C
OMMENTARY
Neurobiology of Anxiety and Fear: Challenges for
Genomic Science of the New Millennium
The recent meeting cosponsored by the Anxiety Disorders Association of America and the National Institute of Mental Health, together with the resulting papers pub-lished in this issue ofBiological Psychiatry,attests to the tremendous current interest in genetics as a powerful tool for exploring how the brain malfunctions in mental disor-ders characterized by fear and anxiety. Many researchers anticipate that an increased understanding of genetic mechanisms will lead to new insights into pathophysiol-ogy and into the role of environmental contributors that interact with genes. The greatest benefit of gene discov-eries for clinical practice is likely to be in the form of genetically informed therapeutics, by which new anxio-lytic compounds are developed and the efficiency of drug regimens enhanced.
Tremendous advances have occurred in mapping rare disorders that are attributable to a single major gene, without prior biological knowledge of how the gene functions to cause disease. In contrast, the complex etiol-ogy of more common diseases like mental disorders, in which vulnerability is produced by the interaction of multiple genes of small effect and nongenetic events, poses major challenges for gene hunters and for neurobi-ologists who hope to use those genes as probes to study pathophysiology.
Genetic Bases of Anxiety Disorders
Data from family and twin studies generally are consistent with the involvement of genetic factors in the familial transmission of anxiety disorders. Molecular genetic stud-ies typically have focused on the detection of association between a disorder and a small number of putative vulnerability genes. These candidate loci were chosen based on the receptor binding profile of anxiolytic com-pounds, or in consideration of the molecules in neurotrans-mitter pathways involved in therapeutic action. Associa-tion analyses of patients with obsessive-compulsive disorder (Karayiorgou et al 1999; Pauls and Alsobrook 1999) have implicated several genes—serotonin (5-HT) transporter, 5-HT2Areceptor, dopamine D4 receptor, and both catechol-O-methyltransferase and monoamine oxi-dase (MAO)-A in males only— but findings typically have not been unambiguously replicated. Likewise, associations between panic disorder and several genes—A2a adenosine receptor, cholecystokinin, and MAO-A— have been re-ported (Deckert et al 1999; Wang et al 1998) but not confirmed. Analyses separating panic disorder families
with a variety of kidney or bladder problems and other medical conditions resulted in evidence for linkage to a DNA marker on chromosome 13q (Weissman et al 2000). Although intriguing, this finding requires replication in independent samples.
The modes of inheritance of anxiety disorders are complex, and if transmission is due to a single major locus in any family, we have not yet detected it. It must be concluded that, for most families, multiple genes of small effect, in interaction with each other and with nongenetic events during brain development, produce a complex vulnerability to the disorder. Emerging tools and technol-ogies for genetic analysis are setting the stage for a new era of gene identification and functional studies in anxiety disorders.
Human Genome Project
Many of these tools and technologies are being provided by the Human Genome Project (HGP), an international effort begun to determine the complete DNA sequence of the human genome. A new plan has been presented (Collins et al 1998), expanding HGP’s goals to include studying human genome sequence variation, developing tools and technologies for functional genomics, and com-pleting the sequence of model systems like the fruit fly (Drosophila melanogaster)and mouse. The 120-megabase gene-rich portion of theDrosophilagenome was recently sequenced (Adams et al 2000). The fly has orthologs to 61% of human disease genes discovered to date (Rubin et al 2000), and this is evidence of the considerable conser-vation of biological processes in the nervous and other organ systems from flies to mammals. Genetic technolo-gies may be applied by neuroscientists to theDrosophila
orthologs of human genes to characterize gene function in the nervous system, and newly identified Drosophila
genes can lead to the isolation of mammalian orthologs and elucidation of mammalian neural pathways. Such explorations will be enhanced with completion of the mouse genome sequence, a draft of which is expected in 2003 or perhaps sooner.
Of even greater importance will be the cataloging of the complete human genome sequence, which will allow comprehensive analysis of the full repertoire of human biological processes. As announced by President Clinton on June 26, 2000 (Clinton et al 2000), a “rough draft” of the human genome has been completed by HGP and a private company (Celera Genomics). Although additional
work is required to produce a “finished,” highly accurate sequence, completion of this rough draft is a landmark scientific achievement that will be a critical milestone in deciphering the specific function of individual genes.
HGP’s sequence production has led to identification of large numbers of single-base variations, or single nucleo-tide polymorphisms (SNPs), which are the most frequent type of variation in the human genome (Collins et al 1998). Construction of an SNP catalog of common gene mutations will permit direct testing of all common gene mutations for association to clinical phenotypes. Of great-est value to researchers studying anxiety disorders may be protein-altering SNPs that occur within genes expressed in brain regions of interest. SNPs will be critical for genetic studies of anxiety disorders because of their abundant distribution across the genome and potential for large-scale automated analysis.
