Activation of spinal dorsal horn inhibitory networks by the CLTMR derived chemokine TAFA4
1,2 1,2 3 1,2 4 1,2
Charline Kambrun Olivier Roca‐Lapirot Chiara Salio Marc Landry Aziz Moqrich Yves Le Feuvre
1 2 3 4
University Bordeaux, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux France CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, 33000 Bordeaux France Department of Veterinary Sciences, University of Turin, 10095 Grugliasco (TO), Italy Aix‐Marseille‐Université, CNRS, Institut de Biologie du Développement de Marseille, UMR 7288, case 907, 13288 Marseille Cedex 09, France.
Introduction:
0 0,2 0,4 0,6 0,8 1 1,2 1 5,0 10,0 15,0 20,0 25,0 Control TAFA4 Wash2‐ TAFA4 increases spontaneous inhibitory activity in dorsal horn
ControlTAFA4 Wash
Control
TAFA4
3‐ TAFA4 modulates c fibers evoked EPSCs
Regarding inhibitory synaptic activity in control condition there is a significant increase of IPSC frequency after 40 min of application of TAFA . This increase is reversible after 30 min of wash.
In attempt to characterise if TAFA4 modulates synaptic transmission between C fibers and lamina IIi interneurons, we recruited nociceptive fibers through high intensity stimulation (500 µA, 500 ms) of dorsal root and measured the amplitude and the paired pulse ratio (PPR) of evoked excitatory synaptic responses in control condition and after application of TAFA4. There is a significant decrease of eEPSCs amplitude as well as an increase of the PPR following a bath application of TAFA4 which thus appears to directly
modulate synapses between primary afferences and neurons of lamina IIi.
To determine if the modulation of synaptic activity by TAFA4 is of presynaptic origin, we recorded m EPSCs (top) and mIPSCs (bottom). We found that the TAFA4 induced decrease of EPSC and increase of IPSCs frequency are preserved in presence of TTX.
These results demonstrate that TAFA4 acts on neuronal networks through a presynaptic mecanisms.
0 0,2 0,4 0,6 0,8 1 1,2 0.00 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Cumula tiv e pr obability
Inter event interval (s)
Control
TAFA4
Wash
1‐ TAFA4 decreases spontaneous excitatory activity in dorsal horn
* ns
1s 20 pA
Wash
4‐ Presynaptic action of TAFA4 on synaptic activity
We measured the occurrence of EPSCs over 3 min recordings in control condition after 40 min of superfusion of TAFA4 (20nM) and after wash. We show that a bath application of TAFA4 induces a significant decrease (reversible in 3 cells upon 7) of the EPSCs frequency.
The objective of this project was to identify
the functionnal consequences of TAFA4 on
lamina Iii neuronal networks using a
pharmacological approach.
Our results demonstrate that TAFA4
interacts with inhibitory interneurons.
TA FA 4 m o d u l a t e s p r e sy n a p t i c a l l y
GABAergic transmission. As a consequence
there is a decrease of evoked and
spontaneous excitatory synaptic activity.
TAFA4 induces a shift of the excitation
/inhibition balance toward increased
inhibition. Thus GABAergic interneurons
are the first relay for the modulation of
painful information in the dorsal horn.
Conclusion:
8‐ TAFA4 decreases spontaneous EPSC frequency in CFA model
11‐ Effect of TAFA4 mediated though GABAergic transmission
We found that the average frequency of spontaneous EPSC frequency is significantly reduced in spinal slices of CFA animals incubated with TAFA4 compared to non‐incubated slices. In inflammatory condition the effect of TAFA4 on EPSCs frequency is
preserved. Control TAFA4
CFA aCSF
CFA TAFA4
1s 20 pA7‐ Antinociceptive action of TAFA4 in CFA inflammatory pain model
1s 100 pA 1s Control (TTX) TAFA4 1 Wash Control (TTX) TAFA4 Wash
Pain is essential to protect animals from potentially
harmful stimulus. Its elaboration results from the
integration within dorsal spinal cord networks of sensory
and nociceptive information conveyed by primary
afferents. These information are then forwarded to the
brain where the pain sensation elaborates. Among
primary afferent neurons, C‐Low threshold Mechano
Receptors (C‐LTMR) have been recently identified as
modulators of pain transmission. These fibres whose soma
are localised in dorsal root ganglia, specifically project
toward the inner Rexed lamina II (IIi). Recent molecular
characterisation revealed that C‐LTMRs express a distinct
set of molecular markers among which the
antinociceptive chemokin TAFA4. The aim of this work is
to investigate the role of this chemokin in the modulation
of synaptic transmission within spinal network using
pharmacological approach.
