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European Journal of Pain 14 (2010) 17–22

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European Journal of Pain

journal homepage: www.EuropeanJournalPain.com

Nitrous oxide and the inhibitory synaptic transmission in rat dorsal horn

Stefan K. Georgiev a, Hiroshi Baba a, Tatsuro Kohno a,b,* a Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi, Chuo ku, Niigata 951-8510, Japan b Pain Mechanism Research Group, 1-757 Asahimachi, Chuo ku, Niigata 951-8510, Japan article info abstract

Article history: The analgesic effect of (N2O) is thought to depend on noradrenaline release in the spinal Received 30 September 2008 cord following activation of descending inhibitory neurons. In addition to this indirect facilitation of inhi- Received in revised form 20 January 2009 bition in the , we previously showed direct inhibition of glutamate receptors in dorsal horn Accepted 27 January 2009 neurons by N O. Since general anesthetics could possibly affect excitatory and/or inhibitory components Available online 3 March 2009 2 of synaptic transmission, we sought to evaluate the direct effect of N2O on inhibitory transmission in spinal cord neurons. Keywords: Using whole-cell patch-clamp recording from rat transversal spinal cord slices, we investigated the Nitrous oxide actions of 50% N O and 0.5% isoflurane (both 0.3 rat MAC; minimum alveolar concentration) on exoge- Analgesia 2 GABA nously applied c-aminobutyric acid (GABA)- and -induced currents in rat dorsal horn lamina II Glycine neurons. The amplitudes and integrated areas of GABA- and glycine-induced currents were not signifi- Spinal cord slice cantly affected by N2O, but were increased in the presence of isoflurane. N2O did not affect the amplitude, Dorsal horn frequency or decay time probability distribution of either GABA or glycine -mediated miniature

Whole-cell patch-clamp postsynaptic currents. We further sought to determine the effect of N2O on focal stimulation-evoked syn- aptic currents mediated by GABA and glycine receptors, and found no effect in the majority of neurons.

These and other findings suggest that N2O has a discrete action in the spinal cord, distinct from the effects of the volatile anesthetics, consisting of inhibition of excitation in SG neurons through an action on ionotropic receptors and potentiation of inhibition through the descending noradrenergic system. Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

1. Introduction the actions of general anesthetics (Collins et al., 1995; Kendig, 2002). Substantia gelatinosa (SG, Rexed’s lamina II) neurons receive

Nitrous oxide (N2O) has been used for analgesia and anesthesia nociceptive inputs from both Ad- and C-fibers and various modula- since the middle of the 19th century and is still widely used today. tory inputs from interneurons, thus playing a central role in noci-

N2O has been reported to affect a variety of different receptors, ceptive information processing. The hypothesis that N2O directly including (Fujinaga, 2005), noradrenergic (Sawamura affects dorsal horn neurons has been tentatively yet repeatedly pro- et al., 2000), acetylcholine (Yamakura and Harris, 2000), c-amino- posed in the past (de Jong et al., 1969; Sugai et al., 1982; Miyazaki butyric acid (GABA) (Hapfelmeier et al., 2000), and N-methyl-D- et al., 1999; Antognini et al., 2001), but most research has focused aspartate (NMDA) (Mennerick et al., 1998) receptors. However, on higher structures. However, in our previous study we demon- the mechanisms underlying its unique pharmacodynamic profile strated a direct inhibitory effect of N2O on glutamatergic excitatory have not yet been fully clarified. Moreover, it is believed that the transmission in rat superficial dorsal horn neurons (Georgiev et al., different effects of N2O (analgesic, anxiolytic, amnestic, hallucino- 2008) that could contribute to the analgesic action of N2O. genic, and sedative among others) are mediated by different path- Because general anesthetics could exert their effects by inhibit- ways. Opioid receptors in the periaqueductal gray, noradrenergic ing excitatory systems and/or potentiating inhibitory systems, in descending inhibitory pathways and inhibitory interneurons in this study we investigated the effects of N2O on inhibitory synaptic the spinal cord (Maze and Fujinaga, 2001) are thought to be in- transmission in rat SG neurons using transverse spinal cord slice volved in the analgesic action of N2O. preparation, which offers the advantage of a preserved local neuro- During the last decade the spinal cord dorsal horn has emerged nal network but remains free of descending influences. as an important site for modulation of nociceptive processing and

