From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors

Abstract

In mammals, identifying the contribution of specific neurons or networks to behavior is a key challenge. Here we describe an approach that facilitates this process by enabling the rapid modulation of synaptic inhibition in defined cell populations. Binding of zolpidem, a systemically active allosteric modulator that enhances the function of the GABAA receptor, requires a phenylalanine residue (Phe77) in the γ2 subunit. Mice in which this residue is changed to isoleucine are insensitive to zolpidem. By Cre recombinase–induced swapping of the γ2 subunit (that is, exchanging Ile77 for Phe77), zolpidem sensitivity can be restored to GABAA receptors in chosen cell types. We demonstrate the power of this method in the cerebellum, where zolpidem rapidly induces significant motor deficits when Purkinje cells are made uniquely sensitive to its action. This combined molecular and pharmacological technique has demonstrable advantages over targeted cell ablation and will be invaluable for investigating many neuronal circuits.

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Figure 1: Strategy to restrict zolpidem sensitivity to selected neural types, using Cre recombinase to drive a γ2I77 to γ2F77 subunit swap.
Figure 2: Summary of mouse genotypes.
Figure 3: Synaptic expression of the γ2F77GFP subunit in Purkinje cells of adult PC-γ2–swap mice.
Figure 4: Potentiation of GABAA receptor–mediated mIPSCs by zolpidem in Purkinje cells of PC-γ2–swap mice.
Figure 5: Motor performance on a rotarod and horizontal beam after systemic administration of zolpidem.

References

  1. 1

    Riedel, G. et al. Reversible neural inactivation reveals hippocampal participation in several memory processes. Nat. Neurosci. 2, 898–905 (1999).

    CAS  Article  Google Scholar 

  2. 2

    Lomber, S.G. The advantages and limitations of permanent or reversible deactivation techniques in the assessment of neural function. J. Neurosci. Methods 86, 109–117 (1999).

    CAS  Article  Google Scholar 

  3. 3

    Pereira de Vasconcelos, A. et al. Reversible inactivation of the dorsal hippocampus by tetrodotoxin or lidocaine: a comparative study on cerebral functional activity and motor coordination in the rat. Neuroscience 141, 1649–1663 (2006).

    CAS  Article  Google Scholar 

  4. 4

    Wulff, P. & Wisden, W. Dissecting neural circuitry by combining genetics and pharmacology. Trends Neurosci. 28, 44–50 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Crick, F. The impact of molecular biology on neuroscience. Phil. Trans. R. Soc. Lond. B 354, 2021–2025 (1999).

    CAS  Article  Google Scholar 

  6. 6

    Rudolph, U. & Mohler, H. Analysis of GABAA receptor function and dissection of the pharmacology of benzodiazepines and general anesthetics through mouse genetics. Annu. Rev. Pharmacol. Toxicol. 44, 475–498 (2004).

    CAS  Article  Google Scholar 

  7. 7

    Farrant, M. & Nusser, Z. Variations on an inhibitory theme: phasic and tonic activation of GABAA receptors. Nat. Rev. Neurosci. 6, 215–229 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Whiting, P.J. GABAA receptors: a viable target for novel anxiolytics? Curr. Opin. Pharmacol. 6, 24–29 (2006).

    CAS  Article  Google Scholar 

  9. 9

    Perrais, D. & Ropert, N. Effect of zolpidem on miniature IPSCs and occupancy of postsynaptic GABAA receptors in central synapses. J. Neurosci. 19, 578–588 (1999).

    CAS  Article  Google Scholar 

  10. 10

    Thomson, A.M., Bannister, A.P., Hughes, D.I. & Pawelzik, H. Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus. Eur. J. Neurosci. 12, 425–436 (2000).

    CAS  Article  Google Scholar 

  11. 11

    Campo-Soria, C., Chang, Y. & Weiss, D.S. Mechanism of action of benzodiazepines on GABAA receptors. Br. J. Pharmacol. 148, 984–990 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Rudolph, U. et al. Benzodiazepine actions mediated by specific γ-aminobutyric acidA receptor subtypes. Nature 401, 796–800 (1999).

