Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons

Journal Pre-proof Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons Hisahiko Kubota, Hironari Akaike, Nobuharu Okamitsu, Il-Sung Jang, Kiku Nonaka, Naoki Kotani, Norio Akaike

PII:

S0361-9230(19)31007-X

DOI:

https://doi.org/10.1016/j.brainresbull.2020.01.016

Reference:

BRB 9849

To appear in:

Brain Research Bulletin

Received Date:

13 December 2019

Revised Date:

20 January 2020

Accepted Date:

22 January 2020

Please cite this article as: Kubota H, Akaike H, Okamitsu N, Jang I-Sung, Nonaka K, Kotani N, Akaike N, Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons, Brain Research Bulletin (2020), doi: https://doi.org/10.1016/j.brainresbull.2020.01.016

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Xenon modulates the GABA and glutamate responses at genuine synaptic levels in rat spinal neurons Hisahiko Kubota a, Hironari Akaike b, Nobuharu Okamitsu c,Il-Sung Jang d, Kiku Nonaka e, Naoki Kotani f, Norio Akaike f, g

a

Department of Pharmacology, Faculty of Medicine, Saga University, 5-1-1

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Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 OeHonmachi, Chuo-ku, Kumamoto 862-0973, Japan

Department of Electrics and Computer Engineering, Faculty of Engineering,

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c

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Nabeshima, Saga, 849-8501, Japan

Hiroshima Institute of Technology, 2-1-1 Miyake, Saeki-ku, Hiroshima 731-5193,

Department of Pharmacology, School of Dentistry, Kyungpook National University,

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d

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Japan

2177 Dalgubeol-daero, Jung-gu, Daegu, 700-412, Republic of Korea Research Division for Life Science, Kumamoto Health Science University, 325

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e

Izumi-machi, Kita-ku, Kumamoto 861-5598, Japan Research Division of Neurophysiology, Kitamoto Hospital, 3-7-6 Kawarasone,

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f

Koshigaya, Saitama 343-0821, Japan Research Division for Clinical Pharmacology, Medical Corporation, Juryo Group,

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g

Kumamoto Kinoh Hospital, 6-8-1 Yamamuro, Kita-ku, Kumamoto 860-8518, Japan

Corresponding author: Corresponding author: Norio Akaike Research Division for Clinical Pharmacology, Medical Corporation, Juryo Group, Kumamoto Kinoh Hospital, 6-8-1 Yamamuro, Kita-ku, Kumamoto 860-8518, Japan 1

Fax: +81 96 345 8188, E-mail address: [email protected]

Highlights:  Effect of Xe on synaptic transmission was studied in rat spinal neuron.  Xe noncompetitively inhibited glutamate and its subtype-induced wholecell current.  Xe inhibited both glutamatergic and GABAergic spontaneous and evoked

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responses.  The inhibition at presynaptic side was greater in the excitatory transmissions.

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 Excitatory response is the main target of anesthesia-induced neuronal

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responses.

Abstract

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Effects of xenon (Xe) on whole-cell currents induced by glutamate (Glu), its three ionotropic subtypes, and GABA, as well as on the fast synaptic glutamatergic and

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GABAergic transmissions, were studied in the mechanically dissociated “synapse bouton preparation” of rat spinal sacral dorsal commissural nucleus (SDCN) neurons.

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This technique evaluates pure single or multi-synapse responses from native functional nerve endings and enables us to quantify how Xe influences pre- and postsynaptic transmissions accurately. Effects of Xe on glutamate (Glu)-, alphaamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, kainate (KA)- and N-methyl-D-aspartate (NMDA)- and GABAA receptor-mediated whole-cell currents were investigated by the conventional whole-cell patch configuration. Excitatory and 2

inhibitory postsynaptic currents (EPSCs and IPSCs) were measured as spontaneous (s) and evoked (e) EPSCs and IPSCs. Evoked synaptic currents were elicited by paired-pulse focal electric stimulation. Xe decreased Glu, AMPA, KA, and NMDA receptor-mediated whole-cell currents but did not change GABAA receptor-mediated whole-cell currents. Xe decreased the frequency and amplitude but did not affect the 1/e decay time of the glutamatergic sEPSCs. Xe decreased the frequency without affecting the amplitude and 1/e decay time of GABAergic sIPSCs. Xe decreased the

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amplitude and increased the failure rate (Rf) and paired-pulse ratio (PPR) without altering the 1/e decay time of both eEPSC and eIPSC, suggesting that Xe acts on the presynaptic side of the synapse. The presynaptic inhibition was greater in eEPSCs

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than in eIPSCs. We conclude that Xe decreases glutamatergic and GABAergic spontaneous and evoked transmissions at the presynaptic level. The glutamatergic

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presynaptic responses are the main target of anesthesia-induced neuronal responses.

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In contrast, GABAergic responses minimally contribute to Xe anesthesia.

1. Introduction

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Keywords; spinal neurons, synaptic transmission, xenon, glutamate, GABA

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Xenon (Xe) has excellent anesthetic and analgesic properties, and if delivered with a low-flow system, it has the potential to become one of the main anesthetics.

