Anxiety Disorders: Macromolecular Pathways and Interactions
Miklos Toth, Department of Pharmacology, Weill Medical College of Cornell University, New York, New York
Fear and anxiety can be a normal adaptive reaction to help cope with stress in the short term, but when the emotional, cognitive, and physical manifestations are long lasting, extreme, and disproportionate to threat, whether real or preceived, anxiety is maladaptive and has become a disabling disorder. Anxiety disorders may be deconstructed to elementary behaviors/symptoms that can be conceptualized as quantitative characters determined by the combined effects of several risk genes and nongenetic factors (e.g., early-life adversity). Progress in neurogenetics, molecular and cellular neuroscience, and neuroimaging is beginning to yield significant insights of how genetic and nongenetic factors contribute to specific manifestations of anxiety disorders. The aim of this overview is to summarize and integrate the current knowledge on anxiety-related macromolecular pathways and mechanisms initiated by genetic risk and envionmental factors. These pathways interact with each other, often during specific periods of development, and could lead to alterations in the formation and function of neuronal circuits that encode emotional behavior.
Anxiety is a state characterized by feelings of fear, apprehension, and worry. Emotionally, anxiety causes a sense of dread or panic, and behaviorally, it can be associated with both voluntary and involuntary behaviors such as escaping or avoiding the source of anxiety. Also, anxiety is associated with specific physical manifestations including increased heart rate and blood pressure. Although anxiety is clinically different from depression, the two disorders are often comorbid. Indeed, it is generally difficult to find individuals with pure anxiety and pure depression. Here we focus on anxiety exclusively but will mention and discuss depression when the separation is not clear. Anxiety, similarly to other psychiatric disorders, has a significant genetic basis, and it is relatively stable during lifetime (trait anxiety). Recent studies implicated candidate genes, each with a small contribution to the risk of anxiety disorders (1). Several of these candidate genes encode proteins that regulate neurotransmitter synthesis and metabolism or correspond to neurotransmitter receptors. Studies implicate an equally important role for environmental factors, including early-life adversity and maternal care, in the development of anxiety and anxiety-like behavior in animals (2-4). Some of these environmental effects also converge on neurotransmitter systems. Because the risk genes or environmental factors individually represent only a small contribution to anxiety, the current view is that interactions between several risk genes and environmental effects are necessary to lead to the symptoms of anxiety. This overview focuses on molecular systems related to anxiety such as neurotransmitters, their receptor, and, when known, downstream intracellular signaling pathways that ultimately regulate the development and/or function of neuronal networks that underlie the behavioral anxiety response.
Neuroticism and Anxiety
Neuroticism is a personality trait characterized by an enduring tendency to experience negative emotional states. Personality traits are underlying characteristics of an individual that can explain the major dimensions of human behavior. Neuroticism represents a continuum between emotional stability and instability, and most people fall in between the extremes (5-9). Neuroticism has a wide individual variability, but it is relatively stable in individuals over time (10). People with a high level of neuroticism respond more poorly to stress and are more likely to interpret ordinary situations as threatening and frustrating. In addition, autonomic arousal is an integral part of neuroticism characterized by increased heart rate and blood pressure, cold hands, sweating, and muscular tension. Extroversion and openness, two other personality traits, are also part of the NEO Personality Inventory (NEO-PI) and the revised (R) NEO-PI (11), and Gray and McNaughton (12) argued that anxiety proneness is primarily captured by measures of neuroticism, together with a smaller contribution from the dimension of extroversion. Specifically, rotating the dimensions of neuroticism and extroversion by 45°, two new dimensions, anxiety (N+, E-) and impulsivity (N+, E+), were proposed (12).
Although neuroticism is not a disease per se, it predisposes individuals to anxiety disorders (12, 13). Neuroticism is a vulnerability factor for all forms of anxiety (14-16). A system established by the Diagnostic and Statistical Manual for Psychiatric Disorders in the United States, currently in its 4th edition (DSM-IV) text revision (TR) (American Psychiatric Association, 2000), sets the boundary at which a particular level of behavior becomes an anxiety disorder—a level often based on the number and the duration of symptoms. DSM is a categorical system based on the qualitative separation of disease states from the state of well-being. The DSM-IVTR category of anxiety disorders currently includes generalized anxiety disorder (GAD), simple phobia, posttraumatic stress disorder (PTSD), panic disorder, social phobia, and obsessive compulsive disorder (OCD) as discrete anxiety disorders. The International Classification of Diseases-10 (IC-10) is a similar system, but it is less frequently used in research (17).
Although DSM-IV and IC-10 are widely used for the clinical diagnosis of anxiety disorders, these categorical models are not based on the underlying biological pathophysiology and may not be optimal for the identification of genetic and environmental factors involved in anxiety disorders. The main reason is the complexity and heterogeneity of anxiety disorders that makes the association of genetic and/or environmental effects with overt anxiety phenotypes difficult. An alternative approach is to deconstruct anxiety disorders to elementary behavioral manifestations or specific symptoms that may represent less complex biological traits and/or environmental influences. The rationale is that a specific phenotype that consists of few elementary behaviors is likely linked to a more limited number of genes and environmental effects than complex disease phenotypes. These elementary behaviors are primarily state-independent (manifest in an individual regardless of whether illness is active). Similar to this principle is the term “endophenotype,” although it usually refers to an internal phenotype along the pathway between the genotype and disease. Evidence suggests that elementary behaviors/endophenotypes of psychiatric disease may be understood as quantitative traits. This model is based on the notion that risk gene variants carried by a given individual in combination with various nongenetic factors such as early-life adversity and nutrition produce anxiety symptoms with variable onset and severity depending on the strengths of the genetic and environmental factors (Fig. 1).
