Supplemental Fig. 1. Schematic representation of experimental procedures used.

(A-I)
Diagram of different protocols used for drug administration where kainate (KA) was the
excitotoxic stimulus and nicotine (1 µM) was applied for 4 h. Two nAChR antagonists, DHβE
and MLA alone or in combination (DHβE+MLA) were used for 4 h.
Supplemental Fig. 2. Changes in polysynaptic reflexes and electrically evoked fictive
locomotion evoked by 1 μM nicotine re-applied on the second day in vitro after first application
of nicotine (4 h) on the previous day. Nico 0 min indicates response immediately prior to
second application of nicotine while sham indicates naive preparations.(A, C) Sample records
of changes in polysynaptic responses (A) and DR evoked fictive locomotion (C) by re-applying
nicotine on day 2 for 15 min. (B) Bar graphs show further decrease in polysynaptic reflex
amplitude and area by re-application of nicotine for 15 min (n = 4) in comparison to sham (n
= 9) (**P = 0.002, U = 68; P = 0.002, U = 76, respectively ; sham vs Nico 15 min) and to
nicotine 4 h treated spinal cords on day 1 (*P = 0.039, U = 58; P = 0.030, U = 59, respectively;
Nico 0 min vs Nico 15 min). Fig. D shows depression in number of oscillations (P = 0.019, U
= 42, sham vs Nico 15 min), cumulative depolarization amplitude (P = 0.006, U = 40, sham vs
Nico 15 min) and area (P = 0.007, U = 36, sham vs Nico 15 min; P = 0.037, U = 32, Nico 0
min vs Nico 15 min) of DR evoked fictive locomotion.
Supplemental Fig. 3. (A-C) No significant change in immunofluorescence intensity (AU) of
astrocytes (green; S100 staining; Dorsal: P = 0.077, F2,12 = 3.581; Central: P = 0.064, F2,12 =
3.655; Ventral: P = 0.061, F2,12 = 3.752; one way analysis of variance test) and general glia
(red; GFAP staining; Dorsal: P = 0.886, F2,12 = 0.123; Central: P = 0.527, F2,12 = 0.684; Ventral:
P = 0.431, F2,12 = 0.915; one way analysis of variance test) by KA together with or followed
by nicotine for 4 h (KA/Nico 4 h and KA+Nico/Nico 4 h, respectively). n = 3-6 spinal cords;
3-10 sections/preparation. Scale bar = 100 μm.
Supplemental Fig. 4. Effect of earlier application of nAChR antagonists on VR reflexes and
fictive locomotion after 24 h in vitro. (A) DHβE (10 μM, n = 5), MLA (10 nM, n = 5) or
DHβE+MLA (n = 4) application depressed monosynaptic reflex which was significant with
DHβE alone (*P = 0.035, t16 = 2.304, DHβE vs sham). (B, C) The polysynaptic area (C) was
increased (**P = 0.007, t16 = -3.012; P = 0.006, t16 = –3.130; P = 0.03, t12 = –2.469; Student’s
t-test; DHβE vs sham; MLA vs sham; DHβE+MLA vs sham, respectively) with no significant
change in amplitude (B) in comparison to sham (n = 13). (D-F) The number of oscillations (D)
during electrically induced fictive locomotion was reduced by DHβE+MLA co-application in
comparison to sham (P = 0.017, t14 = –2.710; Student’s t-test, DHβE+MLA vs sham), with no
significant change in the area (F) and amplitude (E) of cumulative depolarization. (G) The
cycle amplitude of chemically induced fictive locomotion was significantly depressed in MLA
and DHβE+MLA treated preparations (P = 0.02, t16 = 2.590; P = 0.004, t15 = 3.411,
respectively, MLA vs sham, DHβE+MLA vs sham) while the periodicity was increased in
MLA treated spinal cords (P = 0.001, t16 = –6.696, MLA vs sham).
Supplemental Fig. 5. Example (A) of dorsal horn sections showing neurons (stained with
NeuN, red) and cells (stained with DAPI, blue) 24 h after application of KA or KA plus nAChR
antagonists. Plots (B) show average neuronal numbers calculated after 24 h in vitro in sham
condition (no treatment) or after 1 h application of kainate alone or with KA+DHβE+MLA
(Dorsal: ***P = 0.001, U = 0; Central: P = 0.001, U = 0; Ventral: P = 0.001, U = 0, sham vs
KA; Dorsal: P = 0.006, U = 86.5; Central: *P = 0.017, U =68; Ventral; P = 0.377, U = 30, KA
vs KA+DHβE+MLA; Dorsal: P = 0.001, U = 2; Central: P = 0.001, U = 81; Ventral: P = 0.001,
U = 0, sham vs KA+DHβE+MLA). n = 3-5 spinal cords; 3-10 sections/preparation. Scale bar
= 100 μm.