Bogomoletz Institute of Physiology, Kiev,

Ukraine
nana@biph.kiev.ua

Molecular Mechanisms
of Pain
part II

Dr. NANA VOITENKO

AACIMP
KIEV - 2011
1

Part A:
Classification of receptors to glutamate.
Structure and molecular organization of AMPA receptors.
Part B:
Spinal AMPARs-mediated synaptic transmission during inflammation.
Molecular mechanism of GluR2 internalization from synapses.
Part C:
Involvement of spinal extrasynaptic AMPARs in the maintaining of persistent
pain.
Trafficking of Ca2+-permeable AMPARs during inflammatory pain.

Part D:
- Protein kinase Ca as a molecular target for perspective treatment of persistent
pain.
- Antisense oligonucleotides a new strategy of pain treatment

GLUTAMATE RECEPTORS
Ionotropic GluRs

NMDA
NR1

AMPA

NR2A-D

Metabotropic GluRs

Kainate
GluR 5,6,7 KA 1,2

Group I Group II
mGlu1 mGlu5

Group III
mGlu4

mGlu6

mGlu7

mGlu8

mGlu2 mGlu3

NR3A
GluR 1-4

Mediating effects include:

Modulatory effects on:

- Membrane depolarization
- Ca2+ and Na+ influx
- K+ efflux
- Free radical formation

- iGluR
- K+ and Ca2+ channels
- Neurotransmitter release

Drug News Perspect 2003, 16(8): 513

AMPA receptors

is a subtype of ionotropic transmembrane receptor for

glutamate that mediates fast synaptic transmission in the
central nervous system. Its name is derived from its
ability to be activated by the artificial glutamate analog
AMPA. The receptor was discovered by Tage Honore
and colleagues at the School of Pharmacy in
Copenhagen, and published in 1982 in the Journal of
Neurochemistry. AMPARs are found in many parts of the
brain and are the most commonly found receptor in the
nervous system.

AMPARs are trafficked from the dendrite into the

synapse and incorporated through some series of
signaling cascades increasing efficacy of synaptic
transmission.
4

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

Ca2+-permeable and -impermeable AMPARs

Important future of AMPA receptors:

they are not static.

exocytosis

and endocytosis at the postsynaptic membrane

lateral diffusion of glutamate receptors in the plasma membrane
Petrini et al., Neuron. 2009

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

AMPA receptors at dorsal horn synapse

Determination of Sensory Abnormalities after

Complete Freunds Adjuvant (CFA)-induced
Peripheral Inflammation

Increased mechanical hypersensitivity on the

ipsilateral side after CFA, but not saline
injection into a hindpaw (n = 10/time point).

10

Lumbar Spinal Cord with Dorsal Root Region

11

Identification of Ca2+-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.

12

Voitenko group, unpublished data

Inflammation Does Not Change

the Expression of Total GluR1 and GluR2 in Dorsal Horn

Top: representative Western blots showing GluR1 protein (A) and GluR2 protein (B) in total soluble
fraction from the ipsilateral and contralateral dorsal horns of nave 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 (-actin).

Park et al., Mol.13

Pain, 2008

Dorsal Horn GluR2 Internalization during Inflammation

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, statistics 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.

Copyright 2009 Society for Neuroscience

14
Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

Postsynaptic Immunogold Labeling for GluR2

in Superficial DH Synapses

Immunogold 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

15 2009;29:3206-19
Park, J.-S., Voitenko, N. et al. J. Neurosci.

AMPAR-mediated Evoked EPSCs at Synapses

between Primary Afferents and SG Neurons
A

GYKI 52466

SYM 2081

50 pA
10 ms

0.4
0.2

M em brane potential (m V )
-80

-60

-40

**

0.0
-20

20

Saline

1.2

CFA

1
0.8
0.6
0.4

PhTX 433

0.2
0
0

10

12

14

16

Time (min)

A, The evoked EPSCs

were blocked by GYKI
52466, but not by SYM
2081.
B, Increased sensitivity of
EPSC amplitude to PhTx433 after 1 d CFA.
C, IV curves at 1 d postsaline or CFA. Examples of
the EPSCs at 70 mV and
40 mV holding potentials.

40

Saline

-0.2

CFA

-0.4
-0.6
-0.8
-1.0

Copyright 2009 Society for Neuroscience

Normalized current

Saline
CFA

Normalized EPSC amplitudes

1.4

50 pA
10 ms

16
Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

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 stargazin - 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

17
Park, J.-S., Voitenko, N. et al. J. Neurosci. 2009;29:3206-19

Proposed Mechanism for

GluR2 Internalization from Synapses

18

Conclusion
Persistent peripheral inflammation induces
GluR2 internalization from synapses
via NMDA Receptor-triggered PKC activation
in DH neurons

19

Part C:
Involvement of spinal extrasynaptic AMPARs in the
maintaining of persistent pain.
Trafficking of Ca2+-permeable AMPARs during
inflammatory pain.

20

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).

21

AMPARs-mediated Current and [Ca2+]i Transients

in Soma & Dendrites of SG Neurons
SG neurons were identified
according to their pattern of
action potential firing

Kopach et. al. Pain, 2011.

