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The activation of nicotinic acetylcholine receptors enhances the inhibitory synaptic transmission in the deep dorsal horn neurons of the adult rat spinal cord

The activation of nicotinic acetylcholine receptors enhances the inhibitory synaptic transmission... Somatosensory information can be modulated by nicotinic acetylcholine receptors (nAChRs) in the superficial dorsal horn of the spinal cord. Nonetheless, the functional significance of nAChRs in the deep dorsal horn of adult animals remains unclear. Using whole-cell patch-clamp recordings from lamina V neurons in the adult rat spinal cord, we investigated whether the activation of nAChRs could modulate the inhibitory synaptic transmission in the deep dorsal horn. In the presence of CNQX and APV to block excitatory glutamatergic synaptic transmission, bath applications of nicotine (100 µM) significantly increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in almost all neurons tested. The effect of nicotine was mimicked by N-methyl- 4-(3-pyridinyl)-3-butene-1-amine (RJR-2403, 100 µM), an α4β2-nAChR agonist, and was also mimicked by choline (10 mM), an α7-nAChR agonist. The effect of nicotine was completely blocked by the nAChR antagonist mecamylamine (5 µM). In the presence of tetrodotoxin (0.5 µM), nicotine (100 µM) significantly increased the miniature IPSC frequency. On the other hand, RJR-2403 (100 µM) or choline (10 mM) did not affect miniature IPSCs. The application of nicotine (100 µM) also evoked a large inward current in all lamina V neurons tested when cells were held at -60 mV. Similarly, RJR-2403 (100 µM) induced inward currents in the majority of lamina V neurons examined. On the other hand, choline (10 mM) did not elicit any detectable whole-cell currents. These results suggest that several nAChR subtypes are expressed on the presynaptic terminals, preterminals, and neuronal cell bodies within lamina V and that these nAChRs are involved in the modulation of inhibitory synaptic activity in the deep dorsal horn of the spinal cord. Background peripheral nervous system. At least 12 different subunits Neuronal nAChRs are a larger family of ligand-gated ion of nAChRs, including α2–α10, β2–β4, have been identi- channels widely expressed in both the central and the fied so far and these subunits form many different sub- Page 1 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 types of nAChRs with pentameric structures consisting of was to evaluate the effects of nAChR activation on the homomers or heteromers [1]. Homomeric nAChRs are inhibitory synaptic transmission in deep dorsal horn neu- made up of α7, α8 or α9 subunits, while heteromeric rons. nAChRs comprise various combinations of α2–α6 with β2–β4 subunits, α9 with α10 subunits [2,3]. These sub- Results types of nAChRs have different pharmacological and bio- Effects of nicotine and nAChR agonists on spontaneous IPSCs in the lamina V neurons physical properties [1]. It has been shown that nAChRs are involved in a variety of physiological functions includ- Whole-cell patch-clamp recordings were performed from ing learning, reinforcement, development, aging and lamina V neurons of spinal cord slices prepared from nociception [4]. adult rats. Stable recordings could be obtained from slices maintained in vitro for more than 12 hours. Glutamatergic Although Davis et al. (1932) first reported that nicotine excitatory postsynaptic transmission was blocked by has analgesic effects [5], high dosages of nicotine were CNQX (20 µM) and APV (50 µM). All lamina V neurons required to produce antinociception and its effect was rel- tested exhibited spontaneous inhibitory postsynaptic cur- atively modest with a short duration [6-8]. Epibatidine, a rents (sIPSCs) when cells were held at -10 mV. In the pres- potent nAChR agonist isolated from the skin of an Ecua- ence of bicuculline (20 µM) and strychnine (2 µM), dorian frog, was about 100-fold more potent than mor- sIPSCs were completely abolished in all lamina V neurons phine in rodents [9-12]. Unfortunately, the dosage of tested (n = 3; data not shown), indicating these sIPSCs epibatidine to produce antinociception was near that to were mediated by GABA and/or glycine receptors. Per- cause seizure, death, and other side effects [12]. The intol- fusion of nicotine (100 µM) for 1 min resulted in a rapid erable toxic effects of epibatidine were due to its actions and significant increase in sIPSC frequency in all neurons on a broad range of nAChR subtypes. Therefore, the key to tested (Fig. 1A–C). The average sIPSC frequency in con- the development of safe and effective nicotinic agonists as trols was 2.1 ± 0.6 Hz (0.4 – 5.1 Hz, n = 9) and the fre- analgesics is to first understand which nAChR subtypes quency increased to 15.8 ± 2.3 Hz (4.2 – 28.1 Hz, n = 9, P are involved in modulating nociceptive transmission. < 0.05) following the application of 100 µM nicotine (Fig. 1C); the sIPSC frequency increased to 1330 ± 310% of the The spinal dorsal horn is the first site in the central nerv- control (n = 9, P < 0.05). The nicotine-induced increase in ous system where somatosensory information is proc- sIPSC frequency was completely blocked in the presence essed and integrated. Multiple subtypes of nAChRs are of nAChR antagonist mecamylamine (5 µM) adminis- expressed in the spinal dorsal horn and these receptors trated 5 min prior to the application of nicotine (n = 3; have been indicated to modulate sensory inputs from the Fig. 1D, E). After the washout of mecamylamine, a second periphery. Genzen and McGehee (2003) have demon- application of nicotine (100 µM) increased the sIPSC fre- strated that the activation of α7 nAChRs located at the quency in all neurons tested (Fig. 1D, E). central terminals of primary afferents enhances the gluta- matergic excitatory transmission in the spinal dorsal horn We tested RJR-2403, a selective α4β2 nAChR agonist, to [13]. Several subtypes of nAChRs have been shown to see if it also increased sIPSC frequency. Similar to nico- exert tonic or phasic control on the descending inhibitory tine, application of 100 µM RJR-2403 for 1 min markedly serotonergic transmission [14]. Multiple subtypes of increased sIPSC frequency in 13 out of 14 neurons nAChRs are found to be expressed on both inhibitory and recorded (Fig. 2Aa–c). The average sIPSC frequency in the excitatory interneurons in the spinal dorsal horn [15]. The control and following the application of RJR-2403 was 5.8 activation of presynaptic nAChRs facilitates GABAergic ± 1.0 Hz (0.5 – 11.6 Hz, n = 14) and 15.7 ± 1.9 Hz (3.7 – and glycinergic inhibitory synaptic transmission in the 25.2 Hz, n = 14), respectively (Fig. 2Ac). The sIPSC fre- superficial dorsal horn [16-19]. Although the roles of pre- quency following the applications of RJR-2403 signifi- synaptic nAChRs were extensively studied in the superfi- cantly increased to 573 ± 189% of the control (n = 14, P < cial dorsal horn, it is unclear whether nAChRs also 0.05; Fig. 2C). While RJR-2403 alone produced a signifi- mediate sensory modulation in the deep dorsal horn of cant increase in sIPSC frequency, the effects of RJR-2403 the spinal cord in adult animals. A variety of sensory was completely blocked in the presence of dihydro-beta- inputs, including nociceptive and non-nociceptive inputs, erythroidine (DhβE, 1 µM), an α4β2 nAChR antagonist are transmitted into deep dorsal horn [20]. Deep dorsal (97 ± 2% of control, n = 3). Perfusion of choline (10 mM), horn neurons, especially those in the lamina V region, can a selective α7 nAChR agonist, for 1 min also increased the generate long-lasting afterdischarges in response to nocic- sIPSC frequency in 11 out of the 13 neurons examined eptive inputs and this hyperactivity has important impli- (Fig. 2Ba–c). The average sIPSC frequency in the control cations in pathological pain states [21]. Inhibitory and following the application of choline was 7.4 ± 1.2 Hz modulation in this region is critical in preventing the cen- (2.9 – 16.9 Hz, n = 13) and 15.2 ± 2.3 Hz (5.2 – 27.6 Hz, tral hyperactivity and hyperalgesia. The aim of this study n = 13), respectively (Fig. 2Bc). The sIPSC