Abstract

The role of different amygdala nuclei (neuroanatomical subdivisions) in processing Pavlovian conditioned fear has been studied extensively, but the function of the heterogeneous neuronal subtypes within these nuclei remains poorly understood. Here we use molecular genetic approaches to map the functional connectivity of a subpopulation of GABA-containing neurons, located in the lateral subdivision of the central amygdala (CEl), which express protein kinase C-δ (PKC-δ). Channelrhodopsin-2-assisted circuit mapping in amygdala slices and cell-specific viral tracing indicate that PKC-δ+ neurons inhibit output neurons in the medial central amygdala (CEm), and also make reciprocal inhibitory synapses with PKC-δ neurons in CEl. Electrical silencing of PKC-δ+ neurons in vivo suggests that they correspond to physiologically identified units that are inhibited by the conditioned stimulus, called CEloff units. This correspondence, together with behavioural data, defines an inhibitory microcircuit in CEl that gates CEm output to control the level of conditioned freezing.

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Acknowledgements

We thank N. Heinz and X. Gong for generating BAC transgenic mice; C. Saper for providing the Cre-dependent hrGFP AAV construct; C. Xiao for training in slice electrophysiology and preliminary experiments; H. Lester for advice on the GluCl system; L. van Tright for performing in situ hybridizations; W. Lerchner for providing a CAG-driven GluClβ construct; A. Chang for help with behavioural scoring; M. Martinez for tail genotyping; G. Mosconi for laboratory management; and J. Alex, R. Bayon and R. Sauza for animal care. This work was supported by NIH grant 1 R01 MH085082-01A1 and by funds from the Caltech ‘Conscious Mouse’ project. W.H. was supported by a fellowship of the Human Frontier Science Program and P.S.K. by the Jane Coffin Childs Memorial Fund for Medical Research. S.C. and A.L. were supported by the Novartis Research Foundation. D.J.A. is an Investigator of the Howard Hughes Medical Institute.

Author information

Author notes

    • Prabhat S. Kunwar
    • , Haijiang Cai
    •  & Stephane Ciocchi

    These authors contributed equally to this work.

Affiliations

  1. Division of Biology 216-76, California Institute of Technology, Pasadena, California 91125, USA

    • Wulf Haubensak
    • , Prabhat S. Kunwar
    • , Haijiang Cai
    •  & David J. Anderson
  2. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA

    • David J. Anderson
  3. Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland

    • Stephane Ciocchi
    •  & Andreas Lüthi
  4. Systems Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA

    • Nicholas R. Wall
    •  & Edward M. Callaway
  5. Department of Psychology and the Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Ravikumar Ponnusamy
    •  & Michael S. Fanselow
  6. Laboratory for Neuroimaging, University of California, Los Angeles, Los Angeles, California 90095, USA

    • Jonathan Biag
    •  & Hong-Wei Dong
  7. Department of Bioengineering, Stanford University, Stanford, California 94305, USA

    • Karl Deisseroth

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Contributions

W.H. initiated the project, generated BAC constructs, designed experiments, performed anatomical, viral injections and behavioural experiments, and wrote the manuscript. P.S.K. contributed to experimental design, performed viral injections, behavioural experiments, data analysis and interpretation. H.C. contributed to experimental design and performed viral injections and slice electrophysiology experiments, data analysis and interpretation. S.C. and A.L. designed, performed and interpreted in vivo recording experiments (Fig. 5). N.R.W. and E.M.C. performed rabies virus injection experiments. R.P. performed supplementary behavioural experiments and M.S.F. contributed to their interpretation and to statistical analysis. J.B. and H.-W.D. performed supplementary stereotaxic viral injection experiments. K.D. provided Cre-dependent ChR2 constructs and advice. D.J.A. conceived the project, contributed to experimental design and interpretation and wrote the manuscript. P.S.K., H.C. and S.C. contributed equally. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David J. Anderson.

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    Supplementary Information

    This file contains Supplementary Tables 1-6, Supplementary Figures 1- 11 with legends, Supplementary Notes 1-3 and additional references.

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https://doi.org/10.1038/nature09553

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