A, Simultaneous recording of AMPA-induced current (lower row) and [Ca2+]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 [Ca2+]i transients (right) in different
groups of SG neurons.
22
Voitenko group, unpublished data

Inflammation Potentiates
AMPARs-mediated Current and [Ca2+]i Transients in Tonic
but not in Transient SG Neurons

A, AMPA-induced current (lower row) and [Ca2+]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 [Ca2+]i
transient amplitudes and a normalized value of integrated
current in the timeframe of AMPA application.

23
Kopach et. al. Pain, 2011.

Inflammation-induced Increase of the Number of Extrasynaptic

Ca2+-permeable AMPARs
A. Left, AMPA-induced
currents after 5 min preapplication 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 AMPARsmediated 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 salineand CFA-treated rats.
Kopach et. al. Pain, 2011.

24

The Altered Level of GluR1 and GluR2

in the Plasma Membrane Fraction & Cytosol after CFA

Kopach et. al. Pain, 2011.

Park et al.,25
Mol. Pain, 2008

2.5

Saline

CFA

2
1.5
1
0.5
0

Syn Extra Cyto

Syn Extra Cyto

GluR1

GluR2

Ratio (CFA/saline)

Number of immunogold labelings

Peripheral Inflammation Induces

GluA1 Membrane Insertion at Extrasynaptic Sites
of SG Neurons

3
2
1
0

Syn Extra Cyto

GluR1

Syn Extra Cyto

GluR2

A. The number of GluA1- and

GluA2-labeled immunogold
particles at synapses,
extrasynaptic membranes, and
cytoplasm of SG neurons 1 d
post- CFA or saline.
B. Ratios of the number of
GluA1- and GluA2-labeled
particles at extrasynaptic
membranes, synapses and
cytoplasm of SG neurons in
inflamed rats to saline-treated.

A. Surface expression of GluA1 in DH

neuron 1 d post-CFA or saline. Top,
Representative Western blot; bottom,
densitometric analysis. The level of
sample loaded for the total (T) was 10%
of that for the biotinylated surface (S). actin was used as a control.
B. Expression of GluA1 in synaptosomal
fraction from DH.

26

Kopach et. al. Pain, 2011.

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.

27

Conclusion
Increased functional expression of
extrasynaptic Ca2+-permeable AMPARs
contributes to the maintaining of
persistent inflammation

28

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

29

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.
30

Antisense oligonucleotides: scheme of action

31

cmbi.bjmu.edu.cn

Localization of AS ODN in lumbar spinal cord

32

Bourinet et al., THE EMBO JOURNAL (2005) 24, 315 - 324

In Vivo Gene Silencing of PKC

Attenuates Inflammation-induced Hyperalgesia

Effect of anti-sense (AS-) and miss-sense (MS) oligonucleotydes for PKC on CFA-induced hyperalgesia.
Time course of hyperalgesia development following CFA injection, in MS-ODN-treated and AS-ODN-treated rats.
Statistics of paw withdrawal latency value in different groups of rats.

33
Voitenko group, unpublished
data

Paw withdrawal latency and RI values in 4

days of As ODN treatment.
12
PWL (s)

10

8
6
4

Rectification index

14

*
#

2
0

S
C ________________
S
C
S
C
________________
_____________
___i.t. AS-ODN
i.t. MS-ODN
i.t. Saline

Naive

Saline AS-ODN MS-ODN

_________________
CFA

S - saline-injected; C CFA-injected

Voitenko group, 34
under preparation

In Vivo Gene Silencing of PKC Reverses:

CFA-induced Rectification of
Synaptic Currents

CFA-induced Potentiation of
Extrasynaptic AMPARs current and
[Ca2+]i transients in tonic SG neurons
Dendrite
Soma

0.2,
F340/F 380

AMPA 5 M

AS-ODN

I-V curve of evoked AMPA-mediated EPSCs in

AS-ODN-treated and MS-ODN-treated
inflammatory animals

50 pA
MS-ODN

35

Voitenko group, unpublished data

60 s

Inflammation
Increased
GluR1 insertion
into extrasynaptic
sites

Increased
synaptic GluR2
internalization

PKC

Ca2+ influx
through
extrasynaptic
AMPARs

INCREASED EXCITABILITY

Ca2+ influx through

Synaptic AMPARs

IMPAIRED SYNAPTIC EFFICACY

CENTRAL SENSITIZATION

PAIN
36

Future aims

Further investigations of molecular mechanisms of pain

Improvement of AS ODN design
Increasing of bioactivity of AS ODN
Improvement of target delivery of genetic materials
37

Acknowledgements:

Bogomoletz Institute of Physiology,

National Academy of Sciences of Ukraine
Olga Kopach
Andrij Sotnik
Viacheslav Viatchenko-Karpinski
Pavel Belan

Johns Hopkins University School of

Medicine, Baltimore, Maryland, USA
Yuan-Xiang Tao
Ronald S. Petralia
Jang-Su Park
Xiaowei Guan
Ji-Tian Xu
Jordan P. Steinberg
Kogo Takamiya
Richard L. Huganir

Grants:
NIH (NS058886, NS057343) and the JHU Blaustein Pain Research Fund (Y.X.T);
JDRF 1-2004-30 and INTAS 8061 (N.V.); the Intramural Research Program of NIDCD (R.S.P.);
NIH NS036715 and Howard Hughes Medical Institute (R.L.H).

38

THANKS!

39 Vadim Holovanov
Artist: