenil

binding to GABAARs and potentiation of GABA-evokedchloride currents. Potentiation of GABA-gated currentsby 25% occurs at about 35% receptor occupancy fordiazepam and about 45% for triazolam (Facklamet al., 1992b). The benzoquinolizinone Ro 19-8022[(R)-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl)carbonyl]-2-pyrrolidine-methanol], a BZDR partialallosteric modulator, produced 25% potentiation whenreceptor occupancy reached about 95%. However, theimidazobenzodiazepinone bretazenil, or Ro 16-6028[(13aS)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1-c][1,4]benzodiazepine-1-carboxylicacid 1,1-dimethylethyl ester], which also acts as a partialallosteric positive modulator, did not produce 25%potentiation even at 100% receptor occupancy. Thesein vitro observations can be related to in vivo responsesin animal models, which indicate that bretazenil doesnot protect against seizures as well as the full allostericpositive modulator diazepam (Brouillet et al., 1991;Haefely et al., 1992). By contrast, most studies in humansand animals indicate that bretazenil can provide anxio-lytic effects with less sedation and motor impairmentsthan diazepam (Facklam et al., 1992a; Cole and Rodgers,1993; Busto et al., 1994). However, there is an increase insymptoms of sedation and deficits in certain measures ofattention and executive function (digit symbol) amongsubjects receiving 0.5 mg bretazenil (van Steveninck et al.,1996).

Although most compounds possessing the b-carbolinestructure are anxiogenic, the b-carboline abecarnil(isopropyl-6-benzyloxy-4-methoxymethyl-b-carboline-3-carboxylate) produces anxiolytic effects without sub-stantial sedation (Stephens et al., 1990; Ballengeret al., 1992). Abecarnil exhibits high affinity for the BZDrecognition site, although no differences in affinitieswere observed for recombinant receptors expressed inhuman embryonic kidney cells containing a1, a2, or a3

subunits (Hadingham et al., 1993). Another study showsthat abecarnil only partially inhibits diazepam-insensitivebinding of [3H]Ro 15-4513 in the cerebellum, suggestingthat this site can be differentiated into abecarnil-sensitiveand abecarnil-insensitive components (Mehta and Shank,1995).

Electrophysiological studies indicate that abecarnilpotentiates GABA-induced chloride currents less thanfull allosteric modulators. Interestingly, abecarnil ismost effective with the fewest side effects at the lowestdose examined in a double-blind, placebo-controlledclinical trial for treatment of GAD (Ballenger et al.,1991, 1992). However, higher concentrations (.7.5 mg/day)of abecarnil were associated with reports of symptoms andsigns of CNS depression including somnolence, difficultyconcentrating, and dizziness. These observations suggestthat efficacy alone may not account for all of the adverseside effects of BZDs.

The cyclopyrrolone pagoclone, another compoundbelieved to act as a partial positive allosteric modulator


of the GABAAR, has also been investigated for itsanxiolytic effects. A randomized double-blind study ofpatients suffering from panic attacks found that thiscompound provides anxiolytic effects without the typicalside effects associated with BZDs (Sandford et al., 2001),but it has not been approved for use in the treatment ofanxiety disorders (Bateson, 2003; Atack et al., 2006a; deWit et al., 2006).Agents such as imidazenil and SL651498 [6-fluoro-9-

methyl-2-phenyl-4-(pyrolidin-1-yl-carbonyl)-2,9-dihydro-1H-pyrido[3,4-b]indol-1-one], which have a relativelyhigher affinity for specific GABAAR subunits, such asthe a2 subunit, also appear to produce reduced mea-sures of vigilance (a model for anxiolytic effects inhuman) with fewer side effects than the classic BZDs(Sieghart, 1994; Griebel et al., 2001; Williams and Akabas,2001; Collins et al., 2002; Licata et al., 2005). This isconsistent with the hypothesis that the a2 subunit–containing receptor subtype, which is largely expressedin the limbic system, may be an important contributorto the subtle positive modulation of GABAergic in-hibition necessary to induce a reduction in vigilancein rodents and possibly induce anxiolysis in humanswithout undesirable side effects (Pritchett et al., 1989;Pritchett and Seeburg, 1990; Löw et al., 2000; Kaufmannet al., 2003; Licata et al., 2005). Yet bridging the gapbetween convincing results of a therapeutic candidatein animal models for anxiety and its beneficial clinicalutility in humans remains an elusive if not dauntingobjective.Research into the allosteric modulatory effects of com-

pounds such as zolpidem, which act on the BZD site butdo not possess the 7-member ring associated with thestructure of the classic BZDs, has yielded compounds witha preference for specific GABAAR subunits (Ghiani et al.,1994; Serra et al., 1994; Drover, 2004). For example,zaleplon, zolpidem, and zopiclone exhibit a modestpreference of up to 6-fold for GABAARs containing a1

subunits compared with a2 or a3 subunits using modelsystems (Drover, 2004), and are effective sedative/hypnotic agents reported to produce less toleranceand dependence than diazepam (Follesa et al., 2002).Compounds that do not act at GABAARs containing