Figure 1. The quantitative trait model of neuroticism and anxiety.
Genes, Their Protein Products, and Associated Biological Pathways Implicated in Anxiety and Anxiety-Like Behavior
Using the techniques of quantitative behavior genetics, it became clear that roughly 20-60% of the variation in most personality traits has a genetic base, and broad personality traits are under polygenic influence (18, 19). Similarly, genetic epidemiological studies estimate that heritability in anxiety varies between 23 and 50% (1, 20, 21). Recently, genome-wide linkage studies have been performed by using Eysenck personality questionnaire (EPQ) (22, 23) to identify chromosomal regions associated with neuroticism. A two-point and multipoint nonparametric regression identified 1q, 4q, 7p, 8p, 11q, 12q, and 13q (24), whereas another similar study using multipoint, nonparametric allele sharing and regression identified 1q, 3centr, 6q, 11q, and 12p (25), which confirms some links in the previous study. Still another study using a broad anxiety definition instead of the DSM-IV classification identified a linkage at chromosome 14 between 99 and 115 cM (26), and this finding replicated a linkage for a broad anxiety phenotype in a clinically based study (27). However, these studies have not yet identified specific genes.
So far, the candidate gene approach has been more productive than linkage and association studies in implicating gene polymorphisms related to neuroticism and anxiety disorders. The candidate gene approach relies on prior biochemical studies that implicate various molecules, primarily neurotransmitters, their receptors, and signaling in anxiety. Anxiety disorders traditionally have been viewed as disturbances in neurotransmitter systems including the serotonin (5-HT), gamma-aminobutyric acid (GABA), and corticotropin releasing hormone (CRH) systems, among others. Many of these neurotransmitters and their receptors have also been identified as sites of action for anxiolytic drugs. Neurotransmitter systems and corresponding neurobiological pathways that are well established in anxiety both in human studies and animal experiments are discussed below.
Animal studies, especially rodent studies, significantly contributed to the current knowledge on how genes, the environment, and their interaction may produce anxiety. As people with a high level of neuroticism respond more poorly to stress, certain inbred, selectively bred (28, 29), and knockout rodent strains (see below) have increased emotional reactions to stress (emotionality). These behavioral responses include avoidance, hypoactivity/freezing, and autonomic arousal among others. Although substantial similarities are found between human and murine stress responses, complex anxiety phenotypes cannot be reproduced in animals. Nevertheless, emotional stress reactions in animals represent relatively simple behaviors that are evolutionarily conserved and quantifiable. A multitude of tests can measure emotionality in rodents (30-34). Although all behavioral tests use a novel environment and/or fearful situation to produce avoidance, hypoactivity/freezing, and/or autonomic arousal, it seems that the underlying pathophysiology is test specific. Indeed, data with recombinant inbred mouse strains indicate that open field and elevated plus maze behaviors are linked to specific but partly overlapping sets of quantitative trait loci (35, 36). Furthermore, even these relatively simple behaviors can be dissected to more elementary behaviors with a smaller assembly of quantitative trait loci (36). However, it is not clear currently if mouse QTL data can be directly extrapolated to humans.
Many recently developed genetically manipulated mouse strains exhibit increased emotionality, often referred to as anxiety-like behavior. Most strains have constitutive genetic inactivation, but some strains are also available with a conditional allele. Although constitutive gene inactivation may elicit compensatory processes that can complicate the phenotype, this occurrence is not necessarily a disadvantage. Indeed, genetic risk in humans is also constitutive and present from early life. Probably more important is that polymorphisms in risk genes do not cause a complete functional loss; therefore, it may be more appropriate to analyze the behavioral phenotype of mice with a heterozygous inactivation of the risk gene.
The 5-HT pathway and associated genes involved in anxiety
5-HT has long been associated with emotion and anxiety (37). 5-HT is synthesized from tryptophan by the rate-limiting enzyme tryptophan hydroxylase (TPH) in serotonergic neurons in the raphe. Release of 5-HT is controlled by the 5-HT1A and 5-HT1B autoreceptors located at the somatodendritic compartment and axon terminals, respectively. In addition, the synaptic and extracellular levels of 5-HT are regulated by the 5-HT transporter (5-HTT). Genetic risk for anxiety has been associated with all of these macromolecules.
The two isoforms of TPH are as follows: 1) the classical TPH isoform, now termed TPH1, that is detected in the periphery, especially in the duodenum and in blood but not in the brain, and 2) the relatively recently identified TPH2 expressed exclusively in the brain (38). Several common single nucleotide polymorphisms are found around the transcriptional control region of TPH2. T allele carriers of a functional polymorphism in the upstream regulatory region of TPH2 (G-703 T) were found to be overrepresented in individuals with anxiety-related personality traits (39,40). In agreement with these data, T carriers have been shown to exhibit relatively greater activity in the amygdala than do G-allele homozygotes to affective facial expressions (41). Activation of the amygdala, indicated by increased blood flow, is a typical reaction to stress and to unpleasant and potentially harmful stimuli.
