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Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015 1

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SYNAPTIC MODULATION OF EXCITATORY SYNAPTIC TRANSMISSION BY NICOTINIC ACETYLCHOLINE RECEPTORS IN SPINAL VENTRAL HORN NEURONS

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Q2 N. MINE, a W. TANIGUCHI, b* N. NISHIO, b N. IZUMI, a

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transmission in the spinal ventral horn. Ó 2015 Published by Elsevier Ltd. on behalf of IBRO.

N. MIYAZAKI, a H. YAMADA, a T. NAKATSUKA b AND M. YOSHIDA a a Department of Orthopedic Surgery, Wakayama Medical University, Wakayama 641-8510, Japan

Key words: nicotinic acetylcholine receptors, spinal cord, ventral horn, motoneurons, patch-clamp.

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Pain Research Center, Kansai University of Health Sciences, Kumatori, Osaka 590-0482, Japan

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Abstract—Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, including learning, memory, and recognition. However, functions of the cholinergic system in spinal motoneurons remain poorly understood. In this study, we investigated the actions of presynaptic and postsynaptic nAChRs in spinal ventral horn neurons by performing whole-cell patch-clamp recordings on lumbar slices from male rats. The application of nicotine or acetylcholine generated slow inward currents and increased the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs). Slow inward currents by acetylcholine or nicotine were not inhibited by tetrodotoxin (TTX) or glutamate receptor antagonists. In the presence of TTX, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) were also increased by acetylcholine or nicotine. A selective a4b2 nicotinic receptor antagonist, dihydro-b-erythroidine hydrobromide (DhbE), significantly decreased nicotine-induced inward currents without affecting the enhancement of sEPSCs and mEPSCs. In addition, a selective a7 nicotinic receptor antagonist, methyllycaconitine, did not affect either nicotine-induced inward currents or the enhancement of sEPSCs and mEPSCs. These results suggest that a4b2 AChRs are localized at postsynaptic sites in the spinal ventral horn, non-a4b2 and non-a7 nAChRs are located presynaptically, and nAChRs enhance excitatory synaptic

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INTRODUCTION

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15 Nicotinic acetylcholine receptors (nAChRs) are a large 16 family of ligand-gated, ion channels that are widely 17 expressed in the central nervous system and that play 18 major roles in higher brain functions, including learning, 19 memory, and recognition. At least 12 different subunits, 20 including a2–a10 and b2–b4, which form the 21 pentameric nAChR, have been identified. These 22 subunits form many different subtypes of nAChRs, 23 including homomers and heteromers (Changeux and 24 Edelstein, 1998). Homomeric nAChRs are made up of 25 a7, a8, or a9 subunits, while heteromeric nAChRs com26 prise various combinations of a2–a6 with b2–b4 subunits 27 and a9 with a10 subunits (McGehee, 1999; Dani et al., 28 2001). The various nAChR subunits have different phar29 macological and biological properties (Changeux and 30 Edelstein, 1998). The predominant nAChRs are found in 31 most brain regions (McGehee, 1999; Dani et al., 2001). 32 The heteromeric a4b2 is the most common neuronal 33 nAChR and binds nicotine with high affinity (Whiting and 34 Lindstrom, 1986; Couturier et al., 1990; Seguela et al., 35 1993; McGehee and Role, 1995; Buisson and Bertrand, 36 2001; Dani and Bertrand, 2007). The a7 homomer is 37 the other abundant nAChR in the brain (Gotti et al., 2006; Mansvelder et al., 2006). In contrast to a4b2, a7 Q4 38 39 has a low affinity for nicotine. 40 Acetylcholine is involved in the functioning of spinal 41 locomotor networks in several vertebrate systems 42 (Panchin et al., 1991; Fok and Stein, 2002; Quinlan et al., 43 2004). Acetylcholine and nicotine strongly depolarize 44 motoneurons in the Xenopus embryonic spinal cord, and 45 nAChR antagonists can antagonize this effect (Perrins 46 and Roberts, 1994). Previous studies in mammals have 47 shown that neuronal nicotinic receptors can be either pre48 synaptic or postsynaptic [see review (Dani and Bertrand, 49 2007; Marchi and Grilli, 2010]. The presence of functional 50 nicotinic receptors has been reported in spinal motoneu51 rons in immature rats (Blake et al., 1987). In the spinal cord, 52 mRNAs for most nAChR subunits have been identified dur53 ing early developmental stages (Wada et al., 1989, 1990;