    CAS  Article  Google Scholar 

  13. 13

    Crestani, F., Martin, J.R., Mohler, H. & Rudolph, U. Mechanism of action of the hypnotic zolpidem in vivo. Br. J. Pharmacol. 131, 1251–1254 (2000).

    CAS  Article  Google Scholar 

  14. 14

    Crestani, F., Martin, J.R., Mohler, H. & Rudolph, U. Resolving differences in GABAA receptor mutant mouse studies. Nat. Neurosci. 3, 1059 (2000).

    CAS  Article  Google Scholar 

  15. 15

    McKernan, R.M. et al. Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABAA receptor α1 subtype. Nat. Neurosci. 3, 587–592 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Wisden, W., Laurie, D.J., Monyer, H. & Seeburg, P.H. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. I. Telencephalon, diencephalon, mesencephalon. J. Neurosci. 12, 1040–1062 (1992).

    CAS  Article  Google Scholar 

  17. 17

    Duncan, G.E. et al. Distribution of [3H]zolpidem binding sites in relation to messenger RNA encoding the α1, β2 and γ2 subunits of GABAA receptors in rat brain. Neuroscience 64, 1113–1128 (1995).

    CAS  Article  Google Scholar 

  18. 18

    Niddam, R., Dubois, A., Scatton, B., Arbilla, S. & Langer, S.Z. Autoradiographic localization of [3H]zolpidem binding sites in the rat CNS: comparison with the distribution of [3H]flunitrazepam binding sites. J. Neurochem. 49, 890–899 (1987).

    CAS  Article  Google Scholar 

  19. 19

    Sancar, F., Ericksen, S.S., Kucken, A.M., Teissere, J.A. & Czajkowski, C. Structural determinants for high-affinity zolpidem binding to GABAA receptors. Mol. Pharmacol. 71, 38–46 (2007).

    CAS  Article  Google Scholar 

  20. 20

    Buhr, A., Baur, R. & Sigel, E. Subtle changes in residue 77 of the γ subunit of α1β2γ2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site. J. Biol. Chem. 272, 11799–11804 (1997).

    CAS  Article  Google Scholar 

  21. 21

    Wingrove, P.B., Thompson, S.A., Wafford, K.A. & Whiting, P.J. Key amino acids in the γ subunit of the γ-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site. Mol. Pharmacol. 52, 874–881 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Cope, D.W. et al. Abolition of zolpidem sensitivity in mice with a point mutation in the GABAA receptor γ2 subunit. Neuropharmacology 47, 17–34 (2004).

    CAS  Article  Google Scholar 

  23. 23

    Cope, D.W. et al. Loss of zolpidem efficacy in the hippocampus of mice with the GABAA receptor γ2 F77I point mutation. Eur. J. Neurosci. 21, 3002–3016 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Ogris, W. et al. Affinity of various benzodiazepine site ligands in mice with a point mutation in the GABAA receptor γ2 subunit. Biochem. Pharmacol. 68, 1621–1629 (2004).

    CAS  Article  Google Scholar 

  25. 25

    Smeyne, R.J. et al. Local control of granule cell generation by cerebellar Purkinje cells. Mol. Cell. Neurosci. 6, 230–251 (1995).

    CAS  Article  Google Scholar 

  26. 26

    Häusser, M. & Clark, B.A. Tonic synaptic inhibition modulates neuronal output pattern and spatiotemporal synaptic integration. Neuron 19, 665–678 (1997).

    Article  Google Scholar 

  27. 27

    Mittmann, W., Koch, U. & Häusser, M. Feed-forward inhibition shapes the spike output of cerebellar Purkinje cells. J. Physiol. (Lond.) 563, 369–378 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Santamaria, F., Tripp, P.G. & Bower, J.M. Feed-forward inhibition controls the spread of granule cell-induced Purkinje cell activity in the cerebellar cortex. J. Neurophysiol. 97, 248–263 (2007).

    Article  Google Scholar 

  29. 29

    Laurie, D.J., Seeburg, P.H. & Wisden, W. The distribution of 13 GABAA receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J. Neurosci. 12, 1063–1076 (1992).