The serotonin transporter
The transporter removes 5-HT after its release into the synaptic cleft and returns it to the presynaptic terminal, where it is metabolized by monoaminoxidases or stored in secretory vesicles and results in the termination of postsynaptic serotonergic effects (42). 5-HTT is the target of the selective serotonin reuptake inhibitors (SSRIs) that have been shown to be effective in certain anxiety disorders and depression (43).
The 5-HTT transporter belongs to the family of Na+/Cl--dependent transporters, which shows a certain degree of similarity with the GABA and the dopamine transporters (42, 44). The 5-HTT has 12 transmembrane domains (TM) with a large extracellular loop between TMs 3 and 4. Both the N- and C-termini are located within the cytoplasm. Growing evidence suggests that 5-HTT forms a homomultimer in the plasma membrane, although most studies suggest an autonomous function for each monomer (45). Amino acid substitution experiments indicate the importance of TM1 as an important contributor to substrate, ion, and inhibitor interactions (46). In addition to TM1, TM3 has also been shown to play a role in substrate and inhibitor binding (47). Recently, it has been demonstrated that Tyr-95 and Ile-172 in transmembrane segments 1 and 3 of human 5-HTT interact to establish the high-affinity site for antidepressants (48). Although SSRIs have a rapid action on the transporter, their anxiolytic and antidepressant actions are delayed. It is believed that one factor involved in this delay is the activation of 5-HT1A autoreceptors on serotonergic neurons that effectively reduces neuronal firing and 5-HT release rebalancing extracellular 5-HT levels (43). As the SSRI treatment is prolonged, the 5-HTiA autoreceptor desensitizes, and firing activity is restored. The notion that an adaptive change underlies, at least partly, the delayed therapeutic effect of SSRIs is supported by the acceleration of the anxiolytic and antidepressant response by the concomitant administration of the 5-HT1A autoreceptor antagonist pindolol (49). Once 5-HT levels are significantly increased during chronic SSRI administration, it is assumed that the elevated 5-HT levels act on specific 5-HT receptors to elicit anxiolytic and antidepressant effects. Although at least 13 different types of 5-HT receptors exist in mammals, a recent report using a specific behavioral test (novelty induced suppression of feeding) showed that the presence of postsynaptic 5-HT1A receptors is necessary for the anxiolytic effect of the SSRI fluoxetine (50).
Lesch et al. demonstrated that a functional 5-HTT promoter polymorphism is associated with the factor “neuroticism” in the revised NEO personality inventory (51). Specifically, individuals who carry the s/s (short promoter repeat) alleles of the 5-HTT, as compared with individuals with s/l (long) or l/l alleles, have increased neuroticism. Extension of these genetic studies to anxiety disorders by the same authors showed no differences in 5-HTT genotype-distribution between anxiety patients and comparison subjects, but among anxiety patients, carriers of the s/s alleles exhibited higher neuroticism scores (52). Several later reports found association between the 5-HTT promoter polymorphism and anxiety-related traits (53-55). Also, recent meta-analyses of several prior studies found a small but significant association between 5-HTT polymorphism and some but not all measures of neuroticism/anxiety (56-58). Interestingly, imaging data found a significant increase in amygdala activity in subjects who carry the 5 allele, which indicates a functional link between 5-HTT polymorphism and anxiety (59). Consistent with these data, genetic inactivation of the 5-HTT gene in mice results in an increased anxiety-like phenotype (60).
The 5 allele is associated with reduced 5-HTT mRNA expression in vitro and in lymphoblasts (51), but functional imaging studies and postmortem samples generally show no effect of the 5-HTT genotype on 5-HTT expression in adult subjects (61, 62). To resolve this discrepancy, it has been hypothesized that the 5 allele acts primarily during development and less so in adult brain and would primarily affect brain development leading to anxiety in later life (Fig. 2). Consistent with this idea, pharmacological blockade of the 5-HTT in rats and mice during early postnatal life (that mostly corresponds to the third trimester in human) results in changes in emotional behavior (63, 64). However, it is not known how increased 5-HT levels during development lead to lifelong anxiety nor what 5-HT receptors are involved.
Figure 2. Anxiety pathways and their interactions. Two fundamentally different mechanisms involved in anxiety and anxiety-like behavior are proposed. The first mechanism is developmental, and its consequences are manifested later in life. Both genetic and environmental factors linked to anxiety can have a developmental origin, as illustrated by the example of the s allele of the 5-HTT, the deficiency in the 5HTIA receptor, and variability in maternal care. The second possible mechanism is not developmental but based on ''acute'' or current molecular abnormalities that result in anxiety or anxiety-like behavior. Examples include deficiencies in GABAergic neurotransmission and abnormalities in the central CRH system, which have the direct behavioral output of anxiety. It is also proposed that the developmental mechanisms lead to anxiety by converging on the ''acute'' mechanisms.