2. Methods * Corresponding author. Address: Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi, Chuo ku, Niigata 951-8510, Japan. Tel.: +81 25 227 2328; fax: +81 25 227 0790. After approval by the Animal Care and Use Committee at E-mail address: [email protected] (T. Kohno). Niigata University Graduate School of Medical and Dental Sciences,

1090-3801/$36.00 Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2009.01.008 18 S.K. Georgiev et al. / European Journal of Pain 14 (2010) 17–22

Wistar rats (200–300 g) were anesthetized with urethane (1.5 g/kg, (Synaptosoft). Event recognition was performed semi-automati- i.p.). A dorsal laminectomy was performed and a lumbosacral seg- cally, with the detection threshold set to three times the back- ment of the spinal cord with ventral and dorsal roots attached was ground noise value and the period of time was adjusted to allow removed (Wang et al., 2005; Kohno et al., 2008). The rats were then recruitment of a sufficient number of events. The detected events immediately killed by exsanguination and the spinal cord was were further confirmed visually. placed in pre-oxygenated ice-cold Krebs solution. After the arach- Data are presented as means ± SEM. Because most of the data noid membrane and all ventral and dorsal roots were removed, the met the criteria for parametric tests, they were analyzed by the spinal cord was placed on an agar block and mounted on a metal Student’s paired t-test, unless otherwise stated. The effect of N2O stage. A 500-lm-thick transverse slice was cut on a DTK-1500 on the cumulative distribution of mIPSC amplitude, inter-event vibrating microslicer (Dosaka, Japan) and placed on a nylon mesh interval and decay time was analyzed using the Kolmogorov–Smir- in the recording chamber. The slice was perfused with Krebs solu- nov test. P < 0.05 was considered significant. tion (10–15 ml/min) equilibrated with 95% O2,5%CO2 gas mixture at 36 °C. The composition of the Krebs solution was as follows (in 3. Results mM): NaCl 117, KCl 3.6, CaCl2 2.5, MgCl2 1.2, NaH2PO4 1.2, NaHCO3 25 and D-glucose 11. Blind whole-cell patch-clamp recordings were The amplitude and frequency of spontaneous IPSCs recorded made in voltage clamp mode at a holding potential of 0 mV from from SG neurons at 0 mV remained unchanged during administra-

SG neurons with patch-pipettes pulled from thin-walled glass cap- tion of 50% N2O (control, 18.8 ± 6.2 pA vs. N2O, 19.3 ± 7.3 pA; con- illaries (1.5 mm o.d., World Precision Instruments). The patch-pip- trol, 2.8 ± 1.1 Hz vs. N2O, 2.7 ± 1.2 Hz, n = 10, P = 0.24 and 0.63, ette solution contained (in mM): Cs-sulfate 110, CaCl2 0.5, MgCl2 2, respectively). Furthermore, the resting membrane potential was

EGTA 5, HEPES 5, tetraethylammonium (TEA) 5, ATP-Mg salt 5, and also unaffected by N2O (control, À64.7 ± 1.4 mV vs. N2O, had a resistance of 5–8 MX. Lamina II was visually identified as a À65.1 ± 2.1 mV, n = 10, P = 0.52). translucent band and the tip of the recording electrode was direc- ted toward its center. Neurobiotin staining had previously con- 3.1. Effects of N2O on exogenous -elicited currents firmed the locations of the recorded cell bodies to be within the SG (Baba et al., 1999). The reversal potential of excitatory postsyn- Application of a low concentration of GABA (0.1 mM) in the per- aptic currents is around 0 mV (Kohno et al., 2000, 2005); therefore, fusion solution elicited an outward current with an amplitude of only inhibitory postsynaptic currents (IPSCs) were recorded at 41 ± 8 pA (n = 7), which was not affected by application of N2O 0 mV. Synaptic responses were evoked by focal stimulation of SG (101 ± 4% of control, n =7,P = 0.81, not shown). Higher concentra- interneurons with a monopolar silver wire electrode (diame- tion of GABA (1 mM) elicited a current with mean amplitude of ter = 50 lm), insulated except for the tip and located within 152 ± 32 pA and integrated (under the curve) area of 100 lm of the recorded . Stimulus parameters were as fol- 4427 ± 154 pA s. The current amplitude and area did not change lows: intensity, 1–10 mA, duration, 0.1–0.3 ms, and frequency in the presence of N2O (104 ± 3% of control, n =8, P = 0.12 and 0.1 Hz. GABA and -mediated responses were phar- 106 ± 3% of control, n =8,P = 0.18, respectively; Fig. 1), but both in- macologically isolated by adding 6-cyano-7-nitroquinoxaline-2,3- creased in the presence of ISO (118 ± 3% of control, n =9,P < 0.01 dione (CNQX, 10 lM), DL-2-amino-5-phosphonovaleric acid (APV, and 120 ± 4% of control, n =9,P < 0.01, respectively). 50 lM) and strychnine or , respectively.