*Corresponding author. Tel: +81-72-453-8349; fax: +81-72-4530276. E-mail address: [email protected] (W. Taniguchi). Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazoleproQ3 pionic acid; AP5, D-( )-2-amino-5-phosphonopentanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DhbE, dihydro-b-erythroidine hydrobromide; EGTA, ethylene glycol tetraacetic acid; EPSC, excitatory postsynaptic current; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; mEPSCs, miniature excitatory postsynaptic currents; MLA, methyllycaconitine; nAChRs, nicotinic acetylcholine receptors; NMDA, N-methyl-D-aspartate; SCI, spinal cord injury; sEPSCs, spontaneous excitatory postsynaptic currents; TTX, tetrodotoxin. http://dx.doi.org/10.1016/j.neuroscience.2015.01.015 0306-4522/Ó 2015 Published by Elsevier Ltd. on behalf of IBRO. 1

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Hellstro¨m-Lindahl et al., 1998). Numerous nAChR subunits are expressed during the developmental period of motor neuron cell death in the chick and human spinal cord (Keiger et al., 2003). The a3, a4, a5, and b2 subunits show presynaptic localization in the ventral horn during prenatal development in the human spinal cord, particularly in the synaptic varicosities that fluorescently label for cholinergic markers (Hellstro¨m-Lindahl et al., 1998). However, it is not clear which functional subtypes of nAChRs are expressed in the spinal ventral horn after prenatal development. Furthermore, the electrophysiological characteristics of the cholinergic nervous system in spinal motoneurons remain poorly understood. In this study, we investigated the effects of nAChR activation on excitatory synaptic transmission in ventral horn neurons using whole-cell patch-clamp recordings in Rexed’s lamina IX in lumbar spinal cord slices.

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EXPERIMENTAL PROCEDURES

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All experimental procedures involving the use of animals were approved by the Ethics Committee on Animal Experiments, Wakayama Medical University, and were in accordance with the UK Animals (Scientific Procedures) Act, 1986, and associated guidelines.

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Spinal cord slice preparation

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The methods used for obtaining rat spinal cord slice preparations have been previously described (Nakatsuka et al., 2000). In brief, Sprague–Dawley rats (8–12 days of age) were deeply anesthetized with pentobarbital sodium (60 mg/kg, intraperitoneal), and then lumbosacral laminectomy was performed. The lumbosacral spinal cord (L1–S3) was removed and placed in pre-oxygenated Krebs solution at 1–3 °C. Immediately after removal of the spinal cord, the rats were given an overdose of pentobarbital sodium and were sacrificed by exsanguination. The pia–arachnoid membrane was removed after cutting all ventral and dorsal roots near the root entry zone. The spinal cord was mounted on a microslicer, and a 500lm-thick transverse slice was cut. A spinal cord slice was transferred to a recording chamber (1 ml) and placed on the stage of an upright microscope equipped with an infrared–differential interference contrast (IRDIC) system (BX51WI; Olympus, Tokyo, Japan). The spinal cord slice was superfused at a rate of 5–10 ml/min with Krebs solution saturated with 95% O2 and 5% CO2, and maintained at 36 ± 1 °C. The Krebs solution contained (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl2, 1.2 MgCl2, 1.2 NaH2PO4, 25 NaHCO3, and 11 glucose (pH 7.4).

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Patch-clamp recording from lamina IX neurons

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Laminae were identified under low magnification (5 objective), and individual neurons were identified with a 40 objective under the IR-DIC microscope and monitored by CCD camera (C2741–79; Hamamatsu Photonics, Hamamatsu, Japan) on a video monitor. Whole-cell patch-clamp recordings were made from lamina IX neurons with microelectrodes (4–8 MX) made from thin-walled filament-containing glass (1.5-mm o.d.).

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The patch-pipette solution used to examine the presynaptic actions of acetylcholine or nicotine was composed of (in mM) 135 potassium gluconate, 5 KCl, 0.5 CaCl2, 2 MgCl2, 5 EGTA, 5 HEPES, and 5 ATP-Mg (pH 7.2). Signals were acquired with a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Sunnyvale, CA, USA). Data were digitized with an A/D converter (Digidata 1322, Molecular Devices), and then stored and analyzed with a personal computer using the pCLAMP data acquisition program (Version 10.2, Molecular Devices).

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Drug application

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Drugs were dissolved in Krebs solution and applied by perfusion via a 3-way stopcock without any change in perfusion rate or temperature. The time necessary for the solution to flow from the stopcock to the surface of the spinal cord slice was approximately 30 s. The drugs used in this study included acetylthiocholine chloride (Sigma, St. Louis, MO, USA), nicotine (Nic; Wako, Osaka, Japan), D-( )-2-amino-5-phosphonopentanoic acid (AP5; Tocris, Bristol, UK), tetrodotoxin (TTX; Wako, Osaka, Japan), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; Ballowin, MO, USA), dihydro-b-erythroidine hydrobromide (DhbE; Tocris, Bristol, UK), and methyllycaconitine citrate (MLA; Sigma). CNQX was first dissolved in dimethyl sulfoxide at 1000 the concentration to be used. The other drugs were first dissolved in distilled water at 1000 the concentration to be used. These drugs were diluted to final concentrations in Krebs solution immediately before use.

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Statistical analysis

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All numerical data were expressed as mean ± standard error. In electrophysiological data, n refers to the number of neurons studied. In analyzing the change in frequency and amplitude of postsynaptic currents following the application of either acetylcholine or nicotine, the time course of postsynaptic current frequency before and after agonist application was first constructed with a time bin of 10 s using Mini Analysis Program 5.6.7 (Synaptosoft, Decatur, GA, USA). The average response in 30 s around the peak was then used to calculate the percentage change from control. Paired Student’s t-test or Student’s unpaired t-test was used to determine the statistical significance between two groups’ means, and differences between three groups were compared using the Tukey–Kramer method. The Kolmogorov–Smirnov test was used to compare cumulative distributions of excitatory postsynaptic current (EPSC) parameters in the absence and presence of the test drugs. A value of P < 0.05 was considered significant for these tests.

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RESULTS

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Presynaptic and postsynaptic actions of acetylcholine on lamina IX neurons

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After establishing the whole-cell patch-clamp configuration, spontaneous EPSCs (sEPSCs) were detected in

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lamina IX neurons at a holding potential of 70 mV. These sEPSCs were completely blocked in the presence of 10 lM CNQX, a-amino-3-hydroxy-5-methyl4-isoxazolepropionic acid (AMPA) receptor and kainate receptor antagonist, and 50 lM AP-5, as well as an N-methyl-D-aspartate (NMDA) receptor antagonist, indicating that sEPSCs were mainly mediated by glutamate released from presynaptic terminals innervating lamina IX neurons. Perfusion of 100 lM acetylcholine for 2 min generated inward currents (n = 8; average amplitude: 44.6 ± 10.9 pA) and increased the frequency and amplitude of sEPSCs (Fig. 1A). These changes in inward currents (>5 pA) were observed in almost all recorded neurons (7/8 neurons). The average increases in sEPSC frequency and amplitude mediated by acetylcholine were 306.1 ± 89.1% and 139.2 ± 16.2% (n = 8), respectively (Fig. 1C). The effects of acetylcholine on the cumulative distributions of the inter-event interval and amplitude of sEPSCs are shown in Fig. 1B. Acetylcholine increased the proportion of sEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with the control. In the presence of TTX (0.5 lM), a Na channel blocker, the average amplitude of acetylcholine-induced inward currents was 33.0 ± 6.8 pA (n = 6). Acetylcholine significantly increased the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) compared with TTX alone (Fig. 2A). The average increases in mEPSC frequency and amplitude mediated by acetylcholine were 598.3 ± 153.7% and 113.9 ± 3.5% (n = 5), respectively (Fig. 2B). Acetylcholine increased the proportion of mEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with the control. In the presence of 10 lM CNQX and 50 lM AP-5, the average amplitude of acetylcholine-induced inward currents was 24.2 ± 3.4 pA (n = 5) (Fig. 2C). Results from the Tukey–Kramer method showed no significant difference in acetylcholine-induced inward currents between control, in the presence of TTX, and in the presence of CNQX and AP-5. Presynaptic and postsynaptic actions of nicotine on lamina IX neurons We next examined the effects of nicotine, a selective nAChR agonist, on lamina IX neurons. Perfusion of 100 lM nicotine for 2 min generated inward currents (n = 7; average amplitude: 148.4 ± 36.7 pA) and increased the frequency and amplitude of sEPSC (Fig. 3A). These changes in inward currents (>5 pA) were observed in all recorded neurons. The average increases in sEPSC frequency and amplitude mediated by nicotine were 567.4 ± 300.7% and 171.6 ± 28.7% (n = 7), respectively (Fig. 3C). The effects of nicotine on the cumulative distributions of the inter-event interval and amplitude of sEPSCs are shown in Fig. 3B. Nicotine increased the proportion of sEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with

