Abstract

A major challenge in neuroscience is to determine the nanoscale position and quantity of signaling molecules in a cell type– and subcellular compartment–specific manner. We developed a new approach to this problem by combining cell-specific physiological and anatomical characterization with super-resolution imaging and studied the molecular and structural parameters shaping the physiological properties of synaptic endocannabinoid signaling in the mouse hippocampus. We found that axon terminals of perisomatically projecting GABAergic interneurons possessed increased CB1 receptor number, active-zone complexity and receptor/effector ratio compared with dendritically projecting interneurons, consistent with higher efficiency of cannabinoid signaling at somatic versus dendritic synapses. Furthermore, chronic Δ9-tetrahydrocannabinol administration, which reduces cannabinoid efficacy on GABA release, evoked marked CB1 downregulation in a dose-dependent manner. Full receptor recovery required several weeks after the cessation of Δ9-tetrahydrocannabinol treatment. These findings indicate that cell type–specific nanoscale analysis of endogenous protein distribution is possible in brain circuits and identify previously unknown molecular properties controlling endocannabinoid signaling and cannabis-induced cognitive dysfunction.

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Acknowledgements

We are grateful to E. Tischler and G. Goda for their excellent technical assistance, I. Mihály for bouton distribution analysis, and C. Cserép and C. Fekete for their help with immunostaining and antibody labeling protocols. The authors are indebted to A. Zimmer (University of Bonn) for providing the CB1 knockout mouse line and to A. Tímár for his help with software analysis. We also thank N. Hájos, N. Holderith, N. Lenkey and Z. Nusser for their comments on the manuscript. The help of the Nikon Microscopy Center at the Institute of Experimental Medicine, Nikon Europe B.V., Nikon Austria GmbH and Auro-Science Consulting is greatly acknowledged for kindly providing microscopy support. This study was primarily supported by the European Research Council Grant 243153 and by the Momentum Program (LP2013-54/2013) of the Hungarian Academy of Sciences to I. Katona. The project was also funded by the Hungarian Academy of Sciences Equipment Grant (IF-22/2012) for super-resolution microscopy. I. Katona is a recipient of the Wellcome Trust International Senior Research Fellowship (090946/Z/09/Z). Additional support was provided by the US National Institutes of Health (NS74432) to I.S., and by the Italian Ministry of University (Grant PRIN 2009: 200928EEX4) and “Fondazione Banco di Sardegna” to M.P.

Author information

Author notes

    • Barna Dudok
    • , László Barna
    •  & Marco Ledri

    These authors equally contributed to this work.

Affiliations

  1. Momentum Laboratory of Molecular Neurobiology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary.

    • Barna Dudok
    • , László Barna
    • , Marco Ledri
    • , Szilárd I Szabó
    • , Eszter Szabadits
    • , Balázs Pintér
    • , Stephen G Woodhams
    • , Christopher M Henstridge
    • , Gyula Y Balla
    • , Rita Nyilas
    •  & István Katona
  2. School of Ph.D. Studies, Semmelweis University, Budapest, Hungary.

    • Barna Dudok
    •  & Gyula Y Balla
  3. Department of Anatomy and Neurobiology, University of California, Irvine, California, USA.

    • Csaba Varga
    • , Sang-Hun Lee
    •  & Ivan Soltesz
  4. Alfred Renyi Institute of Mathematics, Hungarian Academy of Sciences, Budapest, Hungary.

    • Máté Matolcsi
  5. ImmunoGenes Kft, Budakeszi, Hungary.

    • Judit Cervenak
    •  & Imre Kacskovics
  6. Department of Immunology, Eötvös Loránd University, Budapest, Hungary.

    • Imre Kacskovics
  7. Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan.

    • Masahiko Watanabe
  8. Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, Monserrato Cagliari, Italy.

    • Claudia Sagheddu
    • , Miriam Melis
    •  & Marco Pistis
  9. CNR Neuroscience Institute, Cagliari, Italy.

    • Marco Pistis

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Contributions

B.D. and L.B. designed and performed STORM experiments and corresponding analytical tools, analyzed data, prepared the figures, and wrote the manuscript. M.L. carried out patch-clamp electrophysiological recordings, cell reconstructions, STORM imaging and data analysis. S.I.S. conducted and analyzed in vitro electrophysiological measurements as well as participated in STORM imaging and data analysis. E.S., B.P., S.G.W. and R.N. developed immunostaining, tissue processing and antibody-labeling procedures for STORM imaging and contributed to data acquisition and analysis. C.M.H. performed cell culture and electron microscopy experiments and data analysis. G.Y.B., J.C., I. Kacskovics and M.W. developed and validated antibodies. C.V. performed in vivo electrophysiology. S.-H.L. carried out in vitro electrophysiological recordings. M. Matolcsi developed mathematical analysis tools. C.S., M. Melis and M.P. designed and performed the chronic drug administration experiment. I.S. designed and supervised in vitro and in vivo electrophysiological experiments and contributed to manuscript editing. I. Katona conceived and supervised the project, performed in vitro electrophysiological recordings, and wrote the manuscript.

Competing interests

J. Cervenak is a scientific researcher at ImmunoGenes Ltd.; I. Kacskovics is scientific co-founder of ImmunoGenes Ltd., a company specialized in the generation of FcRn transgenic animals for the production of polyclonal and monoclonal antibodies.

Corresponding author

Correspondence to István Katona.

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    Supplementary Table 1: Physiological and anatomical parameters of CB1-positive regular-spiking, multipolar interneurons in the CA1 subfield of the mouse hippocampus.

    AP, action potential; FWHM, full width at half maximum; AHP, after hyper polarization; BDI, bouton distribution index.

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https://doi.org/10.1038/nn.3892