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Xe has been thought to induce anesthesia via interaction with ligand-gated ion channels, including GABAA and glutamate receptors having NMDA, AMPA, and KA subtypes, which is similar to other general anesthetics. Almost all anesthetics potentiate predominantly the postsynaptic inhibitory responses (Campagna et al. 2003). However, the main sites of Xe’s anesthetic action are the postsynaptic excitatory glutamatergic NMDA receptor (Armstrong et al. 2012; de Sousa et al. 3

2000; Georgiev et al. 2010b; Haseneder et al. 2008; Haseneder et al. 2009a; Haseneder et al. 2009b; Ogata et al. 2006; Weigt et al. 2009; Yamakura and Harris 2000; Yamamoto et al. 2012; Robel et al., 2018) or AMPA/KA receptors (Dinse et al. 2005; Georgiev et al. 2010b; Haseneder et al. 2008; Haseneder et al. 2009b; Plested et al. 2004; Weigt et al. 2009). In contrast, Xe potentiated the postsynaptic inhibitory GABAA receptors (Hapfelmeier et al. 2000; Yamakura and Harris 2000) or did not affect the postsynaptic inhibitory GABAA receptors (de Sousa et al. 2000; Georgiev

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et al. 2010b; Haseneder et al. 2008; Yamamoto et al. 2012). However, the majority of these findings were obtained in combination with in vitro electrophysiological studies using recombinant expression cells or slice preparations. The synaptic

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responses are elicited from many synaptic elements, such as various pre- and postsynaptic components, and influenced by synaptic inputs and/or extra-synaptic

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chemical modulation by surrounding neurons and glia. Therefore, the exact effects

level remain ambiguous.

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of Xe on both excitatory and inhibitory receptor-mediated responses at the synaptic

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Recently, we have been working with the “synapse bouton preparation” of neurons isolated mechanically rather than enzymatically (Akaike and Moorhouse

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2003; Akaike et al. 2002). This preparation preserves the adherent and functional fast glutamatergic, GABAergic, and glycinergic synaptic terminals and completely

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eliminates the confounding effects from surrounding neurons, glia, and autonomic nerve endings (valicosity) (Adachi et al., 2001 a, b; Végh et al., 2017). Additionally, by measuring eEPSCs or eIPSCs under paired-pulse focal electric stimulation of a single glutamatergic, GABAergic, and glycinergic presynaptic nerve endings releasing fast transmitters, such as glutamate, GABA, and glycine, we can precisely evaluate how various anesthetics act on these pre- and postsynaptic transmissions at 4

a single-synapse level (Kotani et al. 2012; Nonaka et al. 2019; Ogawa et al. 2011; Shin et al. 2013; Wakita et al. 2016; Wakita et al. 2013; Wakita et al. 2015a). We recently reported that the glutamatergic presynaptic nerve endings are the main target of Xe under anesthesia in a study using rat hippocampal CA3 neurons. In contrast, GABAergic responses minimally contributed to the anesthesia-induced neuronal responses (Nonaka et al. 2019). However, minimal alveolar concentration (MAC) to evaluate potency of inhalational anesthetics mainly depends on neuronal

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activities in the spinal cord rather than in the brain (Antognini and Schwartz 1993; Rampil et al. 1993). Few investigators (Georgiev et al. 2010b; Yamamoto et al. 2012) have studied the effects of Xe in the spinal cord, and whether Xe induces anesthesia

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through actions in the spinal cord remains unclear. We used SDCN neurons because the population of dorsal commissural neurons is the most basic circuitry for pain

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processing in the dorsal spinal cord. (Comer et al. 2015).

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To understand how Xe affects glutamatergic and GABAergic transmissions of the SDCN neurons in the spinal cord, we studied actions of Xe by measuring the

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frequency, amplitude, and 1/e decay time of sEPSCs and sIPSCs occurring at multi boutons, as well as the amplitude, PPR, Rf, and 1/e decay time of eEPSCs and eIPSCs

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elicited from a single bouton adhered to the “synapse bouton preparation” of rat

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spinal SDCN neurons.

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2. Material and methods 2.1. Ethical approval All experiments were performed in accordance with The Guiding Principles for The Care and Use of Animals in The Field of Physiological Science of The Physiological Society of Japan and were approved by the ethics committee at Kumamoto Kinoh Hospital. All efforts were made to minimize animal suffering and

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to reduce the number of animals used in this study.

2.2. Animals

Mother rats (Wistar/ST) with their newborns were purchased from Japan SLC,

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Inc. (Shizuoka, Japan). The rats were housed on a 12 h light/dark cycle with ad libitum access to water and food. The neonatal rats (11-23 days old, mixed sex) were

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sustained only by breastfeeding from the mother rat until they were used for the

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experiment. The immature rats were decapitated after anesthesia with pentobarbital (50 mg/kg, intraperitoneally). The spinal cord was quickly removed and immersed

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in an ice-cold medium. The spinal cord containing the L5-S2 regions was isolated and cut into 380 µm slices with a microslicer (VR1200S, Leica, Nussloch, Germany)

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in the ice-cold incubation solution. The spinal slices were kept in the incubation solution saturated with 95% O2 and 5% CO2 at room temperature (21-24 ºC) for at

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least 1 h before mechanical dissociation.

2.3. Preparation of single synapse boutons Details of the “synapse bouton preparation” have been described previously.(Akaike and Moorhouse 2003; Murakami et al. 2002). In brief, the tip of the glass pipette coupled to a vibration device (S1-10 Cell Isolator, K.T. Labs, Tokyo, 6

Japan) was placed on the surface of the slice containing the sacral dorsal commissural nucleus (SDCN) region of the spinal cord and was horizontally vibrated at 50 Hz. After mechanical dissociation, the mechanically dissociated SDCN neurons were left to settle and adhere to the bottom of the culture dish for at least 15-30 min.