    CAS  Article  Google Scholar 

  30. 30

    Fritschy, J.M., Panzanelli, P., Kralic, J.E., Vogt, K.E. & Sassoe-Pognetto, M. Differential dependence of axo-dendritic and axo-somatic GABAergic synapses on GABAA receptors containing the α1 subunit in Purkinje cells. J. Neurosci. 26, 3245–3255 (2006).

    CAS  Article  Google Scholar 

  31. 31

    Gunther, U. et al. Benzodiazepine-insensitive mice generated by targeted disruption of the γ2 subunit gene of γ-aminobutyric acid type A receptors. Proc. Natl. Acad. Sci. USA 92, 7749–7753 (1995).

    CAS  Article  Google Scholar 

  32. 32

    Schweizer, C. et al. The γ2 subunit of GABAA receptors is required for maintenance of receptors at mature synapses. Mol. Cell. Neurosci. 24, 442–450 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Barski, J.J., Dethleffsen, K. & Meyer, M. Cre recombinase expression in cerebellar Purkinje cells. Genesis 28, 93–98 (2000).

    CAS  Article  Google Scholar 

  34. 34

    Kittler, J.T. et al. Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras. Mol. Cell. Neurosci. 16, 440–452 (2000).

    CAS  Article  Google Scholar 

  35. 35

    Brickley, S.G., Revilla, V., Cull-Candy, S.G., Wisden, W. & Farrant, M. Adaptive regulation of neuronal excitability by a voltage-independent potassium conductance. Nature 409, 88–92 (2001).

    CAS  Article  Google Scholar 

  36. 36

    Marder, E. & Goaillard, J.M. Variability, compensation and homeostasis in neuron and network function. Nat. Rev. Neurosci. 7, 563–574 (2006).

    CAS  Article  Google Scholar 

  37. 37

    Lima, S.Q. & Miesenbock, G. Remote control of behavior through genetically targeted photostimulation of neurons. Cell 121, 141–152 (2005).

    CAS  Article  Google Scholar 

  38. 38

    Zhang, F. et al. Multimodal fast optical interrogation of neural circuitry. Nature 446, 633–641 (2007).

    CAS  Article  Google Scholar 

  39. 39

    Gosgnach, S. et al. V1 spinal neurons regulate the speed of vertebrate locomotor outputs. Nature 440, 215–219 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Tan, E.M. et al. Selective and quickly reversible inactivation of mammalian neurons in vivo using the Drosophila allatostatin receptor. Neuron 51, 157–170 (2006).

    CAS  Article  Google Scholar 

  41. 41

    Karpova, A.Y., Tervo, D.G., Gray, N.W. & Svoboda, K. Rapid and reversible chemical inactivation of synaptic transmission in genetically targeted neurons. Neuron 48, 727–735 (2005).

    CAS  Article  Google Scholar 

  42. 42

    Yamamoto, M. et al. Reversible suppression of glutamatergic neurotransmission of cerebellar granule cells in vivo by genetically manipulated expression of tetanus neurotoxin light chain. J. Neurosci. 23, 6759–6767 (2003).

    CAS  Article  Google Scholar 

  43. 43

    Wall, M.J. & Usowicz, M.M. Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur. J. Neurosci. 9, 533–548 (1997).

    CAS  Article  Google Scholar 

  44. 44

    McCartney, M.R., Deeb, T.Z., Henderson, T.N. & Hales, T.G. Tonically active GABAA receptors in hippocampal pyramidal neurons exhibit constitutive GABA-independent gating. Mol. Pharmacol. 71, 539–548 (2007).

    CAS  Article  Google Scholar 

  45. 45

    Benavides, J. et al. In vivo interaction of zolpidem with central benzodiazepine (BZD) binding sites (as labeled by [3H]Ro 15–1788) in the mouse brain. Preferential affinity of zolpidem for the omega 1 (BZD1) subtype. J. Pharmacol. Exp. Ther. 245, 1033–1041 (1988).