The 5-HT1A receptor
The G-protein-coupled 5-HT1A receptor emerged as another candidate gene in anxiety as early studies indicated a deficit in the receptor in panic disorder patients (65, 66). These data seem to be consistent with the anxiolytic effect of partial 5-HT1A receptor agonists in the treatment of generalized anxiety disorder, but the pharmacological inhibition of the receptor in animals does not elicit anxiety (67). Then in 1998, it was shown that the genetic inactivation of the 5-HT1A receptor in mice results in enhanced anxiety-like behaviors (68-70) alongside reduced immobility in the forced swim test (70) and tail suspension test (68, 69). Anxiety-related tests in these and follow-up studies included open field, elevated plus maze, zero maze, novelty-induced suppression of feeding, and some fear conditioning paradigms (68-71). More recently, human studies strengthen the association between a 5-HT1A receptor deficiency and certain forms of anxiety. A preliminary neuroimaging study reported a significant negative correlation between 5-HT1A receptor binding in the dorsolateral prefrontal, anterior cingulate, parietal, and occipital cortices and indirect measures of anxiety in healthy volunteers (72). Then, reduced receptor levels were reported in the anterior cingulate, posterior cingulate, and raphe by positron tomography in patients with panic disorder (73). No such association was found in posttraumatic stress disorder (74).
5-HT1A receptors are expressed at both postsynaptic locations in 5-HT target areas (including the amygdala, hippocampus, and cortex) and presynaptic sites on 5-HT neurons in the raphe nuclei as somatodendritic autoreceptors. Because autoreceptors control neuronal firing and consequently 5-HT release, and because an increase in extracellular 5-HT levels during development (see previous section) has been implicated in anxiety, it initially was believed that the anxiety-like phenotype of 5-HT1A receptor-deficient mice was because of increased 5-HT release. However, basal 5-HT levels are not altered, as measured by in vivo microdialysis, in 5-HT1A receptor-deficient mice, presumably because of the compensatory action of presynaptic 5-HT1B receptors (75-77), and expression of 5-HTIAreceptors in forebrain regions rescued the anxiety phenotype of 5-HTIA receptor knockout mice (78), which suggests that the behavioral phenotype results from the absence of postsynaptic rather than the presynaptic 5-HTIA receptors. The anxiety-like phenotype of 5-HTIA receptor-deficient mice has been shown to be associated with genetic background-specific perturbations in the prefrontal cortex GABA-glutamate system and in GABAA receptor subunit expression in this region as well as in the amygdala (79, 80) (Fig. 2). Our recent studies implicate hippocampal abnormalities in some manifestations of the anxiety-like phenotype in 5-HTIA receptor-deficient mice (unpublished data). Presumably the lack of 5-HTIA receptors during development at these postsynaptic areas leads to neuronal network abnormalities that underlie the exaggerated behavioral responses in anxiogenic environments.
Considering the many studies with 5-HTT polymorphism (see above), it is surprising that relatively little is known about 5-HT1A receptor alleles in the context of anxiety. Although the common genetic polymorphism (-1019 C/G) in the promoter region of the 5-HT1A receptor gene has been associated with panic disorder (81), more is known about the link of this polymorphism to depression/suicide (82) (but see Reference 83) and to antidepressant treatment response (84, 85). Nevertheless, the -1019 C/G polymorphism has been found to have functional effects on gene expression. Specifically, the 5-HT1A G(-1019) allele fails to bind Deaf-1 and Hes5, two transcriptional repressors, in raphe-derived cells leading to upregulation of autoreceptor expression. Consistent with these data, an increase in 5-HT1A autoreceptor expression in individuals with the G/G genotype has been observed (86, 87). However, as mentioned earlier, autoreceptors may not be implicated in anxiety-like phenotype, at least not in the mouse model. Deaf-1 has an opposite effect in neurons that model the postsynaptic site as it enhances 5-HT1A receptor promoter activity, and this enhancement is attenuated in the presence of the G allele (88). This cell-specific activity of Deaf-1 in 5-HT1A gene regulation could account for the region-specific decrease of receptor level in postsynaptic 5-HT1A receptor described in anxiety and mood disorders (see above).
The GABA pathway
Abnormalities in GABA levels have been noted in subjects with anxiety disorders. Magnetic resonance spectroscopy studies have shown lower occipital cortex GABA concentrations in subjects with panic disorder (89) and with major depression (90), compared with healthy controls. GABA is synthesized from glutamate by glutamic acid decarboxylase (GAD). Its two isoforms, GAD65 and GAD67, are products of two independently regulated genes, GAD2 and GAD1, respectively. GAD65 is responsible for the synthesis of a small pool of GABA associated with nerve terminals and synaptic vesicles and can be rapidly activated in times of high GABA demand (91, 92). In contrast, GAD67 is responsible for the majority of GABA synthesis, and it is important in developmental processes (92). GABA interacts with the ionotropic GABAA and metabotropic GABAB receptors. GABAA receptors in particular have been associated with anxiety as they control neuronal excitability. The GABAA receptor is a pentameric ion channel composed of subunits from seven families (α1-6, β1-3, γ1-3, δ, ε, θ, and ρ1-3) (93). Receptors that express α1 and α2, together with a β subunit and the γ2 subunit, are the predominant forms in the central nervous system.
GAD and anxiety
GABA neurotransmission has been linked to anxiety disorders, and therefore genes that encode GAD are reasonable candidate susceptibility genes for these conditions. In a multivariate structural equation model, several of the six single-nucleotide polymorphisms tested in the GAD1 region (encoding GAD67) formed a common high-risk haplotype that contributed to individual differences in neuroticism and impacted susceptibility across a range of anxiety disorders, including generalized anxiety disorder, panic disorder, agoraphobia, and social phobia, and also major depression (94). No such association was found with single-nucleotide polymorphisms in GAD2 (encoding GAD65) in this study. Nevertheless, genetic inactivation of GAD65 results in anxiety-like behavior in mice (95). These mice also show increased seizure sensitivity, but otherwise they show no overt developmental phenotype (91). It is not possible to study anxiety in GAD67-/- mice because they die of severe cleft palate during the first morning after birth (92). The less severe and more specific phenotype of GAD65-/- mice is probably because the overall GABA content is normal in GAD65-/- tissues and only the K+stimulated GABA release is reduced, whereas in GAD67-/- mice a severe depletion of GABA occurs.
GABAA receptors and anxiety
A deficit in GABAA receptors has been identified in the hippocampus and parahippocampus of patients with panic disorder and generalized anxiety disorders (96-98). Also, GABAA receptor antagonists elicit anxiety in patients with panic disorder, which suggests an underlying deficit in receptor function in these individuals (99).
Animal studies suggest that a reduction in GABAA receptors that contain α4 subunits is associated with withdrawal-induced anxiety (100, 101). Genetic inactivation of some other subunit genes has a similar effect. For example, heterozygote γ2+/- mice have reduced numbers of GABAA receptors and display anxiety phenotype in the elevated plus maze and the dark-light box tests (100, 102). In addition, γ2+/- mice show increased responses in the passive avoidance paradigm. These behavioral alterations are associated with a lower single channel conductance, a pronounced deficit of functional receptors, and a reduction in α2/gephyrin containing postsynaptic GABAA receptor clusters in cortex, hippocampus, and thalamus. Transgenic mice overexpressing either the mouse γ2L or γ2S subunit of the GABAA receptor showed no difference in anxiety-related behavior as compared with wild-type littermates (103). Compensation at the level of GABAA receptor subunit expression and assembly often occurs when subunit expression is disturbed, which may explain the lack of phenotype in these mice. In contrast to the γ2 subunit, the deletion of the also abundant α1 subunit (104) does not increase anxiety or elicit other behavioral abnormalities (105-107), probably because lack of the α1 subunit is compensated and substituted by other a subunits (105). Although α2 subunit-deficient mice have been generated and a point mutation in this subunit (H101 R) abolishes the anxiolytic effect of diazepam (108, 109), it is not entirely clear whether these mice have a change in anxiety-related behavior. Mice deficient for the α2 subunit show a faster habituation to a novel environment, which is not a typical measure of anxiety (110). As mentioned earlier, a strain of 5-HT1A receptor-/- mice have reduced expression of both the α1 and α2 subunits that could explain, at least partly, their increased anxiety phenotype (79) (Fig. 2). “Knock-in” mice in the α5 subunit (H105R) display enhanced trace fear conditioning to threat cues (102). More analysis showed that these knockin mice exhibit a 33% reduction in hippocampal (CA1 and CA3) α5 receptor subunits (102); thus, the phenotype may be simply because of a partial knockout. Finally, the genetic inactivation of β2, a predominant β subunit, resulted in a more than 50% reduction in the total number of GABAA receptors and increased locomotor activity in open field, which suggests that these receptors may control motor activity (106). Taken together, these studies in general support the notion that a deficiency in GABAA receptors results in anxiety and anxiety-like behavior, but it is difficult to assign specific subunit-containing receptors to anxiety.
The BDNF pathway in anxiety
BDNF is a secretory protein that belongs to the neurotrophin family. A large proportion of neuronal BDNF is secreted in the proform (proBDNF), which is subsequently converted to mature (m) BDNF by extracellular proteases such as plasmin or matrix metalloproteinases (111). A functional single nucleotide polymorphism that produces a valine-(Val)-to-methionine (Met) substitution at aa. 66 in pro-BDNF, first described as altering the intracellular trafficking and activity-dependent secretion of BDNF (112), has been studied extensively in association studies. The outcome of these studies has been variable. One study found that the Met 66 may be a risk allele for anxious temperament as measured by the Tridimensional Personality Questionnaire (113). Another study however concluded that the Val 66 allele is associated with greater neuroticism (114), whereas still another has shown no difference in neuroticism between the Val and Met genotypes (115). A more definitive outcome of the Val-Met polymorphism was reported with mice. Because the mouse does not have the Met allele, it was generated by knockin (116). BDNFMet/Met mice exhibited increased anxiety-related behaviors in a variety of behavioral paradigms including the elevated plus maze, open field, and novelty-induced hypophagia tests (116). Although BDNF expression in BDNFMet/Met mice is equivalent to that in BDNFVal/Val mice, a ~30% deficit in activity-dependent release of BDNF from neurons occurs. This deficit suggests that the anxiety phenotype of BDNFMet/Met mice can be linked to the activity-dependent release of BDNF. Similarly to the BDNFMet/Met mice, BDNF+/- mice have an anxiety-like phenotype and have BDNF levels lower than normal (by 50%); thus, it seems that a partial deficit in BDNF is sufficient to elicit anxiety. The receptor for m-BDNF is trkB, a receptor tyrosine kinase (117). Consistent with the increased anxiety-like phenotype of BDNF+/-, conditional BDNF-/-, and BDNFMet/Met mice (116,118), transgenic mice overexpressing trkB in postmitotic neurons in a pattern similar to that of the endogenous receptor display less anxiety in the elevated plus maze test (119).
Similarly to the serotonin-related genes and their polymorphisms, the Met allele of BDNF causes developmental brain abnormalities. Humans heterozygous for the Met allele have smaller hippocampal volumes (120) and perform poorly on hippocampal-dependent memory tasks (112, 121). Consistent with these data, BDNF+/Met or BDNFMet/Met mice have a significant decrease in hippocampal volume as compared with WT mice (116). Also, a significant decrease in dendritic complexity in dentate gyrus neurons occurs in BDNF+/Met and BDNFMet/Met mice. These data raise the possibility that the Met allele contributes to anxiety by modulating brain development (Fig. 2).
The central CRH-CRH-R pathway and anxiety
CRH is a 41-amino acid neuropeptide in mammals (122), and it is an important mediator of the central stress response (122, 123). The biological function of CRH is determined by the aminated end of the peptide’s C-terminus that binds to the extracellular binding pocket of the receptors CRH-R1 and -R2, whereas its N-terminus contacts other sites on the receptor to initiate signaling (124, 125). CRH-R1 in the anterior pituitary is thought to be the subtype through which hypothalamic CRH primarily initiates its effect on pituitary ACTH release (123, 126). ACTH then induces the secretion of corticosteroids in the adrenal. CRH via CRH-Rs, however, has functions outside the hypothalamic-pituitary-adrenal (HPA) axis, in particular in the amygdala. Indeed, data support the notion that extrahypothalamic CRH, presumably via the central noradrenergic systems, is significantly involved in anxiety (127), whereas dysregulation of the HPA axis via hypothalamic CRH seems to be more characteristic for depression (128).
An association between behavioral inhibition and three single nucleotide polymorphisms in the CRH gene, including one in the coding sequence of the gene, was found in children at risk for panic disorder (129). Behavioral inhibition is a trait that involves the tendency to display fearful, avoidant, or shy behavior in novel situations. In animal experiments, the central administration of CRH produces behavioral effects that correlate with anxiety, such as reduced exploration in a novel environment or enhanced fear response (130). Also, an anxiety-like phenotype has been described in transgenic mice overexpressing CRH (131, 132). However, mice with a deleted CRH gene, although they had significantly decreased basal corticosterone levels, showed no anxiety (133, 134). One possible explanation is that the central CRH system is redundant. Indeed, CRH-like peptides, urocortin 1-3 (that also bind CRH-Rs), are present in the CNS (135-138). Two groups have generated mice with a deletion of the urocortin 1 gene (139, 140), but only one of these studies found an increased anxiety-like phenotype (139). Currently, no obvious explanation is found for the significant difference observed between the two urocortin 1-deficient mice. Finally, deletion of the CRH binding protein, that normally binds and inactivates CRH, resulted in increased anxiety (141). The authors hypothesized that the inactivation of CRH-BP may increase the “free” or unbound levels of CRH or urocortin that lead to anxiety.
As described earlier, hypothalamic CRH regulates the HPA axis. Some reports have indicated the dysregulation of the HPA axis in PTSD and panic disorders. Small but significant decreases in plasma cortisol levels and increased HPA axis sensitivity to low glucocorticoid negative feedback signals have been reported in PTSD (142). In contrast, other studies showed persistent increases in salivary cortisol levels in pediatric PTSD patients (143) and significantly greater CRH-induced ACTH and cortisol responses in women with chronic PTSD (144). In panic patients, abnormal HPA axis regulation, including increased basal cortisol secretion and overnight hypercortisolemia, have also been documented (129). These HPA axis changes, however, may not be specific for anxiety disorders but rather reflect the presence of comorbid depression (145).
No significant association was found between an intron 2 polymorphism in CRH-R1 and the “neuroticism” dimension of personality as assessed by the Revised NEO Personality Inventory in healthy Japanese subjects (146). Similarly, three CRH-R2 gene polymorphisms had no association with panic disorder in another study (147).
The role of CRH-Rs in anxiety-like behavior has been studied extensively by using knockout mice. Whereas mice lacking CRH-R1 display decreased anxiety in the light-dark box and the elevated plus maze (148-150), CRH-R2-deficient mice, generated independently by three groups, exhibit varying degrees of anxiety-related behavior. In one study, increased anxiety was reported in the elevated plus maze and open field but not in the light-dark box test (151); another study found anxious behavior in both the elevated plus maze and light-dark box, but only in males (152). However, this latter study showed an increased time spent by the knockout mice in the center of an open field, which is more consistent with reduced anxiety. Still another report found no significant change in anxiety behavior in the elevated plus maze or open field (153). More recently, a mouse with a conditional deletion of the CRH-R1 in forebrain, hippocampus, and the amygdala, but with normal expression in the anterior pituitary, showed markedly reduced anxiety-like behavioral responses in two avoidance tests, and it exhibited normal ACTH and corticosterone secretion to stress (154).