frequency fol- Page 2 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 lowing the applications of choline significantly increased to 221 ± 22% of control (n = 13, P < 0.05; Fig. 2C). While choline alone produced a significant increase in sIPSC fre- quency, choline did not produce any significant increase in sIPSC frequency in the presence of methyllycaconitine (MLA, 50 nM), an α7 nAChR antagonist (98 ± 2% of con- trol, n = 4). Effects of nicotine and nAChR agonists on mIPSCs in lamina V neurons We examined the effects of nicotine or nAChR agonists on mIPSC frequency in the presence of tetrodotoxin (TTX, 0.5 µM) to determine whether nAChRs might be localized at the presynaptic terminals of GABAergic and/or glyciner- gic inhibitory interneurons. Application of 0.5 µM TTX itself blocked the action potential-driven synaptic trans- mission and decreased the amplitude of IPSCs from 149.9 ± 125.6 pA to 30.0 ± 18.4 pA (n = 4). Under this condi- tion, bath application of nicotine (100 µM) largely increased mIPSC frequency in all neurons recorded, but there was no effect on mIPSC amplitude (n = 10; Fig. 3A). The average mIPSC frequency in the control and follow- ing the applications of nicotine was 1.7 ± 0.4 Hz (0.4 – 5.3 Hz, n = 10) and 15.0 ± 2.3 Hz (1.4 – 25.2 Hz, n = 10), respectively. The mIPSC frequency following the applica- tions of nicotine significantly increased to 1043 ± 153% of control (n = 10, P < 0.05, Fig. 3D). On the other hand, perfusion of 100 µM RJR-2403 (n = 6) or 10 mM choline (n = 7) did not affect mIPSC frequency and amplitude (Fig. 3B, 3C). The average mIPSC frequency following the application of RJR-2403 and choline was 95 ± 3% of con- trol (n = 6) and 98 ± 2% of control (n = 7), respectively (Fig. 3D). E Figure 1 ffects of nicotine on spontaneous IPSCs in lamina V neurons Effects of nicotine on spontaneous IPSCs in lamina V Whole-cell currents directly evoked by nicotine or nAChR neurons. A, A continuous recording of sIPSCs in the con- agonists in lamina V neurons trol and following the application of nicotine (Nic, 100 µM). We determined whether nicotine, RJR-2403 and choline B, A histogram shows the time course of the changes in could evoke whole-cell currents in lamina V neurons. In sIPSC frequency following the application of nicotine; time this set of experiments, cells were held at -60 mV and bin is 10 s. C, The graph shows the individual result from 9 recordings were conducted in the presence of 20 µM lamina V neurons. D. Effects of mecamylamine (Mec) on nic- CNQX, 50 µM APV, 20 µM bicuculline and 10 µM PMBA otine-induced increase in sIPSC frequency. The consecutive (3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine). traces on the left are sIPSCs in the control (upper panel) and following the application of nicotine (lower panel) in the Under this condition, both excitatory and inhibitory post- presence of mecamylamine (5 µM). The consecutive traces synaptic currents were completely disappeared. The bath on the right are sIPSCs in the control (upper panel) and fol- application of nicotine (100 µM) for 1 min evoked an lowing the application of nicotine (lower panel) after the inward current in all neurons tested (Fig. 4A). The average washout of mecamylamine. Note that the bath application of peak amplitude of the inward currents evoked by nicotine nicotine did not affect the sIPSCs in the presence of was 95 ± 19 pA (n = 8; Fig. 4D). The bath application of mecamylamine, but it markedly increased sIPSC frequency RJR-2403 (100 µM) for 1 min also evoked large inward after the washout of mecamylamine. E, Two histograms currents in 6 out of 8 neurons examined (Fig. 4B). The show time courses of changes in sIPSC frequency following average peak amplitude of the inward currents induced by the application of nicotine in the presence of mecamylamine RJR-2403 was 119 ± 42 pA (n = 6; Fig. 4D). In contrast to (left) and after the washout of mecamylamine (right); time bin nicotine and RJR-2403, choline (10 mM) did not elicit is 10 s. any detectable currents (n = 6; Fig. 4C). Page 3 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 