2.4. Electrophysiological measurements Patch pipettes were made from borosilicate capillary glass (Nonaka et al. 2019).

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The resistance of the recording pipettes was 5-8 MΩ when filled with an internal solution. Isolated SDCN neurons were observed on an inverted phase-contrast microscope (Diaphot, Nikon Inc.). Neuronal responses were continuously monitored

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on a computer display and on an oscilloscope (DCS-7040, Kenwood). All membrane currents were filtered at 2 kHz through a low-pass filter (E-3201A Decade Filter: NF

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Electric Instruments, Tokyo, Japan) and stored in a computer using the pCLAMP

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10.2 software (Axon Instruments). Hyperpolarizing step pulses (5 mV, 30 ms duration) were used to monitor access resistance, and if the access resistance of

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neurons changed by more than 20%, the recordings were rejected. Glutamate-, NMDA-, AMPA-, KA-, GABA-induced whole-cell currents (IGlu,

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INMDA, IAMPA, IKA, IGABA) were recorded in the “synapse bouton preparation” of mechanically dissociated SDCN neurons at a holding potentials (VHs) of -65 mV for

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IGlu, IAMPA, and IKA; -40 mV for INMDA; and 0 mV for IGABA. No antagonists of glutamate and GABAA receptors were used in the present experiment (Wakita et al. 2015a). External test solutions were applied using the Y-tube method (Murase et al. 1990). All experiments were performed at room temperature (21-24 ˚C). In the present spinal SDCN “synapse bouton preparation,” fast neurotransmitters, such as glutamate, GABA, and glycine were released spontaneously from the 7

adherent multi nerve terminals. In the present study, the spontaneous glutamatergic and GABAergic postsynaptic currents (sEPSCs and sIPSCs) were recorded from the cell body of SDCN neurons using an amplifier (Multiclamp 700B, Molecular Devices, Sunnyvale, CA) at a VH of -65 mV for sEPSCs and 0 mV for sIPSCs. This isolated “synapse bouton preparation” is not interfered completely with many other neurons, glia, microglia and other possible autonomic neurotransmitters (Adachi et al., 2001 a, b; Végh et al., 2017; Ohshima et al., 2017). The present preparation is

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quite different from the slice preparation, in which neuronal network is well conserved. Therefore, as far as we use “synapse bouton preparation”, we can observe how Xe directly acts at the single (evoked response) and multi synapses (spontaneous

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or miniature responses) levels.

In the “synapse bouton preparation,” a single presynaptic excitatory and

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inhibitory nerve terminal (bouton) was stimulated with paired-pulse focal electric

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shocks to elicit eEPSCs and eIPSCs, respectively (Akaike and Moorhouse 2003; Akaike et al. 2002). When a single bouton attached to isolated neuron was focally

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stimulated by a double-barrel theta (θ) glass tube, the stimulus-response relationships of glutamatergic eEPSCs and/or GABAergic eIPSCs appeared in “all or none”

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fashion like relationship between the stimulus current intensity. The critical location of the stimulation pipette tip upon a bouton was within 0.4 µm in the horizontal shift

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along on neuronal surface membrane. Typical schematic figure of “synapse bouton preparation” and the focal electrical stimulation are shown in our recent study (Shin et al. 2018). Stimulus shocks of 100 µs (duration) and 0.05-0.1 mA (intensity) were delivered every 20 ms (inter-event internal) for glutamatergic eEPSCs, and stimulus shocks of 100 µs (duration) and 0.05-0.1 mA (intensity) were delivered every 40 ms for GABAergic eIPSCs, as described previously (Murakami et al. 2002) 8

2.5. Application of Xe The gas mixture was purchased from a gas supplier (Kumamoto Sanso Co., Kumamoto, Japan). The 70% Xe gas mixture consists of 70% Xe, 28.5% O2, and 1.5% CO2. The Xe gas mixture was continuously bubbled into the external test solution in a 10-ml test tube for at least 3-5 min, and the pH of the Xe gas mixture

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solution was approximately the same as the external control solution (pH = 7.4).

2.6. Solutions

The incubation medium was comprised of the following (in mM): 124 NaCl,

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5 KCl, 1.2 KH2PO4, 2.4 CaCl2, 24 NaHCO3, 1.3 MgSO4, and 10 glucose. The medium was saturated with 95% O2 and 5% CO2. The standard external solution

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contained the following (in mM): 150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose,

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and 10 HEPES. The internal solution was comprised of the following (in mM): 5 CsCl, 135 CsF, 5 TEA-Cl, 2 EGTA, and 10 HEPES. The internal solution was used

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for the recording of IGlu and glutamatergic sEPSCs and eEPSCs. When IGABA and GABAergic sIPSCs or eIPSCs were recorded, the internal pipette solution was

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comprised of the following (in mM): 5 CsCl, 135 Cs-methanesulfonate, 5 TEA-Cl, 10 EGTA, 10 HEPES, and 4 mM ATP-Mg. External test solutions contained the

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following (in mM): 60 NaCl, 100 choline-Cl, 10 CsCl, 10 glucose, 0.01 LaCl3, 5 TEA-Cl, and 10 HEPES for recording INa, and 145 choline-Cl, 5 CsCl, 5 BaCl2, 1 MgCl2, 10 glucose and 10 HEPES for recording IBa. The internal (pipette) solutions for the voltage-dependent currents were comprised of the following (in mM): 105 CsF, 30 NaF, 5 CsCl, 5 TEA-Cl, 2 EGTA, 10 HEPES, and 2 ATP-Mg for INa, and 80 Cs-methanesulfonate, 60 CsCl, 5 TEA-Cl, 2 EGTA, 10 HEPES, and 2 ATP-Mg for 9

IBa. All external and internal (pipette) solutions were adjusted to a pH of 7.4 and 7.2 with Tris-base, respectively.

2.7. Drugs The reagents for external and internal solutions were obtained from Wako Pure Chemicals (Osaka, Japan). Other reagents, such as TEA-Cl, EGTA, L-glutamic acid (glutamate), GABA, NMDA, KA, AMPA, and ATP-Mg, were obtained from Sigma

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(St. Louis, MO, USA).

2.8. Statistical analysis

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Glutamate-, AMPA-, KA-, and NMDA-induced excitatory whole-cell currents (IGlu, IAMPA, IKA, and INMDA) and GABA-induced inhibitory whole-cell current (IGABA)

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were recorded from the postsynaptic SDCN soma membrane. Glutamate, AMPA,

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KA, NMDA, and GABA were exogenously applied using the Y-tube technique. We calculated (IGlu + Xe) / IGlu to evaluate whether the IGlu responses recorded

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at various VHs were voltage-dependent.

