Amygdala circuitry mediating reversible and bidirectional control of anxiety

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Anxiety—a sustained state of heightened apprehension in the absence of immediate threat—becomes severely debilitating in disease states1. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence)2 and contribute to the aetiology of major depression and substance abuse3, 4. Although it has been proposed that the amygdala, a brain region important for emotional processing5, 6, 7, 8, has a role in anxiety9, 10, 11, 12, 13, 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 optogenetics14, 15, 16 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 halorhodopsin15 (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.

At a glance


  1. Projection-specific excitation of BLA terminals in the CeA induces acute reversible anxiolysis.
    Figure 1: Projection-specific excitation of BLA terminals in the CeA induces acute reversible anxiolysis.

    a, Mice were housed in a high-stress environment before behavioural manipulations, and received 5-ms light pulses at 20Hz for all light-on conditions. b, c, ChR2:BLA–CeA mice (n = 8) received selective illumination of BLA terminals in the CeA during the light-on epoch on the EPM; see ChR2:BLA–CeA representative path (b), which induced an increase in open-arm time on photostimulation relative to eYFP:BLA–CeA (n = 9) and ChR2:BLA(somata) (n = 7) controls (c), and an increase in the probability of open-arm entry (see inset). d, e, ChR2:BLA–CeA mice also showed increased centre time on the OFT, as seen in a representative path (d), during light-on epochs relative to light-off epochs and eYFP:BLA–CeA and ChR2:BLA(somata) controls (e).

  2. Projection-specific excitation of BLA terminals in the CeA activates CeL neurons and elicits feed-forward inhibition of CeM neurons.
    Figure 2: Projection-specific excitation of BLA terminals in the CeA activates CeL neurons and elicits feed-forward inhibition of CeM neurons.

    a, Two-photon images of representative BLA, CeL and CeM cells imaged from the same slice, overlaid on a brightfield image. bf, Schematics of the recording and illumination sites for the associated representative current-clamp traces (membrane potential Vm = ~ −70mV). b, Representative BLA pyramidal neuron trace expressing ChR2, all of which spiked for every pulse (n = 4). c, Representative trace from a CeL neuron in the terminal field of BLA projection neurons, showing both sub- and suprathreshold excitatory responses on photostimulation (n = 16). Inset left, population summary of mean probability of spiking for each pulse in a 40-pulse train at 20Hz, dotted lines indicate s.e.m. Inset right, frequency histogram showing individual cell spiking fidelity; y-axis is the number of cells per each 5% bin. d, Six sweeps from a CeM neuron spiking in response to a current step (~60pA; indicated in black) and inhibition of spiking on 20Hz illumination of BLA terminals in the CeL. Inset, spike frequency was significantly reduced during light stimulation of CeL neurons (n = 4; spikes per second before (49±9.0), during (1.5±0.87) and after (33±8.4) illumination; mean±s.e.m.). e, f, On broad illumination of the CeM, voltage-clamp summaries show that the latency of excitatory postsynaptic currents (EPSCs) is significantly shorter than the latency of inhibitory postsynaptic currents (IPSCs), whereas there was a non-significant difference in the amplitude of EPSCs and IPSCs (n = 11; *P = 0.04, see insets). The same CeM neurons (n = 7) showed either net excitation when receiving illumination of the CeM (e) or net inhibition on selective illumination of the CeL (f).

  3. Light-induced anxiolytic effects are attributable to activation of BLA-CeA synapses.
    Figure 3: Light-induced anxiolytic effects are attributable to activation of BLA–CeA synapses.

    a, b, Schematic of the recording site and illumination positions as whole-cell recordings were performed at each illumination location in 100-µm increments away from the cell soma both over a visualized axon and in a direction that was not over an axon (inset). Normalized summary of spike fidelity and depolarizing current (a) to a 20-Hz train delivered at various distances from the soma. b, Representative traces on ~125-µm-diameter illumination at various locations within each slice (n = 7). Illumination of BLA somata elicits high-fidelity spiking (top). Illumination of BLA terminals in CeL elicits strong excitatory responses shown in voltage-clamp in the postsynaptic CeL neuron (middle), but does not elicit reliable antidromic spiking in the BLA neuron itself (bottom), summarized in a frequency histogram (inset, 120 pulses per cell). c, d, A separate group of ChR2:BLA–CeA mice (n = 8) performed the EPM and OFT twice, one session preceded with intra-CeA infusions of saline (red) and the other session with glutamate receptor antagonists NBQX and AP5 (purple), counter-balanced for order. Glutamate receptor blockade in the CeA attenuated light-induced increases in both open-arm time (c) and probability of open-arm entry (inset) on the EPM and centre time on the OFT (d, inset shows pooled summary), without altering baseline performance.