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the control. Repeated nicotine applications (100 lM) showed an incomplete recovery of responses (Fig. 4A). The second nicotine-induced inward current was less than 10% (4.7 ± 6.1%, n = 6), even after a 30-min wash in the normal bath solution (P = 0.028) (Fig. 4D). These results indicate desensitization of ventral horn neurons by nicotine. Additionally, we investigated how lowconcentration nicotine affects ventral horn neurons. Treatment with 1 lM nicotine produced inward currents (n = 5; average amplitude: 15.7 ± 7.7 pA) (Fig. 4B), although they were smaller than with 100 lM nicotine (Fig. 4C). Repeated 1 lM nicotine applications also produced desensitization (25.3 ± 11.2%) (P = 0.043) (Fig. 4B, D). In the presence of TTX, the average amplitude of nicotine-induced inward currents was 137.1 ± 19.5 pA (n = 14), and nicotine significantly increased the frequency and amplitude of mEPSCs (Fig. 5A). The average increases in mEPSC frequency and amplitude mediated by nicotine were 545.2 ± 61.4% and 133.1 ± 10.6% (n = 10), respectively (Fig. 5B). Nicotine increased the proportion of mEPSCs having a significantly shorter inter-event interval (P < 0.05) and a significantly larger amplitude (P < 0.05) compared with the control. In the presence of CNQX and AP-5, the average amplitude of nicotine-induced inward currents was 154.0 ± 60.2 pA (n = 6) (Fig. 5C). Results from the Tukey–Kramer method showed no significant difference in nicotine-induced inward currents between control, in the presence of TTX, and in the presence of CNQX and AP-5. Subsequently, we examined whether nicotine-induced inward currents and nicotine-mediated increases in sEPSC frequency and amplitude were dependent on extracellular Ca2+. In a Ca2+-free bath solution, nicotine-induced inward currents (n = 6; average amplitude: 6.6 ± 4.1 pA) were smaller than in normal Krebs solution (P = 0.0046) (Fig. 6A, B). Moreover, enhancement of sEPSC by nicotine was reduced in a Ca2+-free bath solution. The application of nicotine (100 lM) in a Ca2+-free bath solution increased the frequency and amplitude of sEPSCs by 273.2 ± 105.6% and 112.0 ± 9.1%, respectively (n = 6); this increase in percentage of sEPSC frequency and amplitude was not significantly increased compared with the control (P = 0.074, P = 0.30) (Fig. 6C). Although the increase of sEPSC frequency was comparatively large, this was smaller than the control with normal Krebs solution (545.2 ± 61.4%).

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Effects of selective nAChR antagonists on nicotineinduced actions

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We examined whether selective nAChR antagonists inhibit nicotine-induced excitatory actions on lamina IX neurons. In the presence of DhbE (10 lM), a selective a4b2 nicotinic receptor antagonist, the average nicotineinduced inward currents were 48.6 ± 9.3 pA (n = 13) (Fig. 7A). In contrast, in the presence of MLA (40 nM), a selective a7 nicotinic receptor antagonist, the average nicotine-induced inward currents were 127.1 ± 18.12 pA (n = 17) (Fig. 7C, F). Results from Tukey– Kramer method showed that average nicotine-induced inward currents in the presence of DhbE were

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Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 1. Presynaptic and postsynaptic effects of acetylcholine on lamina IX neurons. (A) Continuous chart recording of inward currents and sEPSCs before and during acetylcholine application (100 lM, upper). Two consecutive traces of sEPSCs are shown in an expanded scale in time, before (lower left) and during acetylcholine application (lower right). Acetylcholine generated inward currents and increased sEPSC frequency. (B) Cumulative distribution of inter-event interval (left) and amplitude (right) of sEPSCs, before (dotted line) and during (continuous line) acetylcholine action. Acetylcholine reduced the inter-event interval and increased the inter-event amplitude (P < 0.05; Kolmogorov–Smirnov test). Data in A and B were obtained from the same neurons. (C) Summary of sEPSC frequency (left) and amplitude (right) under acetylcholine action relative to control. In this and subsequent figures, vertical lines accompanied by bars show SEM. Statistical significance between data shown by bars is indicated by an asterisk; ⁄P < 0.05.