As described previously (Georgiev et al., 2008), N2O was applied by bubbling it through the perfusion solution. The solution was first saturated with the gas mixture for 30 min in an air-tight con- tainer and then applied to the slice for at least 5 min prior to recordings. N2O was mixed at a 1:1 ratio with a prefabricated gas containing 90% O2, 10% CO2 using standard anesthetic machine flowmeters (Acoma, Tokyo, Japan) so that the composition of the gas mixture was: 50% N2O, 45% O2, and 5% CO2. We had previously demonstrated the specificity of N2O’s action by replacing it with an equal concentration of N2, which did not elicit the effects observed following application of N2O. As a positive control we used isoflu- rane (ISO), which was delivered by a commercial vaporizer (Fora- wick, Muraco Medical, Japan), and the gas mixture was similarly dispersed into the perfusion solution using a bubbling stone. The concentrations of the delivered gases (O2,CO2,N2O and ISO) were monitored using a Capnomac Ultima gas monitor from Datex (Finland). GABA (0.1 mM and 1 mM) and glycine (0.1 mM and 1 mM) were applied to the slice by bath superfusion for 30 s. In a subset of experiments, tetrodotoxin (TTX, 1 lM) was used to isolate miniature IPSCs (mIPSCs) allowing us to differentiate presynaptic from postsynaptic actions. The following drugs were purchased: N2O from Japan Fine Products (Kawasaki, Japan); ISO from Dainippon Sumitomo Pharma Co., Ltd. (Japan); GABA, glycine, bicuculline, and APV from Sigma (USA); TTX from Wako Fig. 1. N2O and GABA-induced currents. Representative traces of membrane (Japan); and CNQX from Tocris (USA). current induced by exogenous application of c-aminobutyric acid (GABA) in spinal Data acquisition was performed using pClamp 8.2 software substantia gelatinosa neurons during control conditions and application of 50% (0.3 (Molecular Devices) in combination with an Axopatch 200A ampli- rat MAC) nitrous oxide (N2O) or 0.5% isoflurane (ISO). Drug application is marked with bars. GABA (1 mM, 30 s) induced an outward current, the amplitude of which fier and a Digidata 1322 A/D converter (Molecular Devices). Signals did not change in the presence of N2O (104 ± 3% of control, n = 8), but increased in were filtered at 2 kHz and digitalized at 5 kHz. Data analyses were the presence of an equipotent (0.3 rat MAC) concentration of ISO (118 ± 4% of performed using pClamp and Mini-analysis software 6.0.3 control, n = 9). *P < 0.05 compared with control. S.K. Georgiev et al. / European Journal of Pain 14 (2010) 17–22 19