a1 subunits may be useful as nonsedating anxiolytics.Whereas zolpidem and zaleplon appear to lack signif-icant anxiolytic effects and seem to also not have anyactive metabolites, zopiclone is metabolized in vivoto the active metabolite (S)-desmethylzopiclone, whichhas been shown to produce anxiolysis with fewer neg-ative effects on locomotor activity than diazepam andalprazolam, suggesting that structurally related com-pounds may be useful in the treatment of anxietydisorders (Carlson et al., 2001; Fleck 2002).Ocinaplon (pyrazolo[1,5-a]-pyrimidine) is a novel

pyrazolopyrimidine that potentiates GABAergic neuro-transmission, increases the response of rats and primatesin measures of anxiety (conflict test), and attenuates

symptoms among human subjects with GAD as demon-strated by scores on the Hamilton Anxiety Rating Scales(Lippa et al., 2005). Electrophysiological studies indicatethat ocinaplon has lower potency and efficacy thandiazepam. However, the potentiation of GABA-gatedcurrents in recombinant receptors expressed in Xenopusoocytes observed in this study indicates that ocinaplonhas greater efficacy at a1 subunits than at a2 subunits,a finding that on the surface appears to be in conflictwith the hypothesis that anxiolysis is mediated by a2

subunit–containing GABAARs.A compound with comparable binding affinity but

different efficacies at the various receptor subtypescan exert its effects predominantly at those receptorsubtypes implicated in anxiolysis, rather than havingnonspecific effects and/or producing untoward sideeffects such as sedation (Atack, 2003). Interestingly,although ocinaplon shows selectivity for modulating a1

subunit–containing GABAARs, competitive bindingassays using native GABAAR membrane preparationsderived from rat cerebellum (wherein a1 subunits arehighly expressed) indicate that this compound is also muchless potent at inhibiting the binding of flunitrazepamthan is diazepam (Lippa et al., 2005). Since the clinicalpotency of a drug depends on its binding affinity for thetarget and its efficacy of signal transduction, it is plausiblethat lower receptor occupancy may contribute to theability of ocinaplon to produce anxiolysis without inducingsedation.

Alternatively, some positive modulation of GABAARscontaining a1 subunits may be necessary to effectivelyinduce anxiolysis in the human nervous system com-pared with mouse models, wherein even mild dysreg-ulation of higher-order cognitive processes mediated bythe frontal lobes has been implicated in controllingthreat assessment and anxiety (Cha et al., 2014). Ir-respective of which of these hypotheses or others turnsout to be themost valid, the development of this promisingcompound was halted due to reports of elevated liverenzymes in just a few subjects, underscoring the inherentdifficulties associated with bringing a novel anxiolytic tomarket even using a well defined target.

SL651498 is a pyridoindole that preclinical studiessuggest acts at the BZD binding site to produce anxiolyticeffects qualitatively similar to those of BZDs with re-duced ataxia or sedation. This agent is a full positivemodulator at recombinant rat GABAARs containing a2 ora3 subunits (approximately 95% enhancement) and apartial positive modulator at receptors containing a1 ora5 subunits (approximately 45% enhancement). Binding-affinity assays indicate that SL651498 does not differen-tiate between a1 (17 nM), a2 (73 nM), and a3 (83 nM)subtype–containing GABAARs (Griebel et al., 2001),indicating that binding affinity does not contribute tothe anxiolytic actions of this drug.

2. Natural Product Modulators. Some natural productsmay also provide new platforms for discovery. For example,


chrysin, one of the first flavonoids shown to interactwith the BZD binding site, initiated the search fornatural anxiolytics among traditional medicinal herbs(Medina et al., 1989, 1990, 1997; Wolfman et al., 1994,1996). A number of other naturally occurring flavonoidsand their derivatives, such as K36 (5,7,29-trihydroxy-6,8-dimethoxyflavone) (Huen et al., 2003), hispidulin (Kavvadiaset al., 2004), wogonin (Hui et al., 2000, 2002), and relatedcompounds (Nielsen et al., 1988; Häberlein et al., 1994;Wolfman et al., 1996; Goutman et al., 2003), areallosteric modulators at GABAARs. In a mouse study,wogonin (7.5–30 mg/kg p.o.) was found to produce ananxiolytic response similar to diazepam in the elevatedplus maze without accompanying sedation (Hui et al.,2002). K36, a naturally occurring flavonoid, increasesentries onto the open arms of the elevated plus mazewithout changing locomotor activity, sedation, myorelax-ation, or motor incoordination in mice (Huen et al., 2003).3. Increasing GABA Levels. On the basis of all of