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significantly less than nicotine alone or in the presence of MLA. Moreover, neither DhbE nor MLA significantly affected this nicotine-induced increase in sEPSC

frequency and amplitude (Fig. 7B, D). In the presence of DhbE, the average increase in sEPSC frequency and amplitude was 1060.0 ± 518.6% and 146.7 ± 16.6%,

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 2. Acetylcholine-induced inward currents were not inhibited by Na+ channel or glutamate receptor blockers, while acetylcholine enhanced mEPSCs. (A) In the presence of TTX, acetylcholine-evoked inward currents were not blocked. Moreover, mEPSCs were enhanced by acetylcholine application. (B) Summary of mEPSC frequency (left) and amplitude (right) under acetylcholine action relative to control. (C) The effect of CNQX and AP-5 on acetylcholine-evoked inward currents. CNQX and AP-5 blocked EPSCs in both the absence and presence of acetylcholine.

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respectively (n = 13). These increases were not significantly different compared with the control (Fig. 7E). For MLA, the average increase in rate of sEPSC frequency and amplitude was 1068.5 ± 214.8% and

196.3 ± 10.6%, respectively (n = 14). These increases were not significant compared with the control (Fig. 7E). Subsequently, we examined in the presence of TTX whether DhbE or MLA inhibits nicotine-induced inward

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 3. Presynaptic and postsynaptic effects of nicotine on lamina IX neurons. (A) A continuous chart recording of inward currents and sEPSCs before and during nicotine application (100 lM: upper). Two consecutive traces of sEPSCs are shown in an expanded scale in time, before (lower left) and during nicotine application (lower right). Nicotine generated inward currents and increased sEPSC frequency. (B) Cumulative distribution of inter-event interval (left) and amplitude (right) of sEPSCs, before (dotted line) and during (continuous line) nicotine action. Nicotine reduced the inter-event interval and increased the inter-event amplitude (P < 0.05; Kolmogorov–Smirnov test). Data in A and B were obtained from the same neurons. (C) Summary of sEPSC frequency (left) and amplitude (right) under nicotine action relative to control.

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currents and nicotine-mediated increases in mEPSC frequency and amplitude. In the presence of DhbE and TTX, the average nicotine-induced inward currents were 33.9 ± 10.8 pA (n = 7) (Fig. 8A). However, the average nicotine-induced inward currents in the

presence of MLA and TTX were 119.7 ± 85.8 pA (n = 6) (Fig. 8B). Similar to the condition without TTX, the average nicotine-induced inward currents with TTX in the presence of DhbE were significantly less than nicotine alone or in the presence of MLA (Fig. 8D).

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

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Fig. 4. Nicotine application induced desensitization of ventral horn neurons. (A) Repeated nicotine (100 lM) applications showed an incomplete response recovery. The second nicotine-induced inward current was significantly less than the first nicotine application, even after a 30-min wash. (B) Low nicotine concentrations (1 lM) produced inward currents, although these were less than 100 lM. Low-concentration nicotine application also induced desensitization. (C) Summary of inward current by 100 lM and 1 lM nicotine. (D) Summary of desensitization by repeated application of 100 lM and 1 lM nicotine.

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Moreover, neither DhbE nor MLA significantly affected the nicotine-induced increase in mEPSC frequency and amplitude (Fig. 8A, B). In the presence of DhbE, the average increase in mEPSC frequency and amplitude was 642.0 ± 216.6% and 106.4 ± 34.9%, respectively (n = 6). These increases were not significantly different compared with the control (Fig. 8C). For MLA, the average increase in the rate of mEPSC frequency and amplitude was 602.5 ± 185.4% and 117.2 ± 11.1%, respectively (n = 12). These increases were not significant compared with the control (Fig. 8C).

DISCUSSION

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In the present study, we analyzed the effects of nAChR activation on excitatory synaptic transmission in ventral horn neurons by performing whole-cell patch-clamp recordings in lamina IX neurons in young rat lumbar cord slices. CNQX, a non-NMDA receptor antagonist, and AP-5, an NMDA receptor antagonist, blocked EPSCs at a holding potential of 70 mV, suggesting the involvement of glutamate in excitatory synaptic transmission in ventral horn neurons. Several studies

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Fig. 5. Nicotine-induced inward currents were not inhibited in the presence of Na+ channel or glutamate receptor blockers, while nicotine enhanced mEPSCs. (A) In the presence of TTX, nicotine-evoked inward currents were not blocked. Moreover, mEPSCs enhanced nicotine application. (B) Summary of mEPSC frequency (left) and amplitude (right) under nicotine action relative to control. (C) The effect of CNQX and AP-5 on nicotineevoked inward currents. CNQX and AP-5 blocked sEPSCs in both the absence and presence of nicotine.