E Figure 2 ffects of nicotinic receptor agonists on sIPSCs in lamina V neurons Effects of nicotinic receptor agonists on sIPSCs in lamina V neurons. A, (a) A continuous recording of sIPSCs in the control and following the application of the selective α4β2 nAChR agonist RJR-2403 (100 µM). (b), A histogram shows the time course of changes in sIPSC frequency following the application of RJR-2403; time bin is 10 s. (c), The graph shows the individual result from 14 lamina V neurons. B, The experiment was similar to that shown in (A) except that the selective α7 nAChR agonist choline (10 mM) was tested. Similar results were obtained in 11 out of 13 neurons. C, A histogram shows rel- ative sIPSC frequency following the application of nicotine (n = 9), RJR-2403 (n = 14), or choline (n = 13). sIPSC frequency before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean ± SEM; *P < 0.05. Page 4 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 E Figure 3 ffects of nicotinic receptor agonists on mIPSCs in lamina V neurons Effects of nicotinic receptor agonists on mIPSCs in lamina V neurons. A, The consecutive traces of mIPSCs are in the control (left) and following the application of nicotine (100 µM, right). Two histograms on the bottom panel show the time course of changes in mIPSC frequency (left) and amplitude (right) during the application of nicotine; time bin is 10 s. B, C, Experiments were similar to that shown in (A) except that 100 µM RJR-2403 or 10 mM choline was tested. D, A histogram shows relative mIPSC frequency following the applications of nicotine (n = 11), RJR-2403 (n = 6), or choline (n = 7). mIPSC fre- quency before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean ± SEM; *P < 0.05; n.s., not significant. neurotransmitters, including serotonin, norepinephrine, Discussion The present study demonstrated in lamina V neurons of glutamate, GABA and glycine [22-24]. In the present the adult rat spinal cord that nicotine increased sIPSC fre- study, the activation of nAChRs enhanced the GABA and/ quency when glutamatergic excitatory transmission was or glycine release onto lamina V neurons. A similar blocked in the presence of CNQX and APV and that nico- enhancement of the inhibitory synaptic transmission by tine also increased mIPSC frequency when action poten- the activation of nAChRs has been reported in the super- tial-driven synaptic transmission was not permitted in the ficial layers of the spinal dorsal horn [16,18,19]. In neo- presence of TTX. Interestingly, however, neither the α4β2 natal rats, α4β2 nAChR subtype has been suggested to be nor α7 nAChR agonists increased mIPSC frequency expressed at presynaptic terminals and these receptors although both of them increased sIPSC frequency in lam- mediate significantly increases in the glycinergic [16] and ina V neurons. Together with the findings of our previous GABAergic inhibitory synaptic transmission in the super- study conducted on superficial laminas of the spinal cord ficial lamina of the spinal cord dorsal horn [19]. Interest- of adult rats [18], we have provided electrophysiological ingly, the expression of nAChR subunits in the spinal cord evidence showing that inhibitory synaptic activity in both changes during development [25]. Consistent with the superficial and deep laminas of the spinal cord dorsal development changes of nAChR subunits, our previous horn are modulated by different nAChR subtypes. study demonstrated that a non-α4β2 and non-α7 subtype nAChR mediated an enhancement of both the GABAergic Nicotinic receptors are abundant in different CNS regions, and glycinergic mIPSC frequency in the superficial lami- where they are shown to regulate the release of various nas of the adult rat spinal dorsal horn [18]. In the deeper Page 5 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 RJR-2403- and choline-induced increase in sIPSC fre- quency in our results suggests that α4β2 and α7 nAChRs are not expressed at presynaptic terminals. These receptors are likely to be expressed at the preterminals or other parts of GABAergic and/or