The Lineweaver-Burk plot was applied to detect inhibition mode (competitive

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or non-competitive), as in our recent study (Nonaka et al. 2019). Non-competitive inhibition gives the same X-intercept in the two Lineweaver-Burk plots obtained

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from concentration-response curves with and without Xe. On the other hand, two Lineweaver-Burk plots in the case of competitive inhibition show the same Yintercept. Therefore, we judged how Xe inhibits the glutamate, AMPA, and NMDA responses, as reported in previous studies (Franks et al. 1998; Dinse et al. 2005; Dickinson et al. 2007).

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Glutamatergic sEPSCs and GABAergic sIPSCs were counted and analyzed in pre-set epochs before, during, and after each test condition using the MiniAnalysis Program (Synaptosoft, NJ, USA), as described by Wakita et al. (2015b). The amplitude, frequency, and 1/e decay time of both sEPSCs and sIPSCs were recorded and compared during the control period (2-4 min) and during the period in the presence of Xe. The amplitudes, failure rate (Rf), and paired-pulse ratios (PPR = P2/P1) of both

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eEPSCs and eIPSCs were calculated using the pCLAMP 10.2 software (Kotani et al. 2012). When either P1 or P2 currents failed to appear at the expected latency, the PPR value was not included in the mean PPR data. For the time course plots of PPR in

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which P1 and/or P2 failed, the PPR value was plotted as 0 to indicate a failure in the plots. The effects of 70% Xe on the amplitude, Rf, and PPR of both eEPSCs and

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eIPSCs were normalized as relative changes from their respective controls (Nonaka

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et al. 2019). Data are reported as means ± SEM of these normalized values. Differences in the current amplitude, frequency, Rf, PPR, and 1/e decay time of

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synaptic events were tested by analysis of variance (ANOVA) and post hoc Dunnet’s

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test. A value of p < 0.05 was considered to be statistically significant.

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3. Results 3.1. Effects of 70% Xe on glutamate receptor-mediated whole-cell current (IGlu) Figure 1A shows concentration-response curves of IGlu elicited by glutamate at a wide concentration range between 3 x 10-6 - 3 x 10-4 M in the presence or absence of Xe. Xe inhibited the IGlu of various concentrations but minimally changed the threshold concentrations in two concentration-response curves with and without Xe. The EC50 values with and without Xe were 4.1 x 10-5 M for Xe and 3.4 x 10-5 M for

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control, respectively, where the two values were close to one another. Therefore, a Lineweaver-Burk diagram was generated as shown in Figure 1B, in which R and C represent the relative amplitude of IGlu and the concentration of glutamate (µM),

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respectively. The results suggest that Xe inhibits the IGlu in a non-competitive manner, as the plots for the two parameters intersect on the x-axis. To assess if the inhibition

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is voltage-dependent, the effect of Xe on 3 x 10-5 M IGlu was examined at various

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voltages between -75 and +20 mV (Fig. 1C). As shown in Figure 1D, the inhibition ratios were almost constant at all VHs, and the average inhibition ratio [(IGlu + Xe) / IGlu]

Fig. 1 is here

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induced by Xe at 6 different VHs was 0.72 ± 0.014.

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Xe also inhibited the three ionotropic glutamatergic subtypes (AMPA, KA, and NMDA) receptor-mediated currents (IAMPA, IKA, and INMDA), as well as IGlu (Fig. 2A-

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C). However, Xe did not change their respective threshold concentrations. One should note that INMDA was recorded at a VH of -40 mV, and at this VH, Mg2+ ions do not block NMDA receptor-operated cation channels. In the Lineweaver-Burk diagrams, the plots of IAMPA and INMDA with and without Xe intersected on the x-axis, respectively (Fig. 2A, C). The results suggest Xe non-competitively inhibits the AMPA and NMDA receptors. However, we could not adopt a Lineweaver-Burk 12

diagram for KA concentration-response curves with and without Xe because we needed experimental data points at higher concentrations of KA. However, two threshold concentrations in the concentration-response curves for IKA with and without Xe were very close (Fig. 2B), suggesting that Xe also possibly inhibits KA receptors in a non-competitive manner, as seen in the hippocampal CA3 neurons (Nonaka et al. 2019).

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Fig. 2 is here

3.2. Effects of 70% Xe on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs)

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Figure 3A (a) shows sEPSCs in the presence or absence of Xe. Figure 3A (b) and (c) are current recordings of control and Xe with expanded time scales,

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respectively. Xe significantly decreased the frequency and amplitude but did not

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affect the 1/e decay time, of sEPSCs measured at a VH of -65 mV (Figs. 3A, B). Figure 3B(a-c) represents the cumulative probabilities of the frequency, amplitude,

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and 1/e decay time of the sEPSCs with and without the application of Xe. Figure 3B(d) shows normalized, superimposed, and expanded (along the time scale)

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sEPSCs with and without Xe. There was no kinetic change between the two currents (cont, 2.01 ± 0.57 ms; Xe, 1.93 ± 0.54 ms). Inset histograms of Figure 3Ba-c show

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the relative frequency (a), amplitude (b), and the 1/e decay time (ms) (c) of the sEPSCs with and without Xe. Data were obtained from 7 neurons. Both the frequency and amplitude of sEPSCs significantly decreased after adding Xe; however, the 1/e decay time was not affected. Fig. 3 is here

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3.3. Effects of 70% Xe on glutamatergic excitatory postsynaptic currents (eEPSCs) evoked by action potentials Figure 4A(a) shows typical inwardly-directed glutamatergic eEPSCs in response to paired-pulse focal electric stimulation before (1), during (2), and after (3) application of Xe. Xe markedly decreased the amplitude while increasing both the Rf and PPR of eEPSCs. The Xe effects were completely removed after washing out (wo) Xe with the control solution. Figure 4A(b) shows the normalized, superimposed,

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and expanded P1 amplitudes of eEPSCs with and without Xe. As seen in this figure, the 1/e decay time (ms) of the eEPSCs was not changed by adding Xe. Figure 4B represents a typical time course of P1 (○) and P2 (●) amplitudes of the eEPSCs with

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and without Xe. Figure 4C summarizes the relative P1 amplitude, Rf, and PPR of eEPSCs in the presence of Xe. Xe significantly decreased the P1 amplitude and

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increased the Rf and PPR in eEPSCs. Figure 4D shows the 1/e decay times (ms) with

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and without Xe that were not affected by Xe (control, 1.92 ± 0.12 ms; Xe, 1.96 ± 0.07 ms). All data were obtained from 6 neurons.

