AACIMP 2011 Summer School. Neuroscience Stream. Lecture by Nana Voitenko.

1. Bogomoletz Institute of Physiology, Kiev, Ukraine [email_address] Dr. NANA VOITENKO AACIMP KIEV - 2011 Molecular Mechanisms of Pain part II

5. Molecular Organization of AMPARs : Homomeric & Heteromeric Receptors GluR2 subunit determines functional properties of GluR-channel MRC Centre for Synaptic Plasticity Nature 454(7200):118-121, 2008

6. Ca 2+ -permeable and -impermeable AMPARs

8. Part B: Spinal AMPARs-mediated synaptic transmission during inflammation. Molecular mechanism of GluR2 internalization from synapses.

9. AMPA receptors at dorsal horn synapse

10. Determination of Sensory Abnormalities after C omplete Freund’s A djuvant (CFA)-induced Peripheral Inflammation Increased mechanical hypersensitivity on the ipsilateral side after CFA, but not saline injection into a hindpaw (n = 10/time point) .

11. Lumbar Spinal Cord with Dorsal Root Region

12. Identification of Ca 2+ -permeable AMPARs in DH Neurons by Kainate-induced Cobalt Uptake Loading A, CNQX and GYKI 53655 blocked kainate-induced cobalt loading of DH neurons, whereas AP5 had no effect. B, Immunostaining with neuronal marker NeuN. C, CFA (but not saline) increased cobalt uptake loading in DH on ipsilateral, but not contralateral, side. Right - statistics of the number of cobalt-positive neurons in laminae I-II and laminae III-VII 1 d post-saline and 1 d post-CFA. Voitenko group, unpublished data

13. Inflammation Does Not Change the Expression of Total GluR1 and GluR2 in Dorsal Horn Park et al., Mol. Pain, 2008 Top: representative Western blots showing Glu R 1 protein (A) and Glu R 2 protein (B) in total soluble fraction from the ipsilateral and contralateral dorsal horns of naïve rats (n = 4/time point) and the rats at 2 and 24 h after saline (S) or CFA injection (C ) . Bottom: statistics of the densitometric analysis expressed relative to the control (β-actin).

14. Dorsal Horn GluR2 Internalization during Inflammation Copyright ©2009 Society for Neuroscience Park, J.-S. , Voitenko, N. et al. J. Neurosci. 2009;29:3206-19 GluR2 expression in 150 k-g spin fraction (A) and plasma membrane fraction (B) from ipsilateral DH at 2 h, 1 d, and 3 d after saline or CFA. Top, Representative Western blots; bottom, statistic s of the densitometric analysis ( relative to the naive animals - 0 h). C, Surface expression of GluR2 and NR1 in DH neurons at 1 d after CFA or saline. The amount of sample loaded for the total (T) was 10% of that for the biotinylated surface (S). Actin was used as a control.

15. P ostsynaptic I mmunogold L abeling for GluR2 in S uperficial DH S ynapses I mmunogold labeling for GluR2 (arrowheads) both the synapse and adjacent cytoplasmic structures 1 d after saline and only in cytoplasm 1 d after CFA. Pre, Presynaptic terminal; Post, postsynaptic structure. Scale bars, 100 nm. Copyright ©2009 Society for Neuroscience Park, J.-S. , Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

16. A, The evoked EPSCs were blocked by GYKI 52466 , but not by SYM 2081. B, Increased sensitivity of EPSC amplitude to PhTx-433 after 1 d CF A. C, I–V curves at 1 d post- saline or CFA. Examples of the EPSCs at 70 mV and 40 mV holding potentials. AMPAR-mediated Evoked EPSCs at Synapses between Primary Afferents and SG Neurons Copyright ©2009 Society for Neuroscience Park, J.-S. , Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

17. NMDA Receptors and PKC couple to AMPAR complex PICK1 coimmunoprecipitates with GluR2 and PKC  ( A ) , while GluR2 - with PICK1 and PKC    . PSD-95 coimmunoprecipitates with NR2B and stargazin (C) , while s targazin - with PSD-95 and GluR2 (D) . E, NR1 (5 nm gold, arrowheads) and GluR2 (15 nm gold) colocalize at DH synapses. Scale bar, 100 nm. Copyright ©2009 Society for Neuroscience Park, J.-S. , Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

18. Proposed Mechanism for GluR2 Internalization from Synapses

19. Conclusion Persistent peripheral inflammation induces GluR2 internalization from synap ses via NMDA Receptor- t riggered PKC a ctivation in D H n eurons

20. Part C: Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent pain. Trafficking of Ca 2+ -permeable AMPARs during inflammatory pain.

21. Combined Electrophysiology and Calcium Imaging in Substantia Gelatinosa Neuron Transmitted light image of Substantia Gelatinosa in transverse DH slice (left); SG neuron loaded with 200 mM of fura-2 (right).