  4. Selective inhibition of BLA terminals in the CeA induces an acute and reversible increase in anxiety.
    Figure 4: Selective inhibition of BLA terminals in the CeA induces an acute and reversible increase in anxiety.

    ae, Mice were group-housed in a low-stress environment and received bilateral constant 594-nm light during light-on epochs. a, Selective illumination of eNpHR3.0-expressing BLA terminals suppresses vesicle release evoked by electrical stimulation in the BLA. Schematic indicates the locations of the stimulating electrode, the recording electrode and the ~125-µm diameter light spot. Representative CeL EPSCs before (Off1), during (On) and after (Off2) selective illumination of eNpHR3.0-expressing BLA terminals. Normalized EPSC amplitude summary data from sections containing BLA neurons expressing eNpHR3.0 (n = 7) and non-transduced controls (n = 5) show that selectively illuminating BLA–CeL terminals reduces (*P = 0.006) electrically evoked EPSC amplitude in postsynaptic CeL neurons relative to non-transduced control slice preparations (inset). b, c, Representative eNpHR3.0:BLA–CeA path (b) indicates reduced open-arm time (c) and probability of open arm entry (inset) during illumination, relative to controls. bil., bilateral. d, e, Representative eNpHR3.0:BLA–CeA path (d) reflects reduced centre time on the OFT (e) for the eNpHR3.0:BLA–CeA group during light-on, but not light-off, epochs as compared to controls (inset shows pooled data).


  1. Lieb, R. Anxiety disorders: clinical presentation and epidemiology. Handb. Exp. Pharmacol. 169, 405432 (2005)
  2. Kessler, R. C. et al. Lifetime prevalence and age-of-onset distributions of DSM-IV disorders in the National Comorbidity Survey Replication. Arch. Gen. Psychiatry 62, 593602 (2005)
  3. Koob, G. F. Brain stress systems in the amygdala and addiction. Brain Res. 1293, 6175 (2009)
  4. Ressler, K. J. & Mayberg, H. S. Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic. Nature Neurosci. 10, 11161124 (2007)
  5. LeDoux, J. The emotional brain, fear, and the amygdala. Cell. Mol. Neurobiol. 23, 727738 (2003)
  6. Pare, D., Quirk, G. J. & Ledoux, J. E. New vistas on amygdala networks in conditioned fear. J. Neurophysiol. 92, 19 (2004)
  7. Tye, K. M., Stuber, G. D., de Ridder, B., Bonci, A. & Janak, P. H. Rapid strengthening of thalamo-amygdala synapses mediates cue–reward learning. Nature 453, 12531257 (2008)
  8. Weiskrantz, L. Behavioral changes associated with ablation of the amygdaloid complex in monkeys. J. Comp. Physiol. Psychol. 49, 381391 (1956)
  9. Kalin, N. H., Shelton, S. E. & Davidson, R. J. The role of the central nucleus of the amygdala in mediating fear and anxiety in the primate. J. Neurosci. 24, 55065515 (2004)
  10. Lesscher, H. M. et al. Amygdala protein kinase C epsilon regulates corticotropin-releasing factor and anxiety-like behavior. Genes Brain Behav. 7, 323333 (2008)
  11. Etkin, A., Prater, K. E., Schatzberg, A. F., Menon, V. & Greicius, M. D. Disrupted amygdalar subregion functional connectivity and evidence of a compensatory network in generalized anxiety disorder. Arch. Gen. Psychiatry 66, 13611372 (2009)
  12. Lyons, A. M. & Thiele, T. E. Neuropeptide Y conjugated to saporin alters anxiety-like behavior when injected into the central nucleus of the amygdala or basomedial hypothalamus in BALB/cJ mice. Peptides 31, 21932199 (2010)
  13. Roozendaal, B., McEwen, B. S. & Chattarji, S. Stress, memory and the amygdala. Nature Rev. Neurosci. 10, 423433 (2009)
  14. Boyden, E. S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nature Neurosci. 8, 12631268 (2005)
  15. Gradinaru, V. et al. Molecular and cellular approaches for diversifying and extending optogenetics. Cell 141, 154165 (2010)
  16. Deisseroth, K. Optogenetics: controlling the brain with light. Sci. Am. 303, 4855 (2010)
  17. Woods, J. H., Katz, J. L. & Winger, G. Benzodiazepines: use, abuse, and consequences. Pharmacol. Rev. 44, 151347 (1992)
  18. Ciocchi, S. et al. Encoding of conditioned fear in central amygdala inhibitory circuits. Nature 468, 277282 (2010)
  19. Haubensak, W. et al. Genetic dissection of an amygdala microcircuit that gates conditioned fear. Nature 468, 270276 (2010)
  20. Carlsen, J. Immunocytochemical localization of glutamate decarboxylase in the rat basolateral amygdaloid nucleus, with special reference to GABAergic innervation of amygdalostriatal projection neurons. J. Comp. Neurol. 273, 513526 (1988)
  21. Smith, Y. & Pare, D. Intra-amygdaloid projections of the lateral nucleus in the cat: PHA-L anterograde labeling combined with postembedding GABA and glutamate immunocytochemistry. J. Comp. Neurol. 342, 232248 (1994)
  22. McDonald, A. J. Cytoarchitecture of the central amygdaloid nucleus of the rat. J. Comp. Neurol. 208, 401418 (1982)
  23. Krettek, J. E. & Price, J. L. A description of the amygdaloid complex in the rat and cat with observations on intra-amygdaloid axonal connections. J. Comp. Neurol. 178, 255279 (1978)
  24. Krettek, J. E. & Price, J. L. Amygdaloid projections to subcortical structures within the basal forebrain and brainstem in the rat and cat. J. Comp. Neurol. 178, 225253 (1978)
  25. Davis, M. in The Amygdala: A Functional Analysis (ed. Aggleton, J. P.) 213288 (Oxford Univ. Press, 2000)
  26. Pitkanen, A. in The Amygdala: A Functional Analysis (ed. Aggleton, J. P.) 3199 (Oxford Univ. Press, 2000)
  27. Carola, V., D’Olimpio, F., Brunamonti, E., Mangia, F. & Renzi, P. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav. Brain Res. 134, 4957 (2002)
  28. Davis, M., Walker, D. L., Miles, L. & Grillon, C. Phasic vs sustained fear in rats and humans: role of the extended amygdala in fear vs anxiety. Neuropsychopharmacology 35, 105135 (2010)
  29. Shin, L. M. & Liberzon, I. The neurocircuitry of fear, stress, and anxiety disorders. Neuropsychopharmacology 35, 169191 (2010)
  30. Gray, J. A. & McNaughton, N. The neuropsychology of anxiety: reprise. Nebr. Symp. Motiv. 43, 61134 (1996)