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Fig. 4 is here

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3.4. Effects of 70% Xe on GABAA receptor-mediated whole-cell current (IGABA) When the internal patch pipette solution contains ATP-Mg, the time-

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dependent decrease (run-down) in IGABA is slowed down (Shirasaki et al. 1991). Therefore, an internal solution containing ATP-Mg was used in this study. The effects of Xe on IGABA were tested at a VH of 0 mV, which is the equilibrium potential (Er) of glutamatergic currents. As shown in Figure 5A and B, the run-down of the IGABA was observed during the whole-cell current recordings. The IGABA induced by 3 x 106

or 10-5 M GABA was not affected by the application of Xe. The results were 14

summarized in Figure 5C(a,b), indicating that Xe has no effects on IGABA. All data were obtained from 4 neurons. Fig. 5 is here

3.5. Effects of 70% Xe on GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) A representative time course of GABAergic sIPSCs in the presence and

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absence of Xe is shown in Figure 6A(a), and the current traces at an expanded time scale are shown in Figure 6A(b,c). The cumulative probabilities of the inter-event interval (frequency), amplitude, and 1/e decay time of sIPSCs are shown in Figure

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6B(a-c). The currents shown in Figure 6B(d) are normalized, superimposed, and expanded sIPSCs with and without Xe. The effects of Xe on the relative frequency,

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amplitude, and the 1/e decay time (ms) are summarized in the inset histograms of

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Figure 6B(a-c). Data were obtained from 5 neurons. Xe significantly decreased the frequency but did not affect the amplitude and decay time, of GABAergic sIPSCs,

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suggesting that Xe initiates the inhibitory effects only through the GABAergic presynaptic side (nerve ending).

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Fig. 6 is here

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3.6. Effects of 70% Xe on GABAergic inhibitory postsynaptic currents evoked by action potentials (eIPSCs) Figure 7A(a) shows typical outwardly-directed GABAergic eIPSCs in

response to paired-pulse focal electric stimulation before (1), during (2), and after (3) the addition of Xe. Xe decreased the P1 amplitude of the eIPSCs; however, the inhibition was completely recovered by washing out Xe with the control solution 15

(wo). The currents of Figure 7A1-3 were obtained from Figure 7B1-3. Figure 7A(b) shows normalized, superimposed, and expanded eIPSCs with (gray line) and without (black line) Xe. There was no change in the current kinetics between the two eIPSCs. Representative time courses of the P1 (○) and P2 (●) amplitudes of eIPSCs with and without Xe are shown in Figure 7B. The relative values of the P1 amplitude, Rf, and PPR of eIPSCs in the presence of Xe are summarized in Figure 7C. Xe significantly decreased P1 amplitude and increased Rf and PPR during the

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application of Xe. However, the 1/e decay time (ms) of P1 eIPSCs was not affected by Xe (control, 14.40 ± 0.34 ms; Xe, 16.11 ± 0.71 ms; Fig. 7D). Data were obtained from 6 neurons.

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Fig. 7 is here

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3.7. Effects of 70% Xe on voltage-dependent sodium and calcium channel

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currents

Xe did not affect the voltage-dependent sodium channel current (INa) elicited

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by a depolarizing step pulse from a VH of -70 mV to -20 mV. The peak amplitude and the current activation and inactivation phases of INa were not changed by adding Xe

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(Fig. 8Aa, b). Similarly, Xe did not affect the Ba2+ current (IBa) passing through voltage-dependent Ca2+ channels evoked by a depolarizing step pulse from a VH of -

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60 mV to 0 mV (Fig. 8Ba, b). Data were obtained from 5 and 7 neurons, respectively. Fig. 8 is here

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4. Discussion

4.1. Effects of Xe on whole-cell and synaptic glutamate responses Xe significantly inhibited the concentration-response curve for IGlu (Fig.1). The current-voltage relationship for IGlu at VHs between -75 and +20 mV indicated that Xe-induced inhibition was voltage independent, as the inhibitory ratios calculated from (IGlu + Xe) / IGlu were constant. We also found that Xe did not shift

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the respective threshold concentrations in the concentration-response curves of glutamate and its three ionotropic subtype responses (IGlu, INMDA, IAMPA, and IKA). Furthermore, the X-intercepts of the Lineweaver-Burk plots in these glutamate

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receptors were the same in the respective concentration-response curves with and without Xe. These results clearly indicated that Xe inhibits these receptors in a non-

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competitive manner. Our findings in spinal neurons were comparable with previous

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studies evaluating brain neurons (Dickinson et al. 2007; Dinse et al. 2005; Franks et al. 1998; Nonaka et al., 2019).