HGP will provide neurobiologists with tools and re-agents to study the expression of genes—and eventually proteins—across the entire genome. One exciting devel-opment is the application of DNA microarrays, or DNA (gene) chips, that are fabricated by high-speed robotics on glass or nylon substrates and for which probes are used to determine complementary binding (Watson and Akil 1999). An experiment with a single DNA array can dramatically increase throughput and provide researchers information on thousands of genes simultaneously. Func-tional genomics approaches typically involve isolation of mRNA and then reverse transcribing it into DNA, which in turn is complementary to the mRNA transcript found in the tissue. Full-length complementary DNAs (cDNAs) that contain the entire protein sequence are necessary to understand the function of the protein encoded by the gene. The National Institutes of Health (NIH) has launched an initiative to generate for public distribution a complete set of such full-length cDNA clones and their sequences for all human, mouse, and other mammalian genes (Strausberg et al 1999).
Neural Substrates of Anxiety and Fear
Genomic tools and technologies will provide a global perspective on gene expression and regulation in the nervous system. A powerful complementary approach for genetic studies of anxiety disorders involves the intensive study of genes and proteins implicated in the brain neurocircuitry of anxiety and fear. We now recognize that fear, and fear conditioning in particular, involves path-ways from the thalamus and cortical regions (including the prefrontal cortex) that project to the amygdala. These inputs, in turn, are processed within a complex intra-amygdala circuitry, giving rise to outputs from the central nucleus of the amygdala to the brain stem, hypothalamus,
hippocampus, and other regions (e.g., LeDoux 1998). The amygdala is crucial in the neuorcircuitry of anxiety and fear because of its pivotal role in the assessment of and response to danger. Pathways underlying fear conditioning involve the transmission of information to the amygdala and transmission from the amygdala to various neural networks that control the expression of defensive reac-tions, including behavioral, autonomic nervous system, and hormonal (hypothalamic–pituitary–adrenal [HPA] axis) responses. Amygdala outputs subserving fear versus anxiety might use closely related but independent path-ways, with the central nucleus of the amygdala differen-tially involved in fear and the bed nucleus of the stria terminalis (BNST) differentially involved in anxiety (Davis 1998). Both the amygdala and the BNST exert important modulatory influences on the hypothalamus and brainstem.
The peptide corticotropin-releasing hormone (CRH), which mediates autonomic, immune, and behavioral com-ponents of stress responses, plays an important role in anxiety and fear. Intraventricular administration of CRH produces many behavioral changes indicative of anxiety, including suppression of exploratory behavior, potentia-tion of the acoustic startle reflex, and facilitapotentia-tion of fear conditioning. During the stress response mediated by the HPA axis, neurons of the paraventricular nucleus in the hypothalamus produce CRH, which in turn activates secretion of adrenocorticotropin from the anterior pituitary gland; adrenocorticotropin elicits release of glucocorti-coids from the adrenal cortex.
Genetic research on anxiety disorders will undoubtedly benefit from the continued delineation of identifiable neural circuits involved in anxiety and fear. A new perspective emerges, through which genes and molecules that affect specific neural systems are targeted as candi-dates in presumed pathophysiologic pathways. This ap-proach greatly extends beyond a traditional one of identi-fying candidate genes solely on the basis of clinical observations of a given therapeutic response. A critical complementary development will be the generation of animal models for the functional analysis of these candi-date genes.
Mouse Models
The mouse has emerged as a premier tool to model complex human diseases and to understand fundamental mammalian biology. The clinical observation that partial 5-HT1A receptor agonists have an anxiolytic effect in patients suggested that this receptor may play a role in modulating comparable behaviors in the mouse. Increased anxiety-related responses have been observed in 5-HT1A receptor knockout mice (Julius 1998). Recent promising
Commentary BIOL PSYCHIATRY 1145
work has focused on genes that have known biological relevance in the neurocircuitry of anxiety and fear. Mice heterozygous for deletion of the gene encoding the g2
subunit of the GABAAreceptor showed increased anxiety and reduced synaptic clustering of GABAA receptors in the hippocampus, amygdala, and cortex (Crestani et al 1999). Central nervous system tissue-specific mutations of the glucocorticoid receptor gene in adult mice led to impaired HPA regulation and loss of gene expression in the hippocampus, hypothalamus, pituitary, and adrenal gland (Tronche et al 1999). This in turn led to an impaired behavioral stress response and a reduction in anxiety, implicating the glucocorticoid receptor in anxiety modu-lation. Other studies have found reduced anxietylike be-havior in CRH receptor 1-null mice (Tarantino and Bucan 2000) and increased anxiety-like behavior in CRH recep-tor 2-null mice (Bale et al 2000; Kishimoto et al 2000).
In closing, rapid advances in genomic science will offer the full sequence of human and other mammalian ge-nomes, a catalog of common gene mutations of functional significance, chip-based approaches, and reagents for gene expression and other functional studies. Neurobiologists are thus faced with the unparalleled opportunity to inten-sively study genes and proteins expressed in the mamma-lian nervous system. Cross-talk between basic neuro-science and genomic neuro-science offers a great opportunity to anchor genetic studies of anxiety disorders to the funda-mental brain neurocircuitry of anxiety and fear. Herein lies the promise of neuroscience and genomic science for ultimately directing attention to biochemical pathways that could provide multiple targets for developing new thera-pies for these disabling conditions.
Steven O. Moldin
National Institute of Mental Health Genetics Research Branch
6001 Executive Boulevard, Room 7189 Bethesda MD 20892-9643
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