these results, it would be expected that increasingsynaptic concentrations of GABA, either by inhibitingits reuptake or degradation, could be another approachto enhancing GABAergic neurotransmission and re-ducing anxiety disorders. GABA-transaminase inhib-itors, such as vigabatrin (g-vinyl-GABA) and valproicacid (VPA) (Rando et al., 1981; Gram, 1988), increasesynaptic concentrations of GABA by preventing catab-olism of GABA (Zwanzger et al., 2001a,b; Nemeroff,2003). Vigabatrin raises levels of GABA in nerve terminalsand inhibits striatal dopamine release (Kushner et al.,1997). However, reports of psychosis and depressionassociated with vigabatrin suggest that this particularcompound may have significant clinical limitations(Levinson and Devinsky, 1999; Ashton and Young, 2003).In retrospect, elevating GABA in a way that is pro-portionate to its endogenous levels might be expected toactivate the natural distribution of relevant receptorsubtypes nonselectively, thus increasing the probabilityof side effects. A more nuanced, neural systems–levelapproach might be capable of modulating anxiety ina similar fashion to the effective SSRIs.

B. Modulators of Glutamatergic Transmission

A delicate balance between excitatory and inhibitoryneurotransmission is essential to modulation of anxi-ety and fear responses. Not surprisingly, disturbancesof glutamatergic as well as GABAergic neurotransmis-sion have been implicated in anxiety disorders (Jardimet al., 2005).1. Inhibitors of Glutamatergic Activity. Glutamatergic

neurotransmission in the lateral amygdala is essentialto the acquisition and extinction of fear-related memoriesand behavioral responses associated with re-exposure tofear-inducing stimuli (Sajdyk and Shekhar, 1997). Whena neutral stimulus is paired with an adverse stimulussuch as presentation of a tone paired with delivery ofa foot shock, the previously neutral stimulus is soon able

to elicit a fear response independently. NMDA receptorantagonists such as D,L-2-amino-5-phosphonopentanoicacid, which prevent induction of long-term potentiation,have also been shown to block the acquisition but notthe expression of conditioned fear responses in rodents(Miserendino et al., 1990; Falls et al., 1992). As pre-viously mentioned, the prefrontal cortex appears to bethe brain region most often implicated in attenuation ofanxiety.

Memantine is another NMDA receptor antagonistthat is currently approved for the treatment of Alzheimer’sdisease, but there is some evidence that it may also haveanxiolytic properties. A study in mice found that highstable doses of memantine (10, 30, and 100 mg/kgper day) produced some improvements in measures ofanxiety, including reductions in isolation-induced aggres-sion, escape latency (hidden platform), and wall swimmingtendency in the Morris water maze test (Minkevicieneet al., 2008). In addition, several case studies in humanshave shown some benefit for memantine in reducingsymptoms associated with PTSD, SAD, and GAD(Battista et al., 2007; Schwartz et al., 2012). For example,in a small study of four patients with combat exposure–induced PTSD, treatment with memantine led to consis-tent improvements in memory as well as depressive andhyperarousal symptoms (Battista et al., 2007).

Another study showed that acamprosate, an NMDAand metabotropic glutamate 5 receptor modulator, waseffective as a therapeutic for idiopathic and chemicalwithdrawal–induced anxiety disorders (Kotlinska andBochenski, 2008; Hertzman et al., 2009; Schwartz et al.,2010; Pałucha-Poniewiera and Pilc, 2012; Koltunowskaet al., 2013). Acamprosate seems to be a safe and ef-fective therapeutic option for patients presenting withcomorbid alcoholism. These findings were initiallynot entirely surprising given that enhancing inhibitoryneurotransmission is a well establishedmethod of treatinganxiety; thus, it logically followed that if acamprosateattenuated excitatory neurotransmission, it could yielda similar clinical outcome (Berton et al., 1998). However,subsequent research has revealed both enhancement aswell as inhibition of network synaptic elements underlyingglutamatergic and GABAergic transmission, further em-phasizing the need for a larger-scale understanding ofdrug action in the context of connectivity.

There is also evidence that riluzole, which reducessymptoms of GAD (Pittenger et al., 2008), may exertits effects by inhibiting voltage-gated sodium channelsand enhancing astrocytic uptake of extracellular gluta-mate (Urbani and Belluzzi, 2000; Wang et al., 2004). Inan open-label study of GAD patients, 8 of 15 patientstreated with riluzole had remission of anxiety over aperiod of 8 weeks, with a median time to response of2.5 weeks (Mathew et al., 2005). Thus, therapeutics thatinhibit glutamatergic neurotransmission (e.g., riluzole)may prove to be more useful in treating GAD and thehyperarousal seen in PTSD.