Methods:
In vitro electrophysiology:
C57Bl 6j mice (3‐4weeks) were
used for pharmacological experiment. After removal of the
spinal cord from backbone, acute transversal slices
(300µm) from the lumbar enlargement were obtained.
Whole cell patch‐clamp recording was used to record
synaptic transmission within lamina IIi. To record
excitatory post synaptic currents (EPSCs) the recording
electrode was filled with K‐gluconate solution. To study
inhibitory post synaptic currents (IPSCs) a chloride
enriched solution was used and slices were superfused
with DL‐AP5 (25μM), CNQX (10μM) to block ionotropic
glutamatergic transmission. Miniature EPSCs (mEPSCs)
and IPSCs (mIPSCs) were recorded in presence of
tetrodotoxine TTX (1μM). A glass succion electrode was
used to stimulate spinal dorsal root .
To block inhibitory transmission we used antagonist of
GABA receptor bicuculline (10 μM), of GABA receptor
A BCGP (1 μM) and antagonist of glycinergic receptor
strychnine (50 μM).
Inflammatory model: Mice received a 10 μL subcutaneous
injection of Complete Freund's Adjuvant (CFA, 1 mg/mL
Mycobacterium Tuberculosis, 0.85 mL parafinn oil, 0.15 mL
mannide monooleate) into the left hindpaw. Behavioral
measurement were done before injection, 48h after the
CFA injection and 1h after the intrathecal injection of
TAFA4 (200μg/ml). To test the mechanical sensitivity of
inflammated mice, we used Von Frey test consisting in a
stimulation of plantar surface of the left hindpaw with
calibrated filaments of increasing force (0.07,0 .16, 0.4,1 ,
1.4 g ). Mice were sacrified 48h after the injection of CFA.
In this experiment slices were then incubated either in
artificial cerebro‐spinal fluid (aCSF) or in TAFA4 (20nM)
during 30 min before recordings.
In vivo electrophysiology: C57Bl 6j mice (8 weeks) were
used for these experiments 4 days after CFA injection.
VonFrey filaments were used to stimulate the hindpaw and
thus induce the neuronal discharge recorded with a glass
electrode.
To determine if TAFA4 modulates the nociceptive transmission between primary afferences and the interneurons of lamina II in inflammatory condition, the PPR of evoked responses of interneurons were measured in slices incubated or not with TAFA4. In incubated slices, the average PPR was higher than in non incubated slices (p=0.1), with a clear shift of the population toward higher PPR values. This result indicate that
the effect of TAFA4 on synaptic transmission between C fbers and lamina II interneurons is preserved in inflammatory condition.
9‐ Effect of TAFA4 on C‐fibers evoked EPSCs in CFA condition
Stim 1 Stim 2
As the effect of TAFA4 has not been tested in CFA model, we assessed the antinociceptive action of intrathecally injected TAFA4. Using Von Frey filaments, we found a significant increase in paw withdrawal theshold following TAFA4 injection in inflammed mice.
In attempt to caracterise if TAFA4 acts in inflammatory condition though GABAergic transmission, we assessed the effects of TAFA4 on neuronal discharge in the presence of GABA receptors antagonists. With bicuculline and CGP, TAFA4 is no longer able to decrease the discharge evoked by Von Frey stimulation of the paw, indicating that the effect of TAFA4 on nociceptive transmission is mediated though GABAergic transmission.