Taken together, some of behavioral data obtained with various CRH and CRH-R knockout mice suggest that extrahypothalamic CRH and/or urocortin mediate a dual modulation of anxiety behavior. Activation of CRH-R1 seems to be anxiogenic, whereas activation of CRH-R2 is anxiolytic. Therefore, it may not be surprising that dual CRH-R1/2 knockout mice have only a subtle behavioral phenotype (155).
CRH signaling and anxiety
CRH-R1/2 are coupled to several signaling pathways, including the adenyl cyclase-protein kinase A (PKA)-CREB and the ERK-mitogen-activated protein kinase (MAPK) pathways (156). These pathways can also be linked to anxiety. For example, genetic inactivation of adenylyl cyclase type 8 results in reduced anxiety-like behavior (157). Mice with mutations of the PKA RIIP subunit and CREB also exhibit abnormal anxiety responses (158-160), and activation of CREB in amygdala produces anxiety-like behavior (160). Thus, anxiety-like responses may be initiated and regulated by Gs-coupled CRH receptor signaling, at least partly, via the cyclic AMP-PKA-CREB pathway. The involvement of the ERK-MAPK pathway can also be implicated in CRH-related anxiety-like behavior. ERK1/2 is strongly activated in hippocampal CA1 and CA3 pyramidal cells and basolateral amygdala by the intracerebroventricular administration of CRH (161), and CRH-induced phosphorylation of ERK1/2 was absent in mice with a conditional knockout of forebrain and limbic CRH-R1 exhibiting a low level of anxiety (162). One may hypothesize that processing of anxiogenic stimuli is altered in the amygdala, hippocampus, or other relevant brain region as a result of abnormal CRH signaling that consecutively leads to the behavioral manifestations of anxiety (Fig. 2).
Environmental Factors and Related Biological Pathways Involved in Anxiety
Stressful life events and anxiety
Although most frequently associated with depression (163), stressful life events have also been linked to anxiety disorders (164-166). Kendler et al. attempted to determine if anxiety, specifically GAD symptoms and depression, are associated with different dimensions (humiliation, entrapment, loss, and danger) of stressful life events associated with high contextual threat. Onset of GAD symptoms was predicted by higher ratings of loss and danger, whereas depression was associated with the combination of humiliation and loss, which indicates that event dimensions that predispose to pure GAD episodes versus pure depression can be distinguished with moderate specificity (164). Unfortunately, little is known about the molecular and cellular mechanisms elicited by stressful life events and how these events predispose an individual to anxiety or depression. However, animal studies provided mechanistic insights of how early-life events can contribute to the development of adult anxiety (discussed below).
Maternal care and adult life anxiety in rodents
Brief “handling” of rat pups results in a lifelong decrease to behavioral and endocrine effects of stress, whereas animals separated from their mothers/litters for longer periods of time, for example, for several hours, exhibit increased anxiety (167). Later studies determined that the critical effect of short-term handling is the increase in maternal care (licking and grooming) after the return of the pups to the nest (3).
More studies showed that rat pups nursed by mothers selected for either a high or a low level of licking and grooming (LG) and arched-back nursing (ABN) exhibit a decreased and increased level of anxiety-like behavior in adult life (open field, novelty-suppressed feeding, and shock-probe burying assays), respectively (3, 168-170). Offspring of high LG-ABN mothers (as well as briefly handled pups) show increased glucocorticoid feedback sensitivity, increased hippocampal GR mRNA expression, and decreased hypothalamic CRH mRNA levels (171). Because the activity of the hypothalamic CRH and the function of the HPA axis may not be directly related to anxiety-like behavior (128, 154), it is not clear whether these mechanisms can explain the anxiety-related behavioral consequences of maternal care. Perhaps more plausible mechanisms are the numerous neurochemical changes related to differences in maternal care. For example, rat pups of high LG-ABN dams show altered GABAA receptor subunit expression in the amygdala, locus coeruleus, medial prefrontal cortex, and hippocampus that could contribute to their reduced anxiety-like behavior as compared with pups from low LG-ABN dams (172, 173) (Fig. 2). In addition to the GABAergic system, other potential factors mediating the environmental effects include the glutamatergic system and neurotrophins such as BDNF. Liu et al. found that increased LG-ABN of offspring resulted in increased hippocampal mRNA expression of NR2A and NR2B NMDA receptor subunits at postnatal day 8, a change that was sustained into adulthood (174). Also, increased levels of BDNF, but not NGF or NT-3, mRNA were observed in the dorsal hippocampus of 8-day old high LG-ABN pups (174). Neuronal network changes that could directly explain the behavioral consequences of maternal care have also been found as adult offspring of high LG-ABN dams show increased hippocampal synaptogenesis as compared with low LG-ABN offspring, and it has been found that this change could be normalized when low LG-ABN pups are cross-fostered to high LG-ABN dams (174) (Fig. 2).
