Patch clamp - effect of compounds on glycine-evoked currents

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Effect of compounds on
glycine-evoked currents
of mouse spinal cord

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SUMMARY
Introduction
The aim of the study is to document the functional activity of PAM at Gly receptors
Materials & Methods
Slice preparation
Recordings
Experimental protocol and sample traces
Experiments
Investigation of PAM 1 and PAM 2

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MATERIALS & METHODS
Slice preparation
Transverse spinal cord slices are prepared from C57/BL6 mice (6-8 weeks old, Elevage Janvier, France).
The lumbar region is isolated after sacrificing the animal and transverse slices are prepared after gluing the
lumbar region onto the base of the vibratome chamber.
Slicing is performed in ice-cold sucrose-based solution containing (in mM): sucrose 248, NaH2PO4 1.25, KCl 2,
NaHCO3 26, glucose 11.1, MgSO4 2, kynurenic acid 1, the solution is continuously oxygenated to maintain the
level of oxygen high. Several slices of 250 µm thickness are collected and transferred into the incubation chamber
containing artificial cerebrospinal fluid (aCSF) composed of (in mM): NaCl 126, NaH2PO4 1.25, KCl 2.5, CaCl2 2,
MgCl2 2, glucose 11.1, NaHCO3 26, pH 7.3. The slices were allowed to recover from dissection for at least one
hour at room temperature before electrophysiological experiments. All solutions are bubbled continuously with
carbogen gas (95% CO2, 5% O2).

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MATERIALS & METHODS
Recordings
Slices are transferred into a recording chamber containing about 1 ml of ACSF. The bath solution is oxygenated continuously and
exchanged at a rate of 1.5 ml/min. The recordings are performed at room temperature.
Slices are first visualized using a 10x air objective. For patch-clamp recordings, a 60x water-immersion objective - in combination
with infrared differential interference contrast (IR-DIC) optics - is used.
Whole-cell voltage clamp recordings are performed in lamina II neurons using patch pipettes pulled from borosilicate glass
capillaries. The intracellular pipette solution contains (in mM): CsCl 130, EGTA 10, HEPES 10, MgATP 5, Na3GTP 0.5, pH 7.2.
Voltage clamp is achieved using a Multiclamp 700B amplifier in combination with a Digidata 1440A digitizer (Molecular Devices,
USA). Cells are held at -60 mV. Data are filtered at 2 kHz and digitized at 10 kHz using the pClamp 10 data acquisition software
(Molecular Devices).
10x ventral
dorsal
II III
60x ventral
Lamina II Transverse
spinal mouse cord slice
recording, with the
puffing pipette (bottom
left), the recording
pipette (upper right) .
Magnification: 10x left
panel, 60x right panel.

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MATERIALS & METHODS
Agonist and PAM application
Two successive pressure applications of 20 µM Glycine (2 s puff) are applied to verify the evoked-currents
amplitude stability. Then the PAM is perfused for 10 min onto the slice and two pressure applications of Gly (20
µM, 2 s) are performed again.
Finally, 10 µM strychnine is perfused for 10 min and two pressure applications of Gly (20 µM, 2 s) are performed
again. The full blockade of the Gly-evoked current by strychnine will confirm the “glycinergic “ nature of the
evoked-currents.

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RESULTS
The effect of PAM #1 on the evoked PSCs in lamina II neurons from spinal cord
ACSF*+ xx µM PAM #1
20 µM Glycine
ACSF* =ACSF + 0.5µM TTX + 10µM CNQX + 30µM APV + 10µM bicuculline
50 pA
5s
ACSF*+ xx µM PAM #1
+ strichnine 10µM
ACSF* = control
50 pA
5s
-250
-200
-150
-100
-50
0
Amplitude(pA)
Control PAM # 1 PAM # 1 +
strychnine
N=5
500
400
300
200
100
0
Ratio%
(PAM#1/Control)
Control PAM # 1 PAM # 1 +
strychnine
N=5
YES modulation

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RESULTS
The effect of PAM #2 on the evoked PSCs in lamina II neurons from spinal cord
ACSF*+ xx µM PAM #2
20 µM Glycine
50 pA
5s
ACSF*+ xx µM PAM #2
+ strichnine 10µM
ACSF* = control
50 pA
5s
-100
-80
-60
-40
-20
0
Amplitude(pA)
Control PAM # 2 PAM # 2 +
strychnine
N=5
200
100
0
Ratio%
(PAM#2/Control)
Control PAM # 2 PAM # 2 +
strychnine
N=5
ACSF* =ACSF + 0.5µM TTX + 10µM CNQX + 30µM APV + 10µM bicuculline
NO modulation

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Compound #1 is a potent PAM of Glycine-evoked currents
Compound #2 display no PAM activity on Glycine-evoked currents
CONCLUSION

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