CFA aCSF CFA TAFA4 10 ms 100 pA Stim 1 Stim 2 300 nM
6‐ Direct synaptic contact between C‐LTMRs and GABAergic
terminals
0 5 10 15 EPSCs fr eq. (Hz)Control TAFA4 (20nM) Wash 7 14 mEPSCs fr eq. (Hz) 0 2 4 6 8 10 14
CFA aCSF CFA TAFA4
EPSCs Fr eq. (Hz) 0 5 10 15 19 24
Control CFA CFA TAFA4
0.0 0.5 1.0 1.5 V on Fr ey f or ce ( g) 8 8 8 2.0 0 20 40 60 0 50 100 150 ACSF (n=5) TAFA4 (n=4) Lamina II neur onal dischar ge (%baseline)
Time relative to infusion (min)
Naive mice (15g) 0 20 40 60 0 50 100 150 200 ACSF (n=5) TAFA4 (n=5)
Time relative to infusion (min)
Inflammed mice 0 50 100 150 200 CFA+T AFA4 Lamina II neur onal dischar ge (%baseline) 5 5 CFA aCSF CFA+T AFA4+ CGP+Bicuculline CFA+C GP +Bicuculline
Before TAFA4 After TAFA4 Before TAFA4 After TAFA4
0 2 4 6 8 8
0
50
100
150
TAFA4 Control TAFA4+ CGP+Bicuculline+Strychnine TAFA4+C GP +Bicuculline TAFA4 +Str ychnine Normaliz ed EPSC fr eq. (Hz) 12 12 0 1 2 3CFA aCSF(n=12) CFA TAFA4(n=14)
eEPSC pair
ed pulse r
atio
10 ms
100 pA
5‐ Effect of TAFA4 mediated though inibitory transmission
GFP (10nM)
VGlut3 (30nM) GABA (20nM)
100 ms 100 ms
We hypothesized that the decrease of EPSCs fequency could be a consequence of the increase of the IPSCs frequency. T h u s w e r e c o r d e d E P S C s w i t h a nta go n i st s o f G A BA re c e pto rs (bicuculline and CGP) and antagonist of glycinergic receptor (strychnine). We demonstrate that TAFA4 acts though inhibitory transmission. By blocking either GABAergic or glycinergic transmission, we also demonstrate the
effect of TAFA4 is mediated though the m o d u l a t i o n o f t h e G A B A e r g i c transmsission.
To assess if there is morphological correlation to this functional effect of TAFA4, we took advantage of TAFA4Venus knock‐in mice and performed ultrastructural studies. Quantitative analysis of 295 IR‐profiles showed that there was a net predominance of contacts between Venus+ fibers (blue) and GABA‐IR presynaptic vesicle‐containing dendrites (red) (97.6%).
This result reinforce the action of TAFA4 on GABAergic interneurons. 0.25 0.75 1.25 1.75 2.25 0.25 0.75 1.25 1.75 2.25 0 20 40 60 CFA+aCSF CFA+TAFA4 % of cells
Paired pulse ratio 100pA 0 20 40 60 0 50 100 150 Bicuculline+CGP TAFA4+Bicuculline+CGP
Time relative to infusion (min)
Lamina II neur
onal dischar
ge
(%baseline)
10‐ TAFA4 decreases neuronal discharge in CFA animals
In attempt to caracterise the effect of TAFA4 on sensory input integration within the lamina II in the spinal cord the neuronal discharge was first measured in naive animal (left) by in vivo electrophysiology. Following a local infusion of TAFA4 within lamina II TAFA4 does not alter the neuronal discharge. However in inflammed animals (right), there is a significant decrease of the neuronal discharge 40 min after the infusion of TAFA4 .
These results demonstrate that TAFA4 could modulate the integration of nociceptive input in inflammatory condition.
(15g)
This work was funded by:
C‐LTMR ANR 2014
French Ministry of higher education and research
0 2 4 6 IPSCs fr eq. (Hz) 7 3
Control TAFA4 (20nM) Wash
Cumula
tiv
e pr
obability
Inter event interval (s)
* ns ACSF TAF A 0.0 0.5 1.0 1.5 2.0
eEPSC Paired Pulse Ratio
10 100 1000
ACSF TAF A
eEPSC amplitude (pA)
* * Control TTX TAFA4 Wash * ns Control TTX TAFA4 Wash mIPSCs fr eq. (Hz) ** ns * * ** ns ** *
Von frey stim
* * ** * * ns ns 9 17 9 14 7 14 8 5 5