Bath-applied glycine (0.1 mM) induced an outward current (119 ± 2% of control, n =6, P < 0.01 and 120 ± 4% of control, n =6, with a mean amplitude of 49 ± 12 pA (n = 6), which remained un- P < 0.01, respectively). changed in the presence of N2O (100 ± 2% of control, n =6, P = 0.19, not shown). A higher concentration of glycine (1 mM) in- 3.2. Effects of N2O on mIPSCs duced an outward current with a mean amplitude of 185 ± 39 pA and integrated (under the curve) area of 4973 ± 156 pA s. Similarly, GABAergic mIPSCs were isolated by adding TTX (1 lM) and in the presence of N2O the current amplitude was 105 ± 3% (n =9, strychnine (2 lM) to the Krebs solution. They had a mean ampli- P = 0.24; Fig. 2) and the area was 106 ± 4% (n =9,P = 0.29) of the tude of 14.8 ± 3.8 pA, a frequency of 1.9 ± 0.7 Hz and a decay time control values, but these increased in the presence of ISO of 25.2 ± 8.8 ms. N2O application did not significantly affect mIPSC mean amplitude (106 ± 2% of control, n =8, P = 0.39), frequency (103 ± 3% of control, n =8,P = 0.73) or decay time (104 ± 3% of con- trol, n =8,P = 0.31; Fig. 3A and B). Cumulative histograms of mIPSC amplitude, inter-event intervals (frequency) and decay time were analyzed using the Kolmogorov–Smirnov test, but the cumulative

distributions were not affected in presence of N2O(Fig. 3C). mIPSCs were recorded in the presence of TTX and

bicuculline (20 lM) before and during N2O application (Fig. 4A). They had a mean amplitude of 19.8 ± 6.7 pA, frequency of

1.2 ± 0.6 Hz and decay time of 13.6 ± 5.6 ms. Under N2O applica- tion the amplitude, frequency and decay time were 104 ± 3%, 99 ± 2% and 104 ± 3% of the control values, respectively (n =8, P = 0.36, 0.90, 0.38, respectively; Fig. 4B). The cumulative distribu- tion histograms of mIPSCs amplitude, inter-event intervals and de-

cay time showed no change induced by N2O in the majority of neurons (Fig. 4C).

3.3. Effect of N2O on evoked synaptic responses

Electrical stimulation in the proximity of the recorded neuron evoked postsynaptic currents, which could be divided into two groups based on their kinetics and pharmacological properties. Those with longer durations (50–150 ms), relatively lower average amplitudes (91 ± 11 pA, n = 8) and sensitivity to bicuculline are be- lieved to be GABAergic, and those with shorter durations (10– Fig. 2. N O and glycine-induced currents. Bath-applied glycine (1 mM, 30 s) 2 25 ms), larger amplitudes (206 ± 49 pA, n = 9) and sensitivity to induced outward currents in substantia gelatinosa neurons. Perfusion of 50% N2O did not affect the amplitudes of these currents (105 ± 3% of control, n = 9), but the strychnine are believed to be glycinergic (Fig. 5A). Among the ten amplitudes were increased in the presence of isoflurane (ISO) (119 ± 2% of control, neurons tested, N2O did not change the amplitude of evoked GAB- * n = 6). P < 0.05 compared with control. Aergic IPSCs in five neurons, decreased it in two neurons and

Fig. 3. N2O and GABAergic mIPSCs. GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were isolated using tetrodotoxin (1 lM) and strychnine (2 lM), and recorded in SG neurons before and during N2O perfusion. Fifty percent N2O did not affect the mean amplitude, frequency or decay time (A and B), nor the cumulative distribution of mIPSC amplitude, inter-event interval or decay time (C). 20 S.K. Georgiev et al. / European Journal of Pain 14 (2010) 17–22

Fig. 4. N2O and glycinergic mIPSCs. (A) Glycinergic mIPSCs were isolated using tetrodotoxin (1 lM) and bicuculline (20 lM), and recorded before and during N2O. (B) Mean amplitude, frequency and decay time did not change in the presence of N2O. (C) Cumulative histograms before and during N2O perfusion showed no difference in mIPSC amplitude, inter-event interval and decay time. increased it in one neuron. The average amplitude was 101 ± 5% of in rat SG neurons (Georgiev et al., 2008). In this study we examined the control (P = 0.8, n =8; Fig. 5B-left). Similarly, in five of nine the effects of N2O on inhibitory transmission in the superficial dor- neurons the amplitudes of evoked glycinergic IPSCs were not af- sal horn. Unlike the clear effect on glutamatergic transmission, 50% fected by N2O, but these amplitudes were decreased in two neu- N2O only slightly affected synaptically evoked currents and those rons and increased in another two. The average amplitude was elicited by exogenous application of two major inhibitory trans- 107 ± 8% of control (P = 0.4, n =9;Fig. 5B-right). mitters (GABA and glycine), and did not conceivably alter GABAer- gic or glycinergic mIPSCs. 4. Discussion In previous studies, N2O has shown variable degrees of potenti- ation of GABA-induced currents. Two studies using heteromeric Using the same experimental setting we previously found that receptors expressed in HEK293 cells (Hapfelmeier et al., 2000)