glycinergic neurons whose axons innervate lamina V neurons. Because nicotine could still increase IPSC frequency in the presence of TTX, it suggests that a non-α4β2, non-α7 subtype of nAChR is located at the presynaptic terminals of GABAergic and/or glycinergic neurons that innervate lamina V neurons (Fig. 5). The Wh in lamina V neur Figure 4 ole-cell currents ev ons oked by nicotine and nAChR agonists Whole-cell currents evoked by nicotine and nAChR agonists in lamina V neurons. A, Whole-cell currents evoked by bath application of nicotine (100 µM) in a lamina V neuron. B, Whole-cell currents evoked by RJR-2403 (100 µM) in a different lamina V neuron. C, Choline (10 mM) did not evoke any membrane current in a neuron. C, The histo- gram shows the average amplitude of the whole-cell inward currents induced by nicotine (n = 8), RJR-2403 (n = 6), and choline (n = 6). laminas (lamina III–V) of neonatal rats, a previous study showed that presynaptic α4β2 nAChRs mediate the facili- tation of GABA release [19], a result similar to those shown in superficial laminas of neonatal rats [16,19]. However, the present study revealed that the nAChR- mediated modulation of inhibitory synaptic transmission Schematic diagram of nAChR-me s Figure 5 ory synaptic transmission in the dorsal horn of adu diated modulcation lt rats of sen- in the adult stage is more complicated than that in the Schematic diagram of nAChR-mediated modulcation neonatal stage. We showed that nicotine largely increased of sensory synaptic transmission in the dorsal horn of both sIPSC frequency and mIPSC frequency in the lamina adult rats. In adult rats, α4β2 nAChRs are expressed on the V neurons in adult rats. On the other hand, RJR-2403, a soma of inhibitory interneurons located in the lamina V potent activator of α4β2 nAChR, and choline, a selective region. These nAChRs may be also expressed on pretermi- agonist for α7 nAChR, significantly increased the sIPSC nals but not at presynaptic sites of the lamina V inhibitory frequency, but did not change mIPSC frequency. It has neurons. Lamina V neurons are also synapsed by inhibitory neurons expressing α7 nAChRs at their preterminals and/or been demonstrated that nAChRs are expressed at two cel- on their somas and these inhibitory neurons are likely to be lular locations in the central nervous system [26]. One is located in other lamina regions. The modulation of inhibitory presynaptic sites or synaptic boutons where nAChR activa- activity in lamina V by both α4β2 nAChRs and α7 nAChRs tion modulates transmitter release in a TTX-insensitive depends on membrane depolarization and action potentials. manner. The other is preterminals at terminal axon There is a non-α4β2/non-α7 subtype of nAChRs that are branches where nAChR activation affects transmitter expressed at the presynpatic terminals of inhibitory neurons release by depolarizing axonal membranes to fire action in lamina V region. The modulation of inhibitory transmission potentials. A recent immunohistochemical study revealed in lamina V by non-α4β2/non-α7 subtype of nAChRs is inde- the immunoreactivity of nAChRs in lamina V neurons of pendent of membrane depolarization and action potentials. the spinal dorsal horn at both presynaptic and pretermi- The distribution of nAChR subtypes in the spinal cord lamina nal sites [27]. At the preterminal sites, nAChR-mediated II region of adult rats [18] is also presented in this diagram for a comparison. regulation of transmitter releases is TTX-sensitive and can be blocked in the presence of TTX. The block by TTX of the Page 6 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 presence of a non-α4β2, non-α7 subtype of nAChR in spi- have an antinociceptive effect [37]. Several mechanisms nal cord dorsal horns are supported by previous studies have been proposed to contribute to nAChR-mediated using α4β2 nAChR knock-out mouse [28], in situ hybrid- analgesic effects, including the desensitization of nAChRs ization [29] and the combination of patch-clamp record- on nociceptive primary afferent fibers, the increase of ings with single-cell