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reported that excitatory synaptic transmission induced by motoneurons at motoneuron-Renshaw cell synapses has both glutamatergic and nicotinic components (Mentis et al., 2005; Nishimaru et al., 2005; Lamotte d’Incamps et al., 2008). In the present study, fast EPSCs disappeared completely in the presence of glutamatergic receptor antagonists. In the presence of TTX, the acetylcholine- or nicotine-induced increase in mEPSC frequency was observed. This suggests that nAChRs are expressed on presynaptic terminals and that these nAChRs enhance release of glutamate from the presynaptic terminal in spinal ventral horn neurons. Thus, in presynaptic terminals of the ventral horn neurons, glutamatergic

transmission predominates, while nicotinic transmission is an excitatory modulator of glutamate transmission. The difference between sEPSCs and mEPSCs in the percent increase of frequency by acetylcholine was large in our study. It is unclear why these differences occurred. We suggest that this reflects part of the normal range, as acetylcholine or nicotine can exhibit very strong effects in some neurons. Acetylcholine is also known to affect both nAChR and muscarinic acetylcholine receptors, and there is evidence that muscarinic acetylcholine receptor activation can increase excitability of spinal ventral horn neurons (Nishimaru et al., 2005; Mentis et al., 2005; Barthe´le´my-Requin et al., 2006; Chevallier et al., 2006;

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 6. Nicotine-induced inward currents and nicotine-mediated increases in sEPSC frequency and amplitude were dependent on extracellular Ca2+. (A) In a Ca2+-free bath solution, the nicotine-induced inward currents and enhancement of sEPSCs were inhibited. (B) Summary of nicotineinduced inward currents in a Ca2+-free bath solution. (C) sEPSC frequency (left) and amplitude (right) in a Ca2+-free bath solution relative to control.

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Miles et al., 2007). Although we focused the specific effect of nAChR activation on spinal ventral horn neurons in this study, it is also possible that muscarinic receptors are involved in the high increase of EPSC frequency by acetylcholine. However, muscarinic receptor activation was reported to have inhibitory or both excitatory and inhibitory effects on spinal ventral horn neurons (Jiang and Dun, 1986; Kurihara et al., 1993; Perrins and Roberts, 1994; Quinlan and Buchanan, 2008). Thus, the effect of muscarinic receptor activation in the spinal ventral horn remains controversial. In the presence of TTX, acetylcholine- or nicotineinduced slow inward current and enhancement of mEPSC amplitude were observed. These effects suggest that nAChRs have postsynaptic excitation in spinal ventral horn neurons. Several studies have shown that a4b2 and a7 nAChRs are predominantly expressed in the central nervous system (McGehee, 1999; Dani et al., 2001). To provide insight into which nAChR subtypes are functionally expressed in the ventral horn of the spinal cord, we administered nicotine in the presence of DhbE, an a4b2 receptor antagonist type, or MLA, an a7 receptor antagonist. Compared with a single administration of nicotine, the amplitudes of the nicotineinduced inward currents were reduced in the presence of DhbE. This indicates that a4b2 nAChRs are functionally and postsynaptically expressed in ventral horn neurons. By comparison, the enhancement of sEPSCs or mEPSC frequency induced by nicotine was not affected by DhbE, suggesting that presynaptically localized nAChRs are non-a4b2. The amplitude of the nicotine-induced