2. Potentiators of Glutamatergic Activity. Admin-istration of D-cycloserine, a partial NMDA receptoragonist, has been shown to facilitate extinction of learnedfear responses in rodents and of phobias, SAD, and PTSDamong patients undergoing behavioral exposure therapy(Walker et al., 2002; Ressler et al., 2004; Woods andBouton, 2006; Mao et al., 2008; Aupperle et al., 2009;Difede et al., 2014).A randomized, double-blind, placebo-controlled clinical

trial examining the efficacy of a combination of D-cycloserineor placebo and exposure therapy for treatment of SADrevealed that short-term dosing of partial NMDA receptoragonists may also be effective in treating this disorder(Hofmann et al., 2006). This compound seems to be mosteffective for treating anxiety disorders that respond to“exposure therapy” in which learning not to fear thestimulus facilitates improvement in symptom measures.In a similar study, Difede and colleagues (2014) comparedthe effectiveness of VRE therapy alone versus thecombined treatment with D-cycloserine plus VRE, andfound that D-cycloserine augmented the therapeutic re-sponse to VRE, suggesting that negative memories maybe extinguished using cognitive enhancers. Overall, theseobservations suggest that compounds that increaseglutamatergic neurotransmission are useful in the treat-ment of certain types of anxiety disorders (e.g., specificphobias) and PTSD wherein re-exposure to a specificsalient stimuli can trigger a pathologic response thatinterferes with activities of daily living (Heresco-Levyet al., 2002; Difede et al., 2014).3. Neurosteroids as Modulators of Glutamatergic

Transmission. The observation that stress-inducedanxiety changes glutamatergic neurotransmission (Masneufet al., 2014) raises the possibility that endogenousmechanisms for modulating the balance of excitatoryto inhibitory neurotransmission could underlie thegenesis of anxiety. Neurosteroids such as pregnanolonesulfate, which enhances cognition, positively modulatesNMDAR function, and stimulates NMDAR trafficking tothe cell surface (Wu et al., 1991; Kostakis et al., 2013),may represent one such endogenous mechanism forlinking fearful stimuli and the emotional responsesubject to therapeutic manipulation (Plescia et al.,2013). Neuroactive steroids, such as pregnanolonesulfate and pregnanolone hemisuccinate, which in-hibit glutamatergic neurotransmission by negativelymodulating NMDAR function (Park-Chung et al.,1994) and may also exhibit mixed function by servingas both a negative modulator of the NMDAR anda prodrug for pregnanolone (a positive modulatorof GABAAR) delivery across the blood–brain barrier,may also represent a novel platform for discovery(Weaver et al., 1997, 2000).

C. Dopaminergic Transmission

In a recent study by Zweifel and colleagues (2011),cue conditioning was found to be significantly impaired

in knockout mice lacking functional NMDA receptorson dopaminergic neurons of the ventral tegmentalarea. This impairment was associated with developmentof a GAD-like phenotype, suggesting that dopaminergicneurotransmission plays a role in this anxiety disorder.Interestingly, increased anxiety is among the symptomsassociated with dopamine agonist withdrawal in humans(Nirenberg, 2013). The interplay between the glutamatergicand dopaminergic systems is well studied in drug ad-diction, wherein motivation for drug seeking is mediated,in part, by this relationship. Nucleus accumbens and pre-frontal cortex dopaminergic and glutamatergic systemshave been implicated in anxiety disorders and addiction(Ahmadi et al., 2013) and it has been suggested that theD2/D3 receptor agonist quinpirole may induce compulsivechecking behavior in rodents by enhancing dopaminergicactivity (Alkhatib et al., 2013). The role of motivation inOCD is well established and plays a key role in the abilityof an individual to override his or her pathologic responsesto anxiety-inducing stimuli; it is not uncommon for pa-tients with OCD and phobias to temporarily overcometheir fears to avoid life-threatening events.

In contrast with the D2/D3 receptor agonist quinpirole,the anxiolytic buspirone has been shown to block D3receptors in primates (Kim et al., 2014). However, therole that this mechanism plays in attenuating anxietyis not entirely clear since the compound also acts atserotonin and adrenergic receptors (Skolnick et al.,1984).

D. Epigenetics of Anxiolysis: HistoneDeacetylase Inhibitors

Combining histone deacetylase inhibitors with CBTcan also facilitate extinction of learned fear responses inhumans and animals (Heinrichs et al., 2013; Kuriyamaet al., 2013). Administration of VPA at a dose of 400 mg/kgto human subjects was shown to augment acquisitionand delayed offline consolidation of extinction training(Kuriyama et al., 2013). The unexpected effect of VPAon acquisition could be due to in part to an anxiolyticeffect associated with enhanced GABAergic neurotrans-mission mediated by inhibition of GABA transaminaseby VPA at this dose.

VPA seems to be rather nonspecific in terms of mech-anisms of action, at least at present. For example, inaddition to inhibition of histone deacetylases, VPA alsoincreases levels of brain-derived neurotrophic factorand cAMP response element-binding protein in thehippocampus and amygdala (Jeon et al., 2006; Casuet al., 2007; Jornada et al., 2010). A case of familialbipolar disorder with comorbid anxiety has beenstudied as part of the National Institute of MentalHealth Human Genetics Initiative and was found to beassociated with the genes that code for the extracellu-lar signal-regulated kinase/mitogen-activated proteinkinase and cAMP response element-binding protein–regulated intracellular signaling pathways (Kerner


et al., 2013). Although further research in this area isneeded, these findings suggest that behavioral modifi-cation therapy could be combined with drugs thatmodulate gene expression to facilitate extinction of thefear-inducing stimuli associated with specific phobiasand PTSD.