50% N2O inhibits the glutamatergic NMDA and AMPA receptors and Xenopus laevis oocytes (Yamakura and Harris, 2000) showed

Fig. 5. N2O and eIPSCs. (A) Traces of GABA (left) and glycine (right) receptor-mediated evoked inhibitory postsynaptic currents (eIPSCs) before and during N2O application

(averaged from five consecutive recordings). (B) Despite some variability in the synaptic response, application of 50% N2O did not change the amplitudes of the evoked GABAergic (left) and glycinergic (right) eIPSCs (101 ± 5%, n = 8 and 107 ± 8%, n = 9, respectively). S.K. Georgiev et al. / European Journal of Pain 14 (2010) 17–22 21 potentiation of GABA-induced currents. These two studies used re- 1993; Wakai et al., 2005) and glycine currents, but we failed to ob- combinant a1b2c2 receptors. However, the subunit composition of serve a significant effect of N2O on mIPSC decay phase kinetics. the GABAA-receptors in the different layers of the spinal cord was N2O did not affect focal stimulation-evoked GABAergic or gly- shown to be heterogeneous, with a predominance of a2 and a lack cinergic IPSCs in the majority of neurons. The actions in opposite of a1 subunit in lamina II neurons (Bohlhalter et al., 1996). That directions in the few remaining neurons could be explained by could explain why we could not observe a facilitatory effect on the presence of different functional types of neurons in the SG GABA-induced currents. Reports based on neuronal preparations (Grudt and Perl, 2002; Lu and Perl, 2007) and implies the complex- are contradictory. Two studies using hippocampal cultured neu- ity of neuronal networks at the dorsal horn level. Such variability of rons found a negligible effect on GABA receptors (Jevtovic- N2O effects has been repeatedly reported in the past (Miyazaki Todorovic et al., 1998; Mennerick et al., 1998), which is in et al., 1999; Antognini et al., 2001; Georgiev et al., 2008), thus call- accordance with our results, while a study using acutely dissoci- ing for a functional type-specific approach when investigating dor- ated hippocampal neurons from the CA1 region reported an sal horn neurons. enhancement of the (GABAA agonist)-induced chloride The results of this study confirm the specific nature of N2O’s current in the presence of N2O(Dzoljic and Van Duijn, 1998). Be- pharmacologic effects, differentiating it from other inhalational sides different materials and recording conditions, the latter study anesthetics. Halogenated anesthetics are thought to inhibit neuro- used different type of agonist and much shorter application times, nal excitation and to potentiate inhibition (Campagna et al., 2003), which multiplies the possible reasons for the discrepant results. the later being more important for the anesthetic action in the dor-