inward current was not significantly affected by MLA, while MLA did not block sEPSCs or mEPSC enhancement, suggesting that non-a7 nAChRs are expressed at presynaptic and postsynaptic sites. Overall, these data suggest that the excitatory presynaptic terminals in ventral horn neurons may be modulated by a non-a4b2 and non-a7subtype of nAChRs, and that a4b2 AChRs are localized at postsynaptic sites in the spinal ventral horn. Nicotine was previously reported to produce an inward current via activation of non-a4b2 and non-a7 AChRs in the superficial dorsal horn, and via a4b2 AChRs in the deep dorsal horn, of the adult rat spinal cord (Takeda et al., 2003, 2007). Nicotine was also shown to enhance the frequency of sEPSC or mEPSC without significant changes in amplitude between neurons of the medical habenula nucleus and interpeduncular nucleus, as well as between neurons of the visceral motor nucleus of Terni and the lumbar sympathetic ganglion without inward current (McGehee et al., 1995); this enhancement was blocked by aBgTX, an a7 antagonist. Therefore, in contrast to our data, these studies indicate that presynaptic, rather than postsynaptic, nAChRs mediate this neurotransmission. These different electrophysical responses of nAChRs may be due to the distinct distribution of many nAChR subtypes in the central nervous system (Grove et al., 2011). In this study, the nicotine-induced increase in sEPSC frequency and amplitude was dependent on extracellular Ca2+. Although there was no statistically significant difference in the sEPSC frequency increase without Ca2+, the increased percentage of sEPSC was high.

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 7. Effects of selective nicotinic acetylcholine receptor antagonists on inward currents and EPSC enhancements by nicotine. (A) A continuous chart recording of sEPSCs before and during nicotine application (100 lM: upper) in the presence of dihydro-b-erythroidine hydrobromide (DhbE). Two consecutive traces of sEPSCs are shown in an expanded scale in time, before (lower left) and during nicotine application (lower right). Nicotine generated inward currents and increased sEPSC frequencies and amplitude in the presence of DhbE. (B) Cumulative distribution of the inter-event interval (left) and amplitude (right) of sEPSCs, before (dotted line) and during (continuous line) nicotine action showing that nicotine decreased the inter-event interval and increased the inter-event amplitude (P < 0.05; Kolmogorov–Smirnov test). Data in A and B were obtained from the same neurons. (C) A continuous chart recording of sEPSCs before and during nicotine application (100 lM: upper) in the presence of MLA. Two consecutive traces of sEPSCs are shown in an expanded scale in time, before (lower left) and during nicotine application (lower right). Nicotine generated inward currents and increased sEPSC frequencies and amplitude in the presence of MLA. (D) Cumulative distribution of the inter-event interval (left) and amplitude (right) of sEPSCs, before (dotted line) and during (continuous line) nicotine action showing that nicotine reduced and shifted the inter-event interval and increased the inter-event amplitude (P < 0.05; Kolmogorov–Smirnov test). Data in C and D were obtained from the same neurons. (E) Summary of nicotine-induced sEPSC frequencies (left) and amplitude (right) in the absence (n = 7) or presence (n = 14) of each selective nicotinic receptor antagonist. There were no statistically significant differences between the various treatments. (F) Summary of amplitude of nicotine-induced inward currents in the absence (n = 7) or presence (n = 13) of each selective nicotinic receptor antagonist. 419 420 421

However, the increased percentage of sEPSC without Ca2+ was smaller than with Ca2+. The activation of pre-synaptic a7 nAChRs can increase Ca2+ influx in the

brain. Alternatively, non-a7 nAChRs can initially increase Na+ influx and nerve ending depolarization, and subsequent Ca2+ entry through voltage-gated Ca2+

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Fig. 8. Effects of selective nicotinic acetylcholine receptor antagonists on inward currents and mEPSC enhancements by nicotine. (A) A continuous chart recording of mEPSCs before and during nicotine application (100 lM: upper) in the presence of dihydro-b-erythroidine hydrobromide (DhbE). Nicotine generated inward currents and increased mEPSC frequencies and amplitude in the presence of DhbE. (B) A continuous chart recording of mEPSCs before and during nicotine application (100 lM: upper) in the presence of MLA. Nicotine generated inward currents and increased mEPSC frequencies and amplitude in the presence of MLA. (C) Summary of nicotine-induced mEPSC frequencies (left) and amplitude (right) in the absence (n = 7) or presence of each selective nicotinic receptor antagonist (n = 6 (DhbE), 12 (MLA)). There were no statistically significant differences between various treatments. (D) Summary of amplitude of nicotine-induced inward currents in the absence (n = 7) or presence of each selective nicotinic receptor antagonist (n = 7 (DhbE), 6 (MLA)).