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We found that Xe considerably inhibited the INMDA. This result was supported by results of most previously conducted studies, which have shown the decrease of

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postsynaptic responses in the spinal dorsal horn neurons (Georgiev et al. 2010b), hippocampal neurons (de Sousa et al. 2000), and basolateral amygdala neurons

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(Ranft et al. 2007), as well as in other preparations with recombinant NMDA receptors (Armstrong et al. 2012; Ogata et al. 2006; Yamakura and Harris 2000). In addition, in vivo study, Xe improved the long-term cognitive function and the survival after traumatic brain injury, and reduced the neuronal loss (Campos-Pires et al., 2019; Robel et al., 2018). However, Yamamoto et al. (2012) found that Xe at a clinical concentration had no effect on INMDA in spinal neurons isolated from neonatal 17

rats. The discrepancy among these aforementioned results might be accounted for due to differences in subunit compositions of NMDA receptors between neonatal and adult rats, and the difference may result in different sensitivities of the receptors. Furthermore, the present findings that Xe also inhibited the IAMPA and IKA in this study were comparable with those in previous studies (Dinse et al. 2005; Georgiev et al. 2010b; Yamakura and Harris 2000; Yamamoto et al. 2012). Glutamate first activates AMPA/KA receptors. Depolarization leads to the removal of Mg2+ ions

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that block the NMDA receptor-channel complex and further facilitates the activation of AMPA/KA receptors. Thus, a potential block of AMPA/KA receptors by Xe may synergistically contribute to the inhibition of the NMDA-receptor-mediated signaling

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(Georgiev et al. 2008).

In the present study, Xe decreased the amplitude and increased Rf and PPR

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without altering the decay time of glutamatergic eEPSCs. The results strongly

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suggest that Xe directly inhibits glutamatergic eEPSCs at the synaptic level. Similarly, Hasendeder et al. (2009b) showed that Xe decreased the amplitude but did not alter

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the current kinetics of glutamatergic eEPSCs in both prefrontal cortex and substantia gelatinosa neurons, although the effects of Xe on the PPR and Rf of eEPSCs were

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not analyzed. The decreased amplitude of eEPSCs after the application of Xe was also observed in other studies (Georgiev et al. 2010b; Haseneder et al. 2008;

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Yamamoto et al. 2012). We thus conclude that Xe directly inhibits not only postsynaptic IGlu but also synaptic sEPSCs and eEPSCs. Previous studies have demonstrated that Xe inhibited the IGlu of the spinal

lamina IX (Yamamoto et al. 2012), dorsal horn lamina II (Georgiev et al. 2010b), prefrontal cortex (Haseneder et al. 2009b), substantia gelatinosa (Haseneder et al. 2009b), and basolateral amygdala neurons (Haseneder et al. 2008). Other studies 18

(Georgiev et al. 2010a; Haseneder et al. 2008; Haseneder et al. 2009b; Yamamoto et al. 2012) also revealed that Xe markedly decreased the amplitude of mEPSCs without altering their frequency. Hence, they all concluded that Xe acts predominantly via postsynaptic mechanisms. However, these studies could not quantify the presynaptic contribution of Xe to the glutamatergic transmissions. In contrast, our “synapse bouton preparation” offers the unique and independent evaluation of the effects of anesthetics on both pre- and postsynaptic transmissions

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at single- and multi-synapse levels. At the multi-synaptic level in the present study, Xe significantly decreased both the frequency and amplitude without altering the 1/e decay time of sEPSCs, and at a single synaptic level, Xe considerably decreased the

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amplitude and increased the Rf and PPR without altering the current kinetics of eEPSCs. All of these results clearly suggest that Xe acts directly via a presynaptic

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mechanism at single- and multi-synaptic levels, even though Xe decreased the

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amplitude of the postsynaptic IGlu, INMDA, IAMPA, and IKA. Also, Xe slightly and markedly decreased the amplitude of sEPSC and eEPSC, respectively. Therefore,

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functional effects of Xe on postsynaptic sites cannot be ruled out. In the spinal SDCN neurons, we found a decrease in amplitude of sEPSCs in

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spite of no changes in hippocampal CA3 neurons in our recent study (Nonaka et al. 2019). The decay time of SDCN neurons is 2.7 times shorter than that of the CA3

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neurons. Many studies showed that a different subunit constitution seriously modulated many neuronal current responses, including kinetics, amplitude, and sensitivity in glutamate (Maki et al. 2013; Ren et al. 2017) and GABA (Gingrich et al. 1995; Mercik et al. 2006) receptors. Thus, our results suggest different subunit constitutions between the brain and spinal cord.

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4.2. Effects of Xe on whole-cell and synaptic GABA responses In the present study, Xe did not modulate the IGABA recorded from the neuronal cell body. Previous studies reported controversial findings on the effects of Xe on IGABA. Xe potentiated (Hapfelmeier et al. 2000; Yamakura and Harris 2000) or did not affect IGABA (de Sousa et al. 2000; Georgiev et al. 2010b; Haseneder et al. 2008; Yamamoto et al. 2012). Such discrepancy in the Xe’s effects may be due to the different subunit compositions of GABAA receptors. The pentameric GABAA

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receptors consist of 16 subunits, and more than 20 different types of subunit combinations have been reported (Olsen and Sieghart 2008; Vizi et al. 2010). A few studies examined the relationship between the subunit combination and anesthetic

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sensitivity. The different subunit compositions could explain the different sensitivities of GABAA receptors to Xe in various preparations. For example, one

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study showed that the δ subunit-containing receptors were 10 times more sensitive

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to GABAA agonists than the γ2 subunit-containing receptors (Mortensen et al. 2010). In our present results and those from the majority of previously conducted slice

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studies, the subunit compositions of GABAA receptors were not determined. Therefore, the present data support the hypothesis that the GABA-induced responses

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do not account for Xe-induced anesthesia. At a clinically relevant concentration, we found that Xe significantly

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decreased the frequency of GABAergic sIPSCs and did not change the current amplitude or decay time. Xe also strongly decreased the amplitude, increased the Rf and PPR, and did not change the decay time of GABAergic eIPSCs. These findings suggest that the synaptic GABAergic responses for Xe are different from the wholecell responses (IGABA) and that Xe completely acts only on the presynaptic GABAergic nerve ending. 20

The decrease in the amplitude of GABAergic eIPSCs without alteration of the decay time in our study was comparable with a study evaluated on dorsal horn lamina II neurons (Georgiev et al. 2010b). In contrast, our findings differed from the results of two studies, which used slice preparations from the basolateral amygdala (Haseneder et al. 2008) and from the prefrontal cortex and substantia gelatinosa (Haseneder et al. 2009a). These studies demonstrated that Xe did not change the amplitude and decay time of GABAergic eIPSCs. These inter-study differences are

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likely reflections of the different preparations. Using the “synapse bouton preparation,” we could determine the actions of Xe at the pure synapse levels, whereas other studies seem to potentially include the whole-cell actions, including

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various influences of surrounding neurons and glia. Therefore, it is evident that Xe

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4.3. Effects of Xe on INa and IBa

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has negligible postsynaptic effects on GABAergic transmission.