RT-PCR [15]. All these previous noradrenaline and serotonin release within the spinal studies pointed to the potential presence of other func- cord, the activation of the descending inhibitory pathways tional nAChR subtypes in addition to α4β2 and α7 recep- [14,38], and the increases of GABA and glycine release tors. However, the subunit compositions of the non-α4β2 from inhibitory interneurons in the superficial spinal cord and non-α7 subtype of nAChRs remain to be identified. dorsal horn [39]. Our study suggest that α4β2, α7 nAChRs, and another undefined subtype of nAchRs are We have shown that both nicotine and the α4β2 nAChR involved in regulating GABA and/or glycine release in the agonist RJR-2403 directly evoked whole-cell inward cur- deep lamina of the spinal cord dorsal horn in adult ani- rents in the majority of lamina V neurons. On the other mals. hand, the α7 nAChR agonist choline did not evoke any detectable inward currents in lamina V neurons. Since In this study, the application of nicotine or nicotinic ago- strychnine has been noted to also be an effective antago- nists significantly facilitated GABAergic and/or glycinergic nist at α7 nAChRs [30,31], the effect of choline was exam- inhibitory synaptic transmission in the deep dorsal horn ined in the presence of PMBA, a glycine receptor of the spinal cord. This raises a possibility that acetylcho- antagonist that has no effect on α7 nAChRs [32,33]. The line released endogenously may modulcate inhibitory lack of choline-evoked whole-cell currents raise a possibil- synaptic transmission in a similar fashion. Recently, ity that α7 nAChRs are not expressed on lamina V neu- Rashid et al. (2006) suggested that endogenous acetylcho- rons. If this is the case, then choline-induced increases of line tonically stimulated the GABA and glycine release via sIPSC frequency might be mediated by α7 nAChRs that α4β2 subtype of nAChRs in the superficial dorsal horn in are expressed on the inhibitory interneurons whose cell mice [40]. Acetylcholine may be released from the bodies are located in other lamina regions in the spinal interneurons in the dorsal horn since the cell bodies of cord. In contrast to α7 nAChRs, our results suggest that cholinergic interneurons have been found in lamina III–V α4β2 nAChR expressing inhibitory interneurons are [41]. It appears that in the deep dorsal horn there are no located in lamina V. Consistently, several reports have descending cholinergic systems in the rat [20,42]. Thus, indicated the presence of α4β2 nAChR in the deep dorsal cholinergic interneurons in the dorsal horn [43,44] may horn and α7 nAChRs in other lamina of the spinal cord play an important role in modulating inhibitory synaptic [15,33-35]. Bradia et al. (2002) has reported that α-bun- transmission. garotoxin-sensitive α7 nAChRs are located in the para- sympathetic preganglionic neurons surrounding the In the dorsal horn, GABAergic and glicinergic inhibitory central canal of the spinal cord (lamina X) [33]. A low synapses undergo developmental changes [45-47]. In the level of α7 transcripts were also detected by in situ hybrid- present study, we did not separate inhibitory activity ization in the area around the central canal [35]. Moreo- between those of GABAergic synapses and those of glycin- ver, the single-cell RT-PCR study revealed a more ergic synapses. It would be interesting to further study widespread expression of α7 nAChR subunits in mouse whether nAchR subtype expression on GABAergic and gly- spinal dorsal horn neurons [15]. These findings support cinergic neurons is different in the spinal cord dorsal the idea that α7 nAChR-expressing inhibitory interneu- horn. rons innervate lamina V neurons from other lamina regions in the spinal cord. Conclusion We have demonstrated that several nAChR subtypes are The role of nAChRs in modulating pain transmission has expressed on the presynaptic terminals, preterminals, and been reported by a number of studies. Using α4β2 knock- neuronal cell bodies within lamina V and that they are out mice, Marubio et al. (1999) showed a reduced