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channels activates neurotransmitter release (Marchi and Grilli, 2010). Similarly, in the ventral horn, activation of voltage-gated Ca2+ channels following activation of presynaptic non-a7 nAChRs could contribute to increased glutamate. In the present study, repeated nicotine applications showed an incomplete recovery of response, indicating desensitization. As repeated low- or high-dose nicotine application did not produce the same inward currents in our neonatal spinal cord slice preparation, it is difficult to determine the antagonist profiles on the same neurons. Thus, all experiments in the present study were performed with a single agonist application on a fresh slice without prior drug application. Desensitization is a multiple component complex mechanism that reflects conformational transition of the receptor to multiple inactive states (Dani et al., 2000; Quick and Lester, 2002; Giniatullin et al., 2005). The a7-containing nAChRs are known to desensitize rapidly, making it difficult to observe their actions in the brain (Couturier et al., 1990; Gerzanich et al., 1994). However, in the present study, the a4b2 nAChRs were involved in generating an inward current in ventral horn neurons. It was previously reported

that a4b2 nAChRs desensitize slower than a7 nAChRs at high ACh concentrations (Dani et al., 2000; Quick and Lester, 2002; Wooltorton et al., 2003). Thus, desensitization in the ventral horn may result from slower desensitization of a4b2 nAChRs. As inward currents were produced by acetylcholine or nicotine in the presence of CNQX and AP-5 in our study, the inward currents were not a summation of EPSC. Thus, the mechanism involves a postsynaptic effect. The opening of nAChRs allows the movement of cations (mainly Ca2+) across the cell membrane, which manifests as a depolarizing current (DajasBailador and Wonnacott, 2004). We suggest that this mechanism may provide strong and direct driving forces for inward current as nicotine-induced inward currents were suppressed in a Ca2+-free bath solution. Increased intracellular Ca2+ may activate a large number of intracellular biochemical pathways, which in turn may contribute to activation of AMPA and NMDA receptors. Lamina IX includes both motoneurons and interneuron Renshaw cells. In the present study, the soma of neurons in lamina IX were identified under IRDIC, allowing us to record by visual patch-clamp and analyze these specific neurons. Although we were

Please cite this article in press as: Mine N et al. Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal

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unable to distinguish between motoneurons and Renshaw neurons using the shape of the soma, almost all lamina IX neurons (composed of both motoneurons and Renshaw cells) reacted to acetylcholine or nicotine. This suggests that both motoneurons and Renshaw cells exhibit enhanced excitatory transmissions following nAChR activation. Recent evidence indicates that nicotine may have potent neuroprotective effects in different forms of chronic and acute neurotoxicity (Pauly et al., 2004; Nakamizo et al., 2005; Egea et al., 2007; Huang et al., 2007; Toborek et al., 2007; Picciotto and Zoli, 2008). The mechanisms underlying these protective effects are not fully understood, although nAChR-mediated mechanisms appear to play a major role (Pauly et al., 2004; Nakamizo et al., 2005; Egea et al., 2007; Huang et al., 2007; Toborek et al., 2007; Picciotto and Zoli, 2008). Although, no study has investigated the excitatory function of nAChRs in the spinal ventral horn, nor whether these receptors can impact functional recovery after spinal cord injury (SCI), nicotine can exert beneficial effects on functional recovery after experimental SCI in rats (Ravikumar et al., 2004, 2005). Previous studies indicate that a4b2 and a7 receptors may play the most significant roles in the central nervous system (Nakayama et al., 1995; Hsu et al., 1997; Berg and Conroy, 2002). Other studies suggest that a7 receptors are highly expressed in cultured spinal cord neurons and have an important role in nicotine-mediated neuroprotection (Toborek et al., 2007). However, in the present study, the selective a7 receptor blocker did not inhibit nicotineinduced inward currents and sEPSCs, while the selective a4b2 receptor blocker inhibited nicotine-induced inward currents. These results indicate that a4b2 receptors, and perhaps other receptors, but not a7 receptors, mediate the excitatory actions of acetylcholine on spinal motoneurons. These findings suggest that agonists for these receptors may have therapeutic potential in functional recovery after SCI.

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CONFLICT OF INTEREST STATEMENT

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The authors report no conflicts of interest regarding this study.

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Acknowledgment—This work was supported in part by the 2012 Wakayama Medical Award for Young Researchers to M.N.

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(Accepted 10 January 2015) (Available online xxxx)

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Q1 ventral horn neurons. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.01.015

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Synaptic modulation of excitatory synaptic transmission by nicotinic acetylcholine receptors in spinal ventral horn neurons.

Nicotinic acetylcholine receptors (nAChRs) are distributed widely in the central nervous system and play important roles in higher brain functions, in...
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