The voltage-dependent Na+ and Ca2+ channels trigger the action potential-

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dependent neurotransmitter release. Xe did not alter the INa evoked in an external solution containing 60 mM of Na+. Furthermore, Xe had no effects on IBa. In the rat

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hippocampal neuron, the Ca2+ channel currents participating in glutamatergic eEPSCs mainly depended on P/Q- and N-type types and somewhat on L- and R-

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types (Shin et al. 2018), while the L-, P/Q-, and N-type Ca2+ channels have functional roles in the GABAergic nerve terminal (Murakami et al. 2002). Therefore, these results suggest that Xe might not act on L-, N-, P/Q-, and R-type Ca2+ channel subtypes in the glutamatergic excitatory and GABAergic inhibitory nerve endings of SDCN neurons. The findings also suggest that Xe acts directly in the excitatory and inhibitory presynaptic intra-axonal transmitter release mechanisms. 21

4.4. Comparison to the brain neuron and its clinical implication We found that Xe markedly decreased glutamatergic eEPSCs with a slight decrease in GABAergic eIPSCs. Nearly similar results were obtained compared with results evaluated on the “synapse bouton preparation” of hippocampal CA3 neurons (Nonaka et al. 2019). One difference is that Xe decreased both the frequency and amplitude of sEPSCs in this study. However, we consider that the more pronounced inhibitory effects of glutamate in spinal SDCN neurons are very important to

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elucidate the analgesic property of Xe. Signals from mechanical, thermal, and mechano-thermal nociceptors are transmitted to the dorsal horn of the spinal cord predominantly by Aδ fibers. The Aδ

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fibers terminate in the spinal cord, where they mainly release glutamate and its related agonists (NMDA, KA, AMPA). The inhibition of glutamate responses,

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therefore, decreases nociceptive reception (Zhuo 2017). The SDCN neurons receive

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the glutamatergic inputs (Xu et al. 1996; Xu et al. 1999). The SDCN neurons, the afferent second sensory neuron localized in the dorsal part of spinal central canal, are

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involved in the nociceptive, analgesic, and autonomic actions in the visceral and somatic pathways (Comer et al. 2015; Xu et al. 1999; Xu et al. 1996).

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Xe combines profound analgesic properties even in subanesthetic concentrations (De Hert et al. 2009; Preckel and Schlack 2005). We also found that

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suppression of glutamatergic responses by Xe in the hippocampal CA3 neurons was much greater than that of another gaseous anesthetic, nitrous oxide (Nonaka et al. 2019; Wakita et al. 2015a). As long as anesthetic mechanisms have not been clarified, one cannot apply present findings to elucidate the analgesic potency of Xe. However, the marked suppression of glutamatergic activities by Xe in the spinal cord may explain, in part, the powerful analgesic effects. 22

Funding: This work was supported by the Grant-in-Aids program from the Kumamoto Kinoh Hospital for N. Akaike, and the Kitamoto Hospital for N. Kotani, N. Okamitsu, and N. Akaike.

Declaration of interest: No conflicts of interest, financial or otherwise, are declared

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by the authors.

Declaration of interests

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The authors declare that they have no known competing financial interests or personal

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relationships that could have appeared to influence the work reported in this paper.

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Author statement

Hisahiko Kubota: Methodology, Formal analysis. Hironari Akaike: Validation,

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Visualization. Nobuharu Okamitsu: Formal analysis. Il-Sung Jang: Validation, Visualization, Formal analysis. Kiku Nonaka: Validation, Visualization, Formal

Writing

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analysis. Naoki Kotani: Conceptualization, Methodology, Writing - Original Draft, -

Review &

Editing.

Norio

Akaike:

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Conceptualization, Methodology, Writing - Original Draft,

23

Project

administration,

Author contributions: N. Akaike, N. Kotani, and H. Kubota conceived and designed the experiment; H. Kubota, IS. Jang, H. Akaike, N. Okamitsu, and K. Nonaka collected, analyzed, and interpreted the data. N. Kotani, and N. Akaike drafted and edited the manuscript. All authors have approved the final version of the manuscript and agree to be accountable for all aspects of the work. All persons designated as authors qualify for authorship, and all those who qualify for authorship

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are listed.

Acknowledgments: The authors thank Yushi Ito, Ph.D., Professor emeritus, Kyusyu

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University, for his kind advice. We would like to thank Editage (www.editage.com)

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ur

na

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for English language editing.

24

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Figure legends

Figure 1. Effects of 70% Xe on glutamate-induced whole-cell current (IGlu) in rat SDCN neurons A: Concentration-response curves of glutamate-induced whole-cell current (IGlu) elicited by 3 x 10-6 - 3 x 10-4 M glutamate with (●) and without (○) Xe. All current amplitudes were normalized to the peak current induced by 10-5 M glutamate alone.

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Representative sample currents induced by 3 x 10-5 and 10-4 M glutamate with and without Xe are shown in the inset. All currents were recorded at a holding potential (VH) of -65 mV that is close to the Cl- equilibrium potential (ECl). Each point is the

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mean ± SEM of values obtained from 4-7 neurons.

B: Lineweaver-Burk plots of two curves in A. Two straight lines intersect on the x-

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axis, where R is the IGlu amplitude, and C is the glutamate concentration (μM).