Only a few studies have examined the actions of N2O on glycine sal horn. Inhibition of NMDA and AMPA receptors by volatile anes- receptors. Glycine currents in oocytes are reportedly potentiated thetics has been reported by various studies (Cheng and Kendig, by the gaseous anesthetic (Daniels and Roberts, 1998; Yamakura 2000; de Sousa et al., 2000; Campagna et al., 2003). However, elec- and Harris, 2000), but data from neuronal preparations is not avail- trophysiological studies on dorsal horn neurons failed to demon- able. Although glycine is a major inhibitory in the strate inhibition of glutamate receptors (Wakai et al., 2005)or spinal cord, no discernible effects of N2O on glycinergic synaptic rather suggested inhibition of excitation owing to reduced gluta- transmission could be observed in this study on spinal cord neu- mate release (presynaptic effect) (Haseneder et al., 2004). Haloge- rons. Similarly, subunit heterogeneity might be responsible for nated anesthetics potentiate inhibition by an action on GABA the difference in N2O effect between native and heteromeric receptors (Jones and Harrison, 1993; Banks and Pearce, 1999; receptors. Kitamura et al., 2003), which was confirmed in spinal lamina II The difference in materials and methods used in different stud- neurons (Wakai et al., 2005). There is also evidence for the ies makes a comparison of their results difficult. Nevertheless, a potentiation of glycinergic transmission (Yamashita et al., 2001; difference in the used N2O concentration deserves attention. Most Yamauchi et al., 2002; Zhang et al., 2003). In this and our previous of the above-mentioned studies (Daniels and Roberts, 1998; study, we examined the direct action of N2O on SG neurons. We Dzoljic and Van Duijn, 1998; Mennerick et al., 1998; Hapfelmeier found that N2O blunts the primary afferent stimulation-evoked et al., 2000) used 80% N2O in contrast to the 50% N2O used in our excitatory response through actions on NMDA and AMPA receptors study. We previously found that the ionotropic glutamate recep- of the second-order neuron, but we could not confirm facilitation tors were inhibited even by 50% N2O, which is in agreement with of inhibitory transmission. An interesting pattern of general anes- the fact that N2O possess an analgesic effect even at subanesthetic thetic action on SG neurons emerges from all of these observations concentrations. It could be speculated that a higher N2O concentra- – halogenated anesthetics, known for their good hypnotic, but poor tion is required for a more pronounced effect on GABA and/or gly- analgesic action, affect predominantly GABA receptors. By contrast, cine receptors. Unfortunately, the use of a higher concentration the good analgesic, but poor hypnotic N2O affects predominantly was not possible in this experimental setting owing to technical ionotropic glutamate receptors. However, more experiments are and methodological reasons. Since 50% N2O is approximately 0.3 required to further extrapolate from this observation. rat MAC (Russell and Graybeal, 1992; Leduc et al., 2006), which might be considered to be lower than the sensitivity threshold of 5. Conclusions the method (although in our previous study 50% N2O was proven to be sufficient to modulate glutamate receptors), we tested the ef- In summary, we could not observe any marked direct effect of fect of an equipotent concentration (0.3 MAC) of isoflurane on N2O on inhibitory transmission in SG neurons. It seems that N2O GABA- and glycine-elicited currents and found them to be mildly facilitates inhibition at the spinal cord level not by a direct action potentiated. on dorsal horn neurons, but through activation of descending Theoretically, if the concentration of agonist is high enough to inhibitory pathways and norepinephrine release in the dorsal horn, elicit a maximal saturating response, it would not be further in- as shown in earlier studies (Sawamura et al., 2000). In addition, as creased by the tested drug. The concentrations of GABA and glycine we found previously, N2O inhibits excitation through antagonism used here are derived from previous observations, and even larger of ionotropic glutamatergic receptors in SG neurons, thus exhibit- doses have been used by others (Park et al., 1999; Wu et al., 2008) ing a dual analgesic mechanism. without reaching maximal saturating current. To address this pos- sibility we also used lower concentrations of agonist (0.1 mM Competing Interests GABA and glycine), but these currents were also not affected by N2O. The authors declare that they have no competing interests. We sought to evaluate the effect of N2O on mIPSCs, because changes in mIPSC amplitudes are suggestive of a postsynaptic site of action and changes in the inter-event intervals are suggestive of Acknowledgments a presynaptic action. The lack of an effect on inter-event interval distribution suggests that N2O does not affect inhibitory transmit- The authors thank Yukio Satoh for expert technical assistance. ter release. The lack of an effect on GABA- and glycine-mediated This work was supported by a Grant-in-Aid for Scientific Re- mIPSC amplitudes is consistent with the lack of an effect of N2O search (No. 20390414) from the Ministry of Education, Science, on exogenous agonist-induced current. Inhalational anesthetics Sports and Culture of Japan, Tokyo, Japan and a Yujin Memorial are known to prolong the decay time of GABA (Jones and Harrison, Grant. 22 S.K. Georgiev et al. / European Journal of Pain 14 (2010) 17–22

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