antino- involved in the facilitation of inhibitory synaptic trans- ciceptive effect in a behavior study [28]. In a neuropathic mission. Therefore, the activation of nAChRs in the deep mouse model, epibatidine, a potent agonist of nAChRs dorsal horn of the spinal cord may be capable of inhibit- showed strong analgesic effects. However, the effects of ing nociceptive signaling in physiological and pathologi- epibatidine were not completely prevented by the α4β2 cal pain sensations. nAChR antagonist dihydro-β-erythroidine [36]. These studies suggested that in addition to α4β2 nAChR, other Methods nAChRs were involved in nAChR-mediated analgesic All the experimental procedures involving the use of ani- effects. Consistent with this idea, the intrathecal injection mals were approved by the Ethics Committee on Animal of choline, an α7 nAChR agonist, has been reported to Experiments, Wakayama Medical University, and were in Page 7 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 accordance with the UK Animals (Scientific Procedures) USA), choline (Sigma-Aldrich), mecamylamine (Sigma- Act 1986 and all associated guidelines. Aldrich), 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3- yl]alanine (PMBA, Sigma RBI), bicuculline (Sigma- Spinal cord slice preparation Aldrich), strychnine (Sigma-Aldrich), 6-cyano-7-nitroqui- The method used to prepare adult rat spinal cord slices noxaline-2,3-dion (CNQX, Tocris), D(-)-2-Amino-5- has been described previously [48]. In brief, male adult phosphonopentanoic acid (D-APV, Tocris), and tetrodo- Sprague-Dawley rats (6–8 weeks of age, 200–300 g) were toxin (TTX, Tocris). deeply anaesthetized with isoflurane through a nose cone inhalation, and then lumbosacral laminectomy was per- Statistical analysis formed. The lumbosacral spinal cord (L1-S3) was All numerical data were expressed as the mean ± S.E.M. removed and placed in pre-oxygenated Krebs solution at Statistical significance was determined as P < 0.05 using 1–3°C. Immediately after the removal of the spinal cord, paired Student's t-test. For electrophysiological data, n the rats were killed by exsanguination. The pia-arachnoid refers to the number of neurons recorded. membrane was removed after cutting all the ventral and dorsal roots near the root entry zone. The spinal cord was Abbreviations mounted on a vibratome and then a 600 µm-thick trans- nACh, nicotinic acetylcholine receptor; verse slice was cut. The slice was placed on nylon mesh in the recording chamber, which had a volume of 0.5 ml, IPSC, inhibitory postsynaptic current; and then was perfused at a rate of 10–15 ml/min with Krebs solution saturated with 95% O and 5% CO , and RJR2-403, N-methyl-4-(3-pyridinyl)-3-butene-1-amine; 2 2 maintained at room temperature. A platinum grid was placed on the top of the slice to prevent slice movement. TTX, tetrodotoxin; The Krebs solution contained (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl , 1.2 MgCl , 1.2 NaH PO , 25 NaHCO and 11 GABA, gamma-aminobutyric acid; 2 2 2 4 3 glucose. TEA, tetraethylammonium; Patch-clamp recordings from lamina V neurons Blind whole-cell patch-clamp recordings were made from EGTA, ethyleneglycol bis(2-aminoethylether)tetraacetic lamina V neurons with patch-pipette electrodes having a acid; resistance of 5–10 MΩ [48]. The patch-pipette solution was composed of (in mM) 110 Cs SO , 5 Tetraethylam- HEPES, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesul- 2 4 monium (TEA), 0.5 CaCl , 2 MgCl , 5 EGTA, 5 HEPES, 5 fonic acid); 2 2 ATP-Mg, pH 7.2. Signals were acquired with a patch- clamp amplifier (Axopatch 200B; Axon Instruments, Fos- ATP-Mg, adenosine triphosphate-magnesium; ter City, CA, USA). The data were digitized with an A/D converter (Digidata 1200, Axon Instruments) and stored CNQX,6-cyano-7-nitroquinoxaline-2,3-dion; and analyzed with a personal computer using the pCLAMP data acquisition program (Version 8.2, Axon PMBA,3-[2'-Phosphonomethyl[1,1'-biphenyl]-3- Instruments). Lamina V neurons were viable for up to 24 yl]alanine; h in slices perfused with a pre-oxygenated Krebs solution. All the recordings