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Author information

  1. These authors contributed equally to this work.

    • Kay M. Tye,
    • Rohit Prakash,
    • Sung-Yon Kim &
    • Lief E. Fenno


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

    • Kay M. Tye,
    • Rohit Prakash,
    • Sung-Yon Kim,
    • Lief E. Fenno,
    • Logan Grosenick,
    • Hosniya Zarabi,
    • Kimberly R. Thompson,
    • Viviana Gradinaru,
    • Charu Ramakrishnan &
    • Karl Deisseroth
  2. Neurosciences Program, Stanford University, Stanford, California 94305, USA

    • Rohit Prakash,
    • Sung-Yon Kim,
    • Lief E. Fenno,
    • Logan Grosenick,
    • Viviana Gradinaru &
    • Karl Deisseroth
  3. Department of Psychiatry and Behavioral Sciences, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth
  4. Howard Hughes Medical Institute, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth
  5. CNC Program, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth


K.M.T., R.P., S.-Y.K., L.E.F. and K.D. contributed to study design and data interpretation. K.M.T., R.P., S.-Y.K. and L.E.F. contributed to data collection and K.M.T. coordinated data collection and analysis. K.M.T., S.-Y.K., H.Z. and K.R.T. contributed to immunohistochemical processing, fluorescence imaging and quantitative analyses. K.M.T. and L.G. performed the behavioural and ex vivo electrophysiology statistical analyses. V.G. and C.R. contributed to the design of eNpHR3.0. C.R. cloned all constructs and managed viral packaging processes. K.D. supervised all aspects of the work. All authors contributed to writing the paper.

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  1. Supplementary Information (11M)

    This file contains Supplementary Figures 1-15 with legends, Supplementary Materials and Methods and additional references.


  1. Supplementary Movie 1 (6.4M)

    This movie shows a representative mouse from the ChR2:BLA-CeA group on elevated plus maze. The 15-minute elevated plus maze session is shown at 5x speed; each 5-min epoch is shown in 1 min and the duration of the light epoch is indicated by the appearance of blue text detailing light stimulation parameters. During the light-on epoch, the mouse increased open arm entry and open arm time.

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