E. Modulators of Serotonergic andNoradrenergic Transmission

Several studies reveal the potential benefits of SSRIsover BZD pharmacotherapy of certain types of anxiety.For example, it has been shown that patients whopresent with comorbid symptoms of depression andanxiety may respond better to SSRIs, whereas thosewho suffer from anxiety alone may respond better toBZDs. In addition, patients suffering from GAD re-spond well to BZDs, whereas those with PTSD andOCD have a relatively better clinical response to SSRIs(Rickels and Rynn, 2002; Varia and Rauscher, 2002;Blanco et al., 2003; Davidson, 2003).Paroxetine was the first SSRI to receive approval

from the US Food and Drug Administration for thetreatment of anxiety disorders and was superior toplacebo in short-term clinical trials among patientswith GAD (Allgulander et al., 2003). The SSRI fluoxetineis widely used for the treatment of patients who arecomorbid for depression and anxiety (Cassano et al.,2004). It seems unclear whether the anxiolytic effects offluoxetine may paradoxically be due entirely to enhance-ment of GABAergic tone at the neural-systems level.Electrophysiological results based on recombinantGABAARs transiently expressed in mammalian cellsreveal that fluoxetine increases the response of thesereceptors to submaximal GABA concentrations, althoughit does not alter the maximum current amplitude(Robinson et al., 2003), and this result is of uncertainrelevance to the physiologic effects of SSRIs.Although data are less abundant for SNRIs, several

clinical trials have established the efficacy of venlafaxinein patients with anxiety (Liebowitz et al., 2005; Steinet al., 2005). In one double-blind study of adult patientswith generalized SAD (N = 271, intent to treat), venlafaxineextended release produced significant improvements in theLiebowitz Social Anxiety Scale total score, Clinical GlobalImpressions-Severity of Illness scale, and the Social PhobiaInventory, compared with placebo over 12 weeks (Liebowitzet al., 2005). Similarly positive results were seen over6 months (Stein et al., 2005).A recent meta-analysis compared nine drugs for the

treatment of GAD in the United Kingdom (duloxetine,escitalopram, fluoxetine, lorazepam, paroxetine, pregabalin,sertraline, tiagabine, and venlafaxine). The investigatorsfound that fluoxetine was ranked first for response andremission (probability of 62.9 and 60.6%, respectively),whereas sertraline was ranked first for tolerability (49.3%)(Baldwin et al., 2011). These findings are generally in linewith current practice, in which SSRIs are often the

first-line treatment of a range of anxiety disorders. An-other review in the United States assessed data fromplacebo-controlled clinical trials of BZDs, azapirones,antidepressants, and anticonvulsant and antipsychoticdrugs, and concluded that patients with general anxietydisorder should receive either an SSRI or SNRI as first-line treatment (Reinhold et al., 2011).

F. Neuroactive Steroids as Neuromodulatorsof Transmission

Neuroactive steroid modulation of GABAergic neu-rotransmission in the central amygdala has beenimplicated in anxiety (Wang et al., 2007). There is someevidence that fluoxetine increases brain levels of theneurosteroid allopregnanolone, a positive allosteric mod-ulator of GABAARs (Matsumoto et al., 1999), and it hasbeen suggested that the anxiolytic effects of fluoxetineand possibly other SSRIs (e.g., paroxetine) are related tothis effect on allopregnanolone (Uzunova et al., 1998;Griffin and Mellon, 1999; Khisti and Chopde, 2000). Thishypothesis is supported by a recent study in a mousemodel of social isolation, which found that an SSRI-induced increase in allopregnanolone significantly con-tributes to the anxiolytic effects of SSRI treatment(Pinna, 2010). It is therefore of interest to take a closerlook at the mechanisms of action and modulatory sites ofneuroactive steroids, such as allopregnanolone.

The search for an endogenous system of new targetsfor modulation has been intensive, and neuroactivesteroid-based options offer some promise. Selye (1970)first demonstrated that endogenous steroids and theirmetabolites can produce rapid effects on CNS activity,including modulation of GABAergic neurotransmissionassociated with anxiety (Lambert et al., 2001, 2003;Yang et al., 2002).

The potential effects of neuroactive steroids on thedevelopment of anxiety disorders can be observed duringpuberty, a developmental stage associated with increasedlevels of reproductive hormones and onset of manypsychiatric disorders, including GAD, social anxiety,and panic attacks (Hayward and Sanborn, 2002; Kessleret al., 2005a; Merikangas et al., 2009, 2010; Reardonet al., 2009; Van Oort et al., 2009). Anxiety symptomshave been found to increase from middle to lateadolescence (Van Oort et al., 2009), with a particularlyhigh prevalence of all anxiety disorders reported amongadolescent girls (Merikangas et al., 2009; Leikangeret al., 2012; Legerstee et al., 2013).