22. AMPARs-mediated Current and [Ca 2+ ] i Transients in Soma & Dendrites of SG Neurons Kopach et. al. Pain, 2011 . SG neurons were identified according to their pattern of action potential firing A, Simultaneous recording of AMPA-induced current (lower row) and [Ca 2+ ] i transients (upper row) in soma and dendrites of SG neurons. B-C, AMPARs antagonist, NBQX and GYKI, abolished current and [Ca 2+ ] i transients. D, Statistics of current amplitudes (left) and [Ca 2+ ] i transients (right) in different groups of SG neurons. Voitenko group, unpublished data

23. Inflammation Potentiates AMPARs-mediated Current and [Ca 2+ ] i Transients in “Tonic” but not in “Transient” SG Neurons A, AMPA-induced current (lower row) and [Ca 2+ ] i transients (upper row) in “tonic” neurons 24h after saline or CFA. B-C, A scatter dot plot of currents in SG neurons. D, Statistics of current amplitudes (left) and [Ca 2+ ] i transients (right) in SG neurons. E, A relationship of the [Ca 2+ ] i transient amplitudes and a normalized value of i ntegrated current in the timeframe of AMPA application. Kopach et. al. Pain, 2011 . D E

24. Inflammation-induced Increase of the Number of Extrasynaptic Ca 2+ -permeable AMPARs A. Left, AMPA-induced currents after 5 min pre-application of IEM at 24 h post-CFA or saline. Right, IEM was applied during steady-state current level. Dotted lines are exponential fitting of current. B, Statistics of IEM inhibition of current amplitude. A, I-V relationship of AMPARs-mediated currents in “tonic” neurons at 1 d post-saline and CFA. Note, CFA-induced inward rectification was completely reversed by IEM. B-C, Scatter dot plot illustrated a spread of rectification index (I +30mV /I -50mV ) and statistics in “tonic” neurons from 1 d saline- and CFA-treated rats. Kopach et. al. Pain, 2011 .

25. The Altered Level of GluR1 and GluR2 in the Plasma Membrane Fraction & Cytosol after CFA Park et al., Mol. Pain, 2008 Kopach et. al. Pain, 2011 .

27. Proposed Model for AMPA Receptors Recycling At synapses (green), there are mobile and immobile pools of AMPARs. Mobile receptors leaving synapses can be trapped at EZs (red) either for transient stabilization or for endocytosis (red arrow) and recycling (blue arrow). Newly exocytosed receptors exhibit high mobility and accumulate at synapses.

28. Conclusion Increased functional expression of extrasynaptic Ca 2+ -permeable AMPARs contributes to the maintaining of persistent inflammation

29. Part D: Role of Protein Kinase C  subtype in AMPARs-mediated Pain Plasticity Antisense oligonucleotides – a new strategy of pain treatment

30. AS ODN – Antisense oligonucleotides Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. In the case of antisense RNA they prevent protein translation of certain messenger RNA strands by binding to them. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA. If binding takes places this DNA/RNA hybrid can be degraded by the enzyme RNase H.

31. Antisense oligonucleotides: scheme of action cmbi.bjmu.edu.cn

32. Bourinet et al., THE EMBO JOURNAL (2005) 24 , 315 - 324 Localization of AS ODN in lumbar spinal cord

33. In Vivo Gene Silencing of PKCα Attenuates Inflammation-induced Hyperalgesia Voitenko group, unpublished data Effect of anti-sense (AS-) and miss-sense (MS) oligonucleotydes for PKCα on CFA-induced hyperalgesia. Time course of hyperalgesia development following CFA injection, in MS-ODN-treated and AS-ODN-treated rats. S tatistics of paw withdrawal latency value in different groups of rats.

34. Paw withdrawal latency and RI values in 4 days of As ODN treatment. S - saline-injected; C – CFA-injected Voitenko group, under preparation CFA _________________ 0 2 4 6 8 10 12 14 S C S C S C PWL (s) ________________ i.t. Saline ________________ ________________ i.t. AS-ODN i.t. MS-ODN # * * Rectification index Saline Naive AS-ODN MS-ODN * #

35. In Vivo Gene Silencing of PKCα Reverses: Voitenko group, unpublished data I-V curve of evoked AMPA-mediated EPSCs in AS-ODN-treated and MS-ODN-treated inflammatory animals CFA-induced Rectification of Synaptic Currents CFA-induced Potentiation of Extrasynaptic AMPARs current and [Ca 2+ ] i transients in “tonic” SG neurons

36. Inflammation INCREASED EXCITABILITY IMPAIRED SYNAPTIC EFFICACY CENTRAL SENSITIZATION PAIN Ca 2+ influx through Synaptic AMPARs Increased synaptic GluR2 internalization Ca 2+ influx through extrasynaptic AMPARs Increased GluR1 insertion into extrasynaptic sites PKC 