    CAS  PubMed  Google Scholar 

  46. 46

    Leppa, E. et al. Agonistic effects of the β-carboline DMCM revealed in GABAA receptor γ2 subunit F77I point-mutated mice. Neuropharmacology 48, 469–478 (2005).

    CAS  Article  Google Scholar 

  47. 47

    Lein, E.S. et al. Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007).

    CAS  Article  Google Scholar 

  48. 48

    Zezula, J., Fuchs, K. & Sieghart, W. Separation of α1, α2 and α3 subunits of the GABAA-benzodiazepine receptor complex by immunoaffinity chromatography. Brain Res. 563, 325–328 (1991).

    CAS  Article  Google Scholar 

  49. 49

    Oertel, W.H., Schmechel, D.E., Mugnaini, E., Tappaz, M.L. & Kopin, I.J. Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6, 2715–2735 (1981).

    CAS  Article  Google Scholar 

  50. 50

    Korpi, E.R. et al. Cerebellar granule-cell–specific GABAA receptors attenuate benzodiazepine-induced ataxia: evidence from α6-subunit-deficient mice. Eur. J. Neurosci. 11, 233–240 (1999).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank E. Sigel for pointing out the γ2I77 mutation; J. Oberdick for the L7 expression cassette; S.J. Moss for the γ2F77GFP plasmid; M. Meyer for the L7Cre line; R. Tomioka and E. Mugnaini for antibodies to EGFP and GAD, respectively; H. Monyer for discussion and support; F. Zimmermann for the transgene and stem cell injections; I. Preugschat-Gumprecht for help with mouse genotyping; D. Andersson, T. Karayannis and M. Capogna for contributing to initial electrophysiological recordings; and S. Brickley, M. Capogna, S.G. Cull-Candy, C. De Zeeuw, N. Franks, T. Klausberger and Z. Nusser for comments on the manuscript. This work was funded by the VolkswagenStiftung (grant I/78 554 to W.W., E.R.K., W.S. and P.S.), the Deutsche Forschungsgemeinschaft (grant WI 1951/2 to W.W. and P.W.), the UK Medical Research Council (grant G0501584 to W.W.), the J. Ernest Tait Estate (to W.W. and T.G.), a Heidelberg Young Investigator Award (to P.W.), the Academy of Finland (to E.R.K. and A.-M.L.), the Sigrid Juselius Foundation (to E.R.K. and E.L.), the Institute Pasteur-Fondazione Cenci Bolognetti (to M.R.), the Austrian Federal Government (W.S.), the Medical University Vienna (W.S.) and a Wellcome Trust Programme Grant (to M.F.).

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Contributions

The original idea was conceived by W.S. and developed with P.S., W.W. and E.R.K. Experiments were designed by M.F., E.R.K., P.S., P.W., W.S. and W.W. Experiments were performed by M.F., T.G., A.-M.L., E.L., M.R., J.D.S., P.S., O.Y.V., P.W. and W.W. Behavioral data were analyzed by E.L., A.-M.L. and E.R.K. Electrophysiological data were analyzed by M.F. The manuscript was written by M.F., P.W. and W.W. All authors commented on and helped to revise the text.

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Correspondence to Peer Wulff.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Generation of γ2I77lox and L7γ2F77GFP mice. (PDF 3384 kb)

Supplementary Fig. 2

Confirmation of Purkinje cell–specific Cre activity in the L7Cre line using ROSA26 indicator mice. (PDF 8956 kb)

Supplementary Fig. 3

Expression of the γ2F77GFP subunit in cerebellar Purkinje cells of PC-γ2–swap mice. (PDF 1678 kb)

Supplementary Fig. 4

In slices from PC-γ2–swap mice, potentiation of GABAA receptor–mediated mIPSCs by zolpidem is restricted to Purkinje cells. (PDF 152 kb)

Supplementary Methods (PDF 189 kb)

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Wulff, P., Goetz, T., Leppä, E. et al. From synapse to behavior: rapid modulation of defined neuronal types with engineered GABAA receptors. Nat Neurosci 10, 923–929 (2007). https://doi.org/10.1038/nn1927

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