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Opioids Induce Bidirectional Synaptic Plasticity in a Brainstem Pain Center in

the Rat

Article in The journal of pain: official journal of the American Pain Society · May 2023
DOI: 10.1016/j.jpain.2023.05.001

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7 authors, including:

Ruth Drdla-Schutting Roni Hogri

Medical University of Vienna Medical University of Vienna
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SUPPLEMENTARY INFORMATION

Supplementary figures

Figure S1. A, In control experiments, aCSF application did not affect EPSC amplitudes. In total, LTD and

LTP were observed in 1 out of 9 and 0 out of 9 cells, respectively. B, Illustration of the LPBN and the

localization of the recorded neurons receiving either 0.5 µM (LTD: black dot, no LTD: grey dot) or 10

µM DAMGO (LTP: green dot; no LTP: white dot). LPBN parts: r = rostral; m = middle; c = caudal; 1

internal, 2 dorsal, 3 crescent, 4 external, 5 central, 6 ventral. C, The paired pulse ratio (PPR) was not

affected by either 0.5 µM, 10 µM DAMGO or aCSF application. D,E, MOR activation is necessary for the

induction of low-DAMGO LTD and high-DAMGO LTP. The application of CTOP (5 µM) 5 min before 0.5

µM (D, n=6) and 10 µM DAMGO (E, n=6) blocked the effects of DAMGO on EPSC amplitudes. F, The

application of 5 µM CTOP alone had no effect on EPSC amplitudes (n=7). Scale bars: 100 pA, 20 ms.

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Figure S2. Following washout, DAMGO concentration-dependently induces LTD or LTP in the LPBN of

female rats. A-B, Application of 0.5 µM DAMGO resulted in an acute depression of EPSC amplitudes,

and CTOP-precipitated washout revealed a low-DAMGO LTD, in 8 out of 9 cells. C, In slices treated with

10 µM DAMGO, the acute DAMGO-induced depression of EPSC amplitude was followed by an overall

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return to baseline upon washout; moreover, in 4 out of 11 cells, high-DAMGO LTP was observed (D).

E, In control experiments, aCSF application did not affect EPSC amplitudes in female LPBN neurons

(n=8). Insets show example traces recorded at the indicated time points (average of 3 sweeps); scale

bars: 100 pA, 20 ms (A, B), 200 pA, 10 ms (E). *** p < 0.001 Holm-Sidak corrected post hoc comparisons

with baseline.

Figure S3. mGluRI/II and NMDAR blockade does not affect basal synaptic transmission in the LPBN.

EPSC amplitudes remained stable during S-MCPG (A) and D-AP5 (B) application. Insets show example

traces recorded at the indicated time points (average of 3 sweeps); scale bars: 200 pA, 20 ms.

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Figure S4. Application of 0.5 µM (A) and 10 µM (B) DAMGO upon astrocytic inhibition with

fluoroacetate induced LTD in the majority of cells tested (n=8/9 in both groups). C, Incubation with

fluoroacetate did not affect EPSC amplitudes. Insets show example traces recorded at the indicated

time points (average of 3 sweeps); scale bars: 200 pA, 20 ms.

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Figure S5. Basal synaptic transmission in LPBN neurons receiving input from spinal (A) or PAG axons

(B). The amplitude of photo-evoked EPSCs was stable over time following aCSF application. Insets show

example traces recorded at the indicated time points (average of 3 sweeps); scale bars: 50 pA, 10 ms.

C, Illustration of the LPBN and the localization along the rostro-caudal axis of the recorded neurons

with spinal (grey dot) or PAG input (black dot). LPBN parts: r = rostral; m = middle; c = caudal; 1 internal,

2 dorsal, 3 crescent, 4 external, 5 central, 6 ventral.