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C: Inhibitory effects of Xe on 3 x 10-5 M IGlu at VHs between -75 and +20 mV. Horizontal bars of each open (○) and filled (●) circle show mean ± SEM of

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values obtained from 6 neurons.

D: Relative inhibition ratio of (IGlu+Xe) / IGlu. Data were quoted from C. There was

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almost constant inhibition of IGlu by Xe throughout the wide voltage ranges used.

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No voltage-dependency was seen.

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Figure 2. Effects of 70% Xe on the whole-cell inward currents elicited by three subtypes of ionotropic glutamate receptors

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A-C: Concentration-response curves of AMPA-, KA-, and NMDA-induced currents (IAMPA, IKA, and INMDA) with (●) and without (○) Xe. Each point shows the mean

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± SEM of values obtained from 4-7 neurons for both IAMPA and IKA and from 4-5 neurons for INMDA. Xe did not change the threshold concentrations in the

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concentration-response curves of respective subtype currents with and without Xe.

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In the Lineweaver-Burk plots with and without Xe shown in A and C, the two straight lines intersect on the x-axis. VHs were -65 mV for IAMPA and IKA recordings, and -40 mV for INMDA recordings.

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Figure 3. Effects of 70% Xe on glutamatergic sEPSC

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A: Typical current traces (a) of sEPSCs with and without Xe. The current traces with

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an expanded time scale are shown in b (control, Cont) and c (Xe). VH = -65mV. B: Effects of Xe on the cumulative probabilities of the inter-event interval (frequency,

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a), amplitude (b), and 1/e decay time (c) of sEPSC. Black lines, control; dashed lines, Xe. B(d) shows normalized, superimposed, and expanded (along the time

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scale) sEPSCs with (gray line) and without (black line) Xe. Each inset histogram of B(a-c) is relative frequency (a), relative amplitude (b), and 1/e decay time (ms)

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(c) of sEPSCs with and without Xe. Each histogram in B(a-c) is the average value

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± SEM. Data were obtained from 7 neurons. **p < 0.01; ***p < 0.001; ns., not significant.

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Figure 4. Effects of 70% Xe on glutamatergic eEPSCs

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A(a): Typical glutamatergic eEPSC before (1), during (2), and after (3) application

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of Xe. The current traces 1, 2, and 3 in A(a) were obtained at time points 1, 2, and 3 in B, respectively. Arrowheads on P1 (◅ ) and P2 (◄) show the peak of the

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first and second eEPSCs elicited by paired-pulse focal electric stimuli, respectively. (b): The inset figure is normalized, superimposed, and expanded

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eEPSCs with (gray line) and without (black line) Xe. Data were obtained from the same neuron. VH = -65mV.

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B: Typical time course of peak amplitude of P1 (○) and P2 (●) in eEPSCs with and

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without Xe. The time of Xe application is shown by a filled horizontal bar. C: Relative P1 amplitude, Rf, and PPR in the presence of Xe. Data were obtained from 6 neurons.

D: 1/e decay time (ms) of eEPSCs with and without Xe. Data were obtained from 6 neurons. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

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Figure 5. Effects of 70% Xe on GABA-induced whole-cell current (IGABA)

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A: 10-5 M GABA-induced outward whole-cell currents that show gradual run-down

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in spite of the presence and absence of Xe. VH is 0 mV. A(a-c) were obtained from B(a-c), respectively.

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B: Time course in run-down of IGABA induced by alternate application of 10-5 M GABA with (●) and without (○) Xe. Each point is the average value ± SEM

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obtained from 4 neurons.

C: Histograms (a, b) show the relative IGABA induced by 3 x 10-6 and 10-5 M GABA

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with and without Xe. Data were obtained from 4 neurons.

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Figure 6. Effects of 70% Xe on GABAergic sIPSC

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A(a): Representative current traces of GABAergic sIPSCs before, during, and after

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application of Xe. Xe was applied at the horizontal filled bar. The current traces with an expanded time scale are shown in (b) (control, Cont) and c (Xe). VH = 0

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mV.

B: Cumulative probabilities for the inter-event intervals (a), amplitude (b), and 1/e

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decay time (c) of sIPSCs. Black lines, control; Dotted lines, Xe. (d): The current traces show normalized, superimposed, and expended (along the time scale)

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sIPSCs with (dotted line) and without (black line) Xe. Inset histograms of B(a-c)

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represent the relative frequency, relative amplitude, and 1/e decay time (ms), respectively. Data were obtained from 5 neurons. ***p < 0.001; ns, not significant.

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Figure 7. Effects of 70% Xe on GABAergic eIPSC

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A(a): Typical GABAergic eIPSCs before (1), during (2), and after (3) application of

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Xe. Current traces 1, 2, and 3 were obtained at time points 1, 2, and 3 in B, respectively. (b): Normalized, superimposed, and expanded (along the time scale)

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eIPSCs with (gray line) and without (black line) Xe. VH = 0 mV. B: Time course of the peak current amplitudes of P1 (○) and P2 (●) in eIPSCs before,

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during, and after adding Xe, which was applied during the period indicated by the dark horizontal bar.

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C: Relative P1 current amplitude (Amp), Rf, and PPR in the presence of Xe. Data

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were obtained from 6 neurons. D: 1/e decay time (ms) of eIPSCs with and without Xe. Data were obtained from 6 neurons. *p < 0.05; **p < 0.01; ns, not significant.

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Figure 8. Effects of 70% Xe on voltage-dependent Na+ and Ca2+ channel

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currents (INa and IBa)

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A: Effects of Xe on INa. The typical INas with (gray line) or without (black line) Xe are shown in (a). The summarized data obtained from 5 neurons are shown in (b).

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No differences were observed between the two current peaks. B: The representative IBa with (gray line) or without (black line) Xe are shown in (a).

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The summarized data obtained from 7 neurons are shown in (b).

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