Of particular interest in this setting are the neuroac-tive steroids allopregnanolone (in humans and rats) andpregnanolone (in humans only), metabolites of thereproductive hormone progesterone that are also pro-duced in the brain in response to stress (Purdy et al.,1991; Gïrdler et al., 2001). Allopregnanolone decreasesin the cerebral spinal fluid of women with PTSD(Rasmusson et al., 2006), but increases in depressedpatients receiving SSRIs (Uzunova et al., 1998). Moreover,


administration of sodium lactate and cholecystokinin-tetrapeptide to persons with panic disorder–inducedattacks decreases plasma concentrations of bothpregnanolone and allopregnanolone, and increases theconcentration of the functional antagonistic isomer3b,5a-tetrahydroprogesterone (Ströhle et al., 2003).Together these findings suggest that changes in endoge-nous brain levels of neuroactive steroids associated withage, sex, stress, and administration of SSRIs may playa role in the onset and clinical response to treatment incertain anxiety disorders.Supporting this hypothesis are findings related to

the effects of allopregnanolone on gonadotrophin hormone-releasing hormone, the primary chemical messengerimplicated in the onset of puberty and sexual matura-tion (Moguilevsky and Wuttke, 2001). Allopregnanolonehas been found to suppress the release of hypothalamicgonadotrophin hormone-releasing hormone via alloste-ric modulation of GABAARs (Calogero et al., 1998; Simet al., 2001). Although this inhibition is increased byallopregnanolone administration before puberty and inadulthood, it is paradoxically reduced during puberty,leading to increased excitability of pyramidal cells inhippocampal region CA1. This effect appears to be duein part to inhibition of a4-containing GABAARs, whichare expressed at higher levels than normal in the CA1region of the hippocampus during puberty.It has also been shown that GABAARs of the a4b2d

subtype, which have a d subunit instead of a g subunit,play a role in tonic inhibition in areas such as the dentategyrus and cortex (Wisden et al., 1992). GABAAR-mediatedconductance is normally inhibitory; however, the reversalpotential of GABAAR-mediated postsynaptic current indentate gyrus granule cells is “positive” to the restingmembrane potential, making membrane hyperpolar-ization of GABAARs unlikely. Inhibition of shuntingappears to play a role in overcoming this process, such

that nonhyperpolarizing inhibitory conductance re-duces the depolarizing effect of postsynaptic poten-tials by decreasing proximal membrane resistance(Staley and Mody, 1992).

In the dentate gyrus and cortex, the GABAergiccurrent is inward (i.e., chloride flux is outward) (Staleyand Mody, 1992; Gulledge and Stuart, 2003); thus,inhibition in these areas is enhanced by allopregnanolone.However, in the CA1 region, the current is normallyoutward (Alger and Nicoll, 1982); thus, increasedexpression of a4b2d GABA receptors paradoxicallyresults in allopregnanolone attenuating rather thanenhancing inhibition. The reduction in current generatedby allopregnanolone at a4b2d GABAAR is dependentupon the presence of arginine 353 in the intracellularloop of a4, where it may serve as a chloride modulatorysite (Shen et al., 2007). This polarity-dependent decreasein inhibition mediated by allopregnanolone may haveimportant implications for how we approach the treat-ment of anxiety disorders in the future.

The modulation of GABAergic neurotransmission byneuroactive steroids is mediated by interactions withallosteric sites on GABAAR (Lambert et al., 2001,2003; Yang et al., 2002; Hosie et al., 2006). The in-teraction of certain neuroactive steroids with GABAARis stereoselective, suggesting that the binding sitesfor these compounds are of a specific dimension andshape (Harrison and Simmonds, 1984; Park-Chung et al.,1994; Covey et al., 2000). There is also evidence that thetransmembrane domains play a role in neuroactivesteroid–mediated modulation of GABAARs (Hosie et al.,2006) (Fig. 9), and that the effects of neuroactive steroids onGABAARs may be associated with its actions at d subunit–containing receptors (Mihalek et al., 1999, 2001; Spigelmanet al., 2003; Stell et al., 2003). Although changes in thesereceptors have not been associated with alterations inbehaviors considered indicative of vigilance or possibly

Fig. 9. Neurosteroid activity at the GABAAR is determined by a-subunit M1 domain residues. (A) The M1 through the end of M2 murine a1 and b2subunits were replaced with the corresponding sequence from the resistance to dieldrin (RDL) subunit, forming the chimeras aR and bR, respectively,which were previously shown to have very low sensitivity to potentiation and to lack direct activation by micromolar concentrations of neurosteroids.Polar residues present only in a1 (blue) are in bold, with Thr236 and Gln241 (red) highlighted. The transmembrane domains are boxed. Thepharmacology of receptors containing the modified a subunit was then compared with that of wild-type receptors using whole-cell recordings fromhuman embryonic kidney cells. (B) THDOC concentration–response curves for direct activation (red) and for potentiation (blue) of EC10 (concentrationcausing 10% of maximal response) GABA currents expressed as a percentage of the maximum GABA response, at wild-type a1b2g2 (squares) andchimeric aRb2g2 (circles) receptors showing that potentiation and direct activation by THDOC and allopregnanolone is abolished on receptorsincorporating aR. (C) Structures of ALLOP and THDOC. ALLOP, allopregnanolone; THDOC, tetrahydrodeoxycorticosterone. Reprinted withpermission from Hosie et al. (2006).


