Scientific Understanding of Consciousness
Consciousness as an Emergent Property of Thalamocortical Activity

Amygdala Circuitry Controlling Anxiety




Nature, Volume: 471, (17 March 2011) Pages: 358–362

Amygdala circuitry mediating reversible and bidirectional control of anxiety

Kay M. Tye, Rohit Prakash, Sung-Yon Kim, Lief E. Fenno,  Logan Grosenick, Hosniya Zarabi,  Kimberly R. Thompson, Viviana Gradinaru,  Charu Ramakrishnan  & Karl Deisseroth

Department of Bioengineering, Stanford University, Stanford, California 94305, USA

Neurosciences Program, Stanford University, Stanford, California 94305, USA

Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California 94305, USA

Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

CNC Program, Stanford University, Stanford, California 94305, USA


Anxiety—a sustained state of heightened apprehension in the absence of immediate threat—becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)—achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA—exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA–CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.

Despite the high prevalence of anxiety disorders, the underlying neural circuitry is incompletely understood. Available treatments are inconsistently effective or, in the case of benzodiazepines, addictive and linked to significant side effects including cognitive impairment and respiratory suppression, pointing to the need for a deeper understanding of anxiety control mechanisms in the mammalian brain.

Although amygdala microcircuitry for conditioned fear has been optogenetically dissected, the causal underpinnings of unconditioned anxiety have not yet been investigated with cellular precision. Pointing to the need for precise optogenetic exploration, the amygdala is composed of functionally and morphologically heterogeneous subnuclei with complex interconnectivity. The BLA is primarily glutamatergic (~90%) whereas the CeA, which encompasses the centrolateral (CeL) and centromedial (CeM) nuclei, consists of ~95% GABAergic medium spiny neurons. The primary output region of the amygdala is the CeM, which (when chemically or electrically excited) mediates autonomic and behavioural responses associated with fear and anxiety via projections to the brainstem. Because patients with generalized anxiety disorder may have abnormal activity arising from the BLA and CeM, and as BLA neurons excite GABAergic CeL neurons that provide feed-forward inhibition onto CeM ‘output’ neurons, we considered that the BLA–CeL–CeM circuitry could be causally involved in anxiety. However, BLA pyramidal neurons as a whole could have varied and antagonistic roles in diverse projections throughout the brain, with targets including the bed nucleus of the stria terminalis (BNST), nucleus accumbens, hippocampus and cortex.

We therefore developed a method to selectively control BLA terminals in the CeA. BLA glutamatergic projection neurons were transduced with an adeno-associated virus serotype 5 (AAV5) carrying codon-optimized channelrhodopsin (ChR2)–eYFP under control of the CaMKIIα promoter followed by unilateral implantation of a bevelled guide cannula to allow preferential illumination of the non-transduced CeL. In vivo electrophysiological recordings were used to determine illumination parameters for selective control of those BLA terminals in the CeA without nonspecific control of all BLA somata.

To investigate the functional role of the BLA–CeA pathway in anxiety, we probed freely moving mice under projection-specific optogenetic control in two well-validated anxiety assays: the elevated-plus maze (EPM) and the open-field test (OFT). Mice display anxiety-related behaviours in open spaces; therefore, increased time spent in the open arms of the EPM or in the centre of the OFT is interpreted as reduced anxiety. To test whether anxiety-related behaviours could be related to activation of the BLA–CeA projection, and not all BLA somata as a whole, we compared mice receiving projection-specific photostimulation (ChR2:BLA–CeA) to a group with identical illumination parameters transduced with a control virus (eYFP:BLA–CeA), and to another control group expressing ChR2 in the BLA receiving direct illumination of the BLA (ChR2:BLA(somata)). Photostimulation of BLA terminals in the CeA (ChR2:BLA–CeA) increased open-arm time (F1,42 = 69.09, P < 0.00001; Fig. 1b, c) and the probability of open-arm entry from the maze centre (F1,42 = 24.69, P < 0.00001) on the EPM, as well as increasing centre time in the OFT (F1,105 = 24.46, P < 0.00001), reflecting anxiety reduction without impaired locomotion. In contrast, the ChR2:BLA(somata) group showed reduced open-arm time (F1,42 = 6.20, P = 0.02) and probability of open-arm entry (F1,42 = 5.15, P = 0.03) during photostimulation relative to eYFP:BLA–CeA controls, reflecting a distinct anxiogenic effect. Thus, selective illumination of BLA projections to the CeA, but not of BLA somata nonspecifically, produced an acute, rapidly reversible anxiolytic effect.

Here, we have identified the BLA–CeL pathway as a neural substrate for real-time bidirectional modulation of the unconditioned expression of anxiety. The observation that selective illumination of specific BLA terminals produces distinct, and even opposite, behavioural responses from illumination of all glutamatergic BLA somata nonspecifically, points to the essential value of optogenetic control in causally dissecting intact neural circuitry, and indicates that multiple subpopulations or projections of BLA neurons can act in opposition (for example, direct excitation of CeM along with feed-forward inhibition of CeM). Neural circuitry arranged in this way provides many opportunities for modulation of expression of anxiety phenotypes; for example, this microcircuit is well-positioned to be influenced by top-down cortical control from regions important for processing fear and anxiety, including the prelimbic, infralimbic, anterior cingulate and insular cortices that provide robust innervation to the BLA and CeL.

These data are consistent with reports implicating CeA involvement in anxiety but it is important to note that our findings do not exclude downstream or parallel circuits including the BNST, the insular and prefrontal cortices, and the septal–hippocampal circuit; for example, stress induces CeL release of corticotropin releasing hormone (CRH) in the BNST. In the course of providing insight into native anxiogenic and anxiolytic processes, these findings demonstrate that anxiety is continuously regulated by balanced antagonistic pathways within the amygdala, and illustrate the importance of resolving specific projections in the study of neural circuit function relevant to psychiatric disease.

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