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 Supplementary Tables

Post hoc comparisons

Rats ANOVA
(N) DAMGO CTOP
F p p p Figure
0.5 µM DAMGO (n=12) 9 F (1.87, 20.75) = 29.30 0.0001 <0.0001 0.0001 1B
LTD (n=10/12) F (1.69, 15.21) = 47.26 0.0001 <0.0001 <0.0001 1C
Males
10 µM DAMGO (n=14) 11 F (1.17, 15.21) = 31.60 0.0001 <0.0001 0.20 1E
LTP (n=6/14) F (1.03, 5.13) = 66.08 0.0004 0.0003 0.0016 1F
0.5 µM DAMGO (n=12) 9 F (1.76, 19.33) = 1.15 0.33 - - S1B
PPR 10 µM DAMGO (n=14) 11 F (1.35, 17.60) =0.88 0.39 - - S1B
aCSF (n=9) 8 F (1.37, 11.00) = 1.73 0.22 - - S1B
0.5 µM DAMGO (n=6) 6 F (1.61, 8.05) = 0.424 0.63 - - S1D
CTOP
10 µM (n=6) 5 F (1.68, 8.41) = 1.047 0.38 - - S1E
0.5 µM (n=9) 7 F (1.60, 12.83) = 31.59 0.0001 <0.0001 0.0002 S2A
LTD (n=8/9) F (1.82, 12.73) = 45.76 0.0001 0.0002 0.0002 S2B
Females
10 µM DAMGO (n=11) 7 F (1.52, 15.19) = 15.16 0.0005 0.0001 0.61 S2C
LTP (n=4/11) F (1.31, 3.92) = 16.64 0.014 0.13 0.020 S2D
0.5 µM DAMGO (n=10) 7 F (1.59, 14.35) = 5.56 0.021 0.0042 0.47 2A
LTD (n=2/10) Too few cells for ANOVA 2B
S-MCPG
10 µM DAMGO (n=10) 6 F (1.21, 10.85) = 8.821 0.010 0.0010 0.1394 2C
LTP (n=4/10) F (1.16, 3.48) = 27.16 0.0089 0.0470 0.047 2D
0.5 µM DAMGO (n=9) 8 F (1.68, 13.40) = 51.94 0.0001 <0.0001 <0.0001 2E
LTD (n=7/9) F (1.25, 7.48) = 118.30 0.0001 <0.0001 <0.0001 2F
D-AP5
10 µM DAMGO (n=9) 9 F (1.90, 15.21) = 32.63 0.0001 <0.0001 0.0009 2G
LTP (n=0), LTD (n=5/9) F (1.43, 5.74) = 21.03 0.0030 0.0058 0.0039 2H
0.5 µM DAMGO (n=9) 8 F (1.79, 14.32) = 21.17 0.0001 0.0011 0.0011 3A
Fluoro- LTD (n=8/9) F (1.81, 12.64) = 36.76 0.0001 <0.0001 0.0004 S4A
acetate 10 µM DAMGO (n=9) 7 F (1.74, 13.90) = 35.52 0.0001 <0.0001 0.0006 3B
LTP (n=0), LTD (n=8/9) F (1.68, 10.06) = 40.39 0.0001 0.0006 0.0006 S4B
0.5 µM DAMGO (n=10) 9 F (1.38, 12.41) = 36.44 0.0001 0.0002 0.0004 4C
Spinal LTD (n=9/10) F (1.19, 9.55) = 60.01 0.0001 <0.0001 <0.0001 4D
input 10 µM DAMGO (n=9) 7 F (1.51, 12.06) = 23.18 0.0002 0.0002 0.15 4E
LTP (n=4/9) F (1.06, 3.17) = 41.28 0.0063 0.039 0.0022 4F
0.5 µM DAMGO (n=9) 8 F (1.91, 15.29) = 9.31 0.0025 0.013 0.0039 5C
LTD (n=6/9) F (1.88, 9.41) = 12.71 0.0023 0.012 0.0074 5D
PAG input
10 µM DAMGO (n=9) 9 F (1.63, 13.02) = 13.35 0.0011 0.0055 0.023 5E
LTP (n=0), LTD (n=1/9) Too few cells for ANOVA 5F
aCSF (males) (n=9) 8 F (1.32, 10.58) = 0.07 0.85 - - S1A
aCSF (females) (n=8) 5 F (1.66, 11.58) = 1.46 0.27 - - S2E
CTOP (n=7) 6 t(6)=0.497 0.64 (paired t-test) S1F
Control MCPG (n=6) 6 F (1.23, 6.13) = 0.42 0.58 - - S3A
Recordings d-AP5 (n=6) 4 F (1.10, 5.48) = 0.51 0.52 - - S3B
Fluoroacetate (n=6) 5 F (1.31, 6.56) = 0.61 0.50 - - S4C
aCSF (spinal input) (n=6) 6 F (1.27, 6.36) = 0.68 0.47 - - S5A
aCSF (PAG input) (n=8) 8 F (1.53, 10.68) = 1.41 0.28 - - S5B

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 Supplementary Table 1. Statistics of electrophysiological experiments (EPSC amplitudes and PPR).

Unless otherwise specified, data were analysed using a one-way mixed-model ANOVA, followed by

Holm-Sidak corrected post hoc comparisons with baseline.

Post hoc comparisons

Low-
Low- High- DAMGO vs
Figure
DAMGO vs DAMGO vs High-
ANOVA Vehicle Vehicle DAMGO
F p p p p
% of responding DAMGO F (2, 84) = 10.97 <0.0001 0.0005 0.0003 0.74 3E
cells CTOP F (2, 85) = 6.65 0.0021 0.023 0.0029 0.44 3F
DAMGO F (2, 70) = 3.85 0.026 0.15 0.025 0.32 3G
Event rate
CTOP F (2, 78) = 9.82 0.0002 0.049 <0.0001 0.049 3H
DAMGO F (2, 70) = 1.55 0.22 - - - 3I
Peak amplitude
CTOP F (2, 78) = 1.57 0.10 - - - 3J

Supplementary Table 2. Statistics of calcium imaging experiments. Data were analysed using a one-

way ANOVA, followed by Holm-Sidak corrected post hoc comparisons. Vehicle group n=36, N=10

animals, low-DAMGO group n=26, N=9 animals, high-DAMGO group n=25 slices, N=10 animals.

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