anxiety in animals (Mihalek et al., 1999), evidencestrongly suggests that a change in subunit subtype froma1bxg2 to a4bxg2 increases receptor desensitization anddecreases GABAergic inhibition, a process that mayplay a role in epilepsy (Roberts et al., 2005, 2006; Lundet al., 2008; Grabenstatter et al., 2012). Could sucha change in subunit composition also underlie increasedanxiety and serve as a biomarker to identify patientsresistant to existing anxiolytic therapeutics? Couldswitching of receptor subunit subtypes—perhaps involvingthe a4 subunit, which is insensitive to BZD positivemodulators—be a control point in anxiolysis as in epilepsy?Both allopregnanolone and pregnenolone have been

implicated in the prevalence of anxiety disorders inmen and women (Le Mellédo and Baker, 2004), and arange of preclinical studies suggest potential mechanisticrationales for the anxiolytic effects of these neuroactivesteroids. It has been shown that allopregnanoloneexhibits anticonflict effects pharmacologically at 8 mg/kgin rats (Brot et al., 1997), and that it attenuates certaineffects of caffeine in rats indicative of vigilance or anxiety(Jain et al., 2005). Pregnanolone potentiates electro-physiological responses to and binding of muscimol toGABAARs (Majewska et al., 1988; Wu et al., 1993;Park-Chung et al., 1994), and exhibits sedative hypnoticproperties via its likely interaction with GABAARs(Wang et al., 2001). Overall, these observations suggestthat allopregnanolone and pregnanolone act as endoge-nous modulators of GABAergic neurotransmission invivo (Lambert et al., 2001, 2003; Yang et al., 2002;Ströhle et al., 2003). In support of the hypothesis thatallopregnanolone and pregnanolone have anxiolyticproperties, two small studies suggest that the plasma

concentrations of pregnenolone sulfate, a precursor ofthese steroids, is lower in males with generalized socialphobia or GAD (Semeniuk et al., 2001; Heydari and LeMellédo, 2002). Administration of pregnenolone is asso-ciated with an increase in allopreganolone and reducedactivity in the amygdala and insula, as well as a reductionin self-reported anxiety (Sripada et al., 2013) (Fig. 10).

Because they have poor bioavailability and canpotentially be metabolized to hormonally active ste-roids, endogenously occurring neuroactive steroidshave limited therapeutic use in patients. Syntheticanalogs of neuroactive steroids, which are more re-sistant to metabolism and better able to cross theblood–brain barrier, are now being investigated forpotential use as anxiolytics, anesthetics, and anticon-vulsants (Weaver et al., 1997; Gasior et al., 1999). Onesynthetic neurosteroid, pregnanolone hemisuccinate,produces sedation in mice and rats (Weaver et al.,1997), raising the possibility that it acts either byinhibiting the NMDA receptor or by crossing the blood–brain barrier and undergoing metabolism to pregnanolone.Synthetic neurosteroids bearing a hemisuccinate groupare more resistant to hydrolysis than the correspond-ing sulfate esters and are partly unionized at physiologicpH, allowing ready passage across the blood-brainbarrier.

Ganaxolone (3-hydroxy-3-methyl-5-pregnane-20-one),an orally active synthetic analog of allopregnanolone, isa positive allosteric modulator of GABAAR that hasshown promising basic and early stage clinical outcomesand is currently under investigation as a novel treat-ment of epilepsy and PTSD (Carter et al., 1997; Gasioret al., 1999; Reddy and Rogawski, 2012). Another

Fig. 10. Functional MRI studies showing that administration of PREG (400 mg) is associated with a reduction in activity in regions associated withgeneration of negative emotion. (A) Compared with placebo, pregnenolone administration decreased activation in right amygdala and right insula. (B)Compared with placebo, pregnenolone administration increased dorsal medial prefrontal cortex activation during appraisal. The percent signal changeis displayed next to each figure. PBO, placebo; PREG, pregnenolone administration group. Reprinted with permission from Sripada et al. (2013).


synthetic analog of allopregnanolone, Co 3-0593 (3b-ethenyl-3a-hydroxy-5a-pregnan-20-one), was found to have anxi-olytic effects comparable to BZDs after both subcutane-ous and oral administration in rodents. An absence oftolerance to this drug is suggested by the fact that itseffects were maintained even after chronic administration(Wieland et al., 1997).Although these data suggest that anxiety disorders

may be amenable to treatment with synthetic neuro-steroids or through modulation of the synthesis ofendogenous neuroactive steroids, the more compellingvalue of these agents to date has been the insights theyhave provided into the pathophysiology of anxiety, bothinfluencing and being influenced by the GABAergic,serotonergic, and other neural systems in a range ofanxiety disorders.