Interaction Between Genetic and Environmental Factors
Although the interaction of genes and environment in shaping behavior is well accepted, direct experimental evidence to support their role in the pathogenesis of psychiatric diseases has been difficult to obtain. However, recent association studies with the 5-HTT polymorphism have indicated, at least in depressive disorders, that genetic and environmental factors act together, enhancing the phenotype beyond the level established by either factor alone. In 2003, Caspi et al. reported that carriers of the 5, transcriptionally less active, allele of 5-HTT are more likely to develop depression after stressful life events or childhood abuse than individuals homozygous for the l allele (175). The actual interpretation of these data was that the l allele moderates the environmental effect. The majority of replication studies have provided results consistent with such effect (176). It seems, however, that this environment x gene interaction is specific for depressive symptomatology as it was not identified for generalized anxiety disorder or anxiety symptoms (177,178). However, the combined effects of early-life adversity (nursery rearing as opposed to maternal rearing) and 5-HTT polymorphism in anxiety behavior have been found in primates. Rhesus monkeys have a 5-HTT polymorphism similar to that of the human, and it was shown that although both mother- and nursery-reared heterozygote (l/s) animals demonstrate increased affective responding (a measure of temperament) relative to l/l homozygotes (a genotype effect), nursery-reared, but not mother-reared, l/s infants exhibited lower orientation scores (a measure of visual orientation, visual tracking, and attentional capabilities) than their l/l counterparts (an environment x genotype interaction) (179). Also, monkeys with deleterious early-rearing experiences could be differentiated by genotype in cerebrospinal fluid concentrations of the 5-HT metabolite, 5-hydroxyindoleacetic acid, whereas monkeys reared normally were not (180).
In rodents, several environment x gene interaction studies related to anxiety behavior have been conducted. For example, early-life handling or cross-fostering of genetically highly neophobic BALB/c mice to less neophobic C57BL/6 mice equalizes both the behavioral and the benzodiazepine receptor expression differences between these two strains (170,181-184) and, thus, indicates the moderating effect of the environment on genetic disposition to anxiety-like behavior. Also, 5HT1A receptor-deficient mice have increased ultrasonic vocalization (USV) when reared by 5HT1A receptor-/-instead of 5HT1A receptor+/+ dams (185), which indicates that the maternal environment, presumably in interaction with the offspring genotype, alters offspring behavior. However, contrary to the expectation, 5HT1AR+/- offspring reared by 5HT1AR-/- mothers have decreased measures of anxiety in the elevated plus maze as adults when compared with 5HT1AR+/- offspring raised by 5HT1AR+/+ dams. Although it is difficult to consolidate these conflicting results, it seems that the level of anxiety in 5HT1A receptor-deficient mice can be altered by the maternal environment.
In addition to early environmental influences, later-life or adult environment can also influence the expression of a genetic effect on emotionality, as demonstrated in mouse models. Lack of the nociceptin/orphanin FQ gene leads to an enhanced anxiety phenotype in mice (186, 187), but the degree of this phenotype is dependent on environmental influences such as social interactions. Ouagazzal et al. found that homozygous mutant animals, when housed alone, performed similarly to their wild-type controls on tests of emotional reactivity. Enhanced emotionality became apparent only when the singly-housed animals were introduced to group housing (five animals/cage) that induced greater levels of aggression and increased anxiety responses (188).
Convergence of Anxiety-Related Pathways and Mechanisms
Two fundamentally different mechanisms associated with anxiety and anxiety-like behavior seem to exist: one that has a developmental origin with the cause and the adult manifestations of the phenotype separated in time and another mechanism that presents itself in “acute” settings (Fig. 2). Typical examples for developmental anxiety are those caused by the s allele of the 5-HTT, the deficiency in the 5HT1A receptor and BDNF, and the variability in maternal care. All of these produce their effect in adults only when present during prenatal and/or early postnatal life. Indeed, genetic inactivation of the 5HT1A receptor during adult life is not accompanied by anxiety. Because the individual genetic risk factors mentioned above have subtle effects, a combination of them may be necessary to lead to a significant level of anxiety. For example, BDNF+/- and 5-HTT+/- double mutants show a more pronounced anxiety phenotype as compared with singly heterozygous mice (189).
In other anxiety forms and models, developmental mechanisms do not seem to play a role. A typical example is represented by a deficient GABAergic neurotransmission (as a result of less GABA availability or altered composition of the GABAA receptor) that results in reduced neuronal inhibition and increased excitation. The direct behavioral output of these neuronal and neuronal network changes is anxiety (Fig. 2). Another example is the central CRH system as its pharmacological manipulation acutely alters anxiety levels. Because many developmentally relevant anxiety genes and environmental effects and their corresponding mechanisms (see above) modulate GABAergic and glutamatergic transmission as well as central CRH signaling, it is possible that the developmental mechanisms converge on and are interconnected sequentially to the “acute” mechanisms that ultimately lead to the anxiety behavior (Fig. 2).
In summary, it seems that anxiety in many cases has a developmental origin whether it is elicited by genetic polymorphisms or environmental effects. Because individual genetic influences have small effects, it is believed that multiple genetic risk genes, together with adverse environment, are required to have a large enough impact to cause anxiety. Although some early signs of anxiety may be manifested during childhood, anxiety becomes more apparent in predisposed individuals as the brain and behavior mature during adolescence. Although relatively little is known about how early developmental mechanisms lead to anxiety in later life, data suggest that long-lasting alterations in neurotransmitter systems (GABA, glutamate, and CRH) and/or the morphology and function of neuronal networks (amygdala, hippocampus, etc.) are involved. Additional environmental influences during adolescence and adulthood can increase the incidence and severity of anxiety.
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Brain Development, Neurochemistry of
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