VI. Conclusions

“Personalized medicine” is now a familiar concept, notonly in the scientific literature but also in the popularmedia. The concept is so familiar, in fact, that some arenow questioning whether the term has outlived itsusefulness, implying both too much and too little: toomuch in the sense that unique treatments are not beingdeveloped for every individual patient, and too little inthe sense that personalized medicine is what cliniciansalready aspire to every time they treat a patient(Katsnelson, 2013). Regardless of the term we use todescribe this aspiration, it is certainly true that the morewe are able to tailor treatments to a patient’s individualprofile, the greater the chance of clinical success. In thecases of mental disorders such as anxiety or depression,the idea of individualized approaches to therapy take oneven greater urgency, given the uniquely personal andcomplex mental processes that underlie the disorders.We believe that an adequate response to these

challenges will require a systems-based approach toresearch. Although traditional target-based discoveryhas shown success in identifying new drugs againstpreviously validated targets, the reductionist nature ofthis approach often leads to late-stage failure when thedrug is assessed in increasingly complex systems. Bycontrast, a systems-based approach may speed the drugdiscovery process by focusing less on drug interactionswith single targets and more on systems-level assaysrepresentative of the disease (Desbiens and Farb, 2012).A “pharmacologic connectome” is emerging based

upon which disorder-specific novel therapeutic agentsfor anxiety can be developed (Fig. 11). Functionalneuroimaging studies are revealing the brain regionsand nuclei implicated in specific anxiety disorders andare providing important clues about which pharmaco-logical interventions are the most appropriate for whichdisorders. The use of drugs that enhance extinction isgaining acceptance for the treatment of specific phobiasand the intrusive memories associated with PTSD

(Rothbaum et al., 2014). Compounds that inhibit excit-atory neurotransmission or enhance inhibitory neuro-transmission are proving to be more effective for treatingGAD and the hypervigilance associated with PTSD(Mathew et al., 2005). At the same time, we are beginningto understand that because of genetic polymorphisms,some patients may not respond to SSRIs and SNRIs(Zanardi et al., 2001). The opportunity to apply thisgrowing body of knowledge to the development of noveltherapeutics for anxiety disorders has not been greater.

Neuroactive steroids, which modulate various neuro-transmitter pathways and are a locus for the neuralcrosstalk involved in anxiety disorders, may prove to be animportant tool for advancing a systems-based approach todrug development. The unique place of neuroactivesteroids in this setting is given further support bya growing body of evidence showing that these agentsare important for both improving the survival of neuronsand increasing the number of newly formed neurons(Charalampopoulos et al., 2008).

Fig. 11. A pharmacologic connectome that depicts how specific brainregions and nuclei respond to therapeutic agents via connectivity mapsactivated in anxiety is beginning to be assembled as the human brain isdeconstructed. Episodic memories associated with anxiety-inducing eventsare stored in the parietal lobes (precuneus). The amygdala assigns an“appropriate” emotional-response tag to a stimulus, such as a smiling or anangry face. The frontal lobes in turn provide for the “top-down regulation”of emotional responses to these same stimuli. The cingulate and insulaform the major association pathways between the various regions involvedin anxiety, whereas the BNST connects the amygdala to the HPA axis,which in turn regulates how the endocrine system responds to the stimulus.In anxiety disorders that are triggered by a specific stimulus (e.g., phobiasand PTSD), the amygdaloid-mediated response to the stimuli is highlymemory-dependent. In disorders of increased apprehension and persever-ative cognition (e.g., GAD), the frontal lobe and top-down modulationof amygdaloid nucleus function would be altered.


The results reviewed in this article suggest thatcurrent research into brain activation and therapeutictargeting of GABA, 5-HT, and NMDA receptor subunitshas not produced a silver bullet for treating anxiety thatoptimizes efficacy and reduces risk of adverse events.Moreover, true personalized medicine in patients suffer-ing from various anxiety disorders would require acomprehensive and multifaceted approach beyond anal-ysis of neurologic activation. This could include assess-ment of patient demographics, clinical presentation,environmental factors, family history, relevant geneticpolymorphisms, and biomarkers, as well as treatmentoptions encompassing not only pharmacotherapy butalso psychotherapeutic approaches such as CBT andbehavioral activation therapy. Nevertheless, researchinto the brain regions and variations in receptorsubunits engaged in anxiety provides reason for opti-mism that the pathophysiology underlying this complexmosaic of perceptions will lead to treatments bettertailored to the individual needs of patients sufferingfrom a range of anxiety disorders based on results fromnoninvasive brain imaging and improved therapeutics.

Acknowledgements

The authors thank Dr. Cynthia Czajkowski for Fig. 7 and Dr. ConorC. Smith for artwork in Fig. 8.

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Farb, Ratner.

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