described in this study were made D-APV, D(-)-2-Amino-5-phosphonopentanoic acid; within 12 h. Whole-cell patch-clamp recordings were sta- ble for up to 4 h. All of the neurons had membrane poten- Competing interests tials more negative than -50 mV. Unless otherwise noted, The author(s) declare that they have no competing inter- all the recordings in this study were performed in the pres- ests. ence of CNQX (20 µM) and APV (50 µM). Acknowledgements This work was supported by The General Insurance Association of Japan, Drug Applications The Japanese Health Sciences Foundation, and Grants-in-Aid for Scientific Drugs were dissolved in Krebs solution and then were Research from the Ministry of Education, Science, Sports and Culture of applied by perfusion via a three-way stopcock without any Japan to T.N. change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the References surface of the spinal cord slice was approximately 20 s. 1. Changeux JP, Edelstein SJ: Allosteric receptors after 30 years. The drugs used in this study were nicotine (Sigma-Aldrich, Neuron 1998, 21:959-980. 2. 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Page 9 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 47. Ataka T, Gu JG: Relationship between tonic inhibitory currents and phasic inhibitory activity in the spinal cord lamina II region of adult mice. Mol Pain 2006, 2;2:36. 48. Nakatsuka T, Ataka T, Kumamoto E, Tamaki T, Yoshimura M: Alter- ation in synaptic inputs through C-afferent fibers to substan- tia gelatinosa neurons of the rat spinal dorsal horn during postnatal development. Neuroscience 2000, 99:549-556. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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The activation of nicotinic acetylcholine receptors enhances the inhibitory synaptic transmission in the deep dorsal horn neurons of the adult rat spinal cord

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Springer Journals
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Copyright © 2007 by Takeda et al; licensee BioMed Central Ltd.
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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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Abstract

Somatosensory information can be modulated by nicotinic acetylcholine receptors (nAChRs) in the superficial dorsal horn of the spinal cord. Nonetheless, the functional significance of nAChRs in the deep dorsal horn of adult animals remains unclear. Using whole-cell patch-clamp recordings from lamina V neurons in the adult rat spinal cord, we investigated whether the activation of nAChRs could modulate the inhibitory synaptic transmission in the deep dorsal horn. In the presence of CNQX and APV to block excitatory glutamatergic synaptic transmission, bath applications of nicotine (100 µM) significantly increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in almost all neurons tested. The effect of nicotine was mimicked by N-methyl- 4-(3-pyridinyl)-3-butene-1-amine (RJR-2403, 100 µM), an α4β2-nAChR agonist, and was also mimicked by choline (10 mM), an α7-nAChR agonist. The effect of nicotine was completely blocked by the nAChR antagonist mecamylamine (5 µM). In the presence of tetrodotoxin (0.5 µM), nicotine (100 µM) significantly increased the miniature IPSC frequency. On the other hand, RJR-2403 (100 µM) or choline (10 mM) did not affect miniature IPSCs. The application of nicotine (100 µM) also evoked a large inward current in all lamina V neurons tested when cells were held at -60 mV. Similarly, RJR-2403 (100 µM) induced inward currents in the majority of lamina V neurons examined. On the other hand, choline (10 mM) did not elicit any detectable whole-cell currents. These results suggest that several nAChR subtypes are expressed on the presynaptic terminals, preterminals, and neuronal cell bodies within lamina V and that these nAChRs are involved in the modulation of inhibitory synaptic activity in the deep dorsal horn of the spinal cord. Background peripheral nervous system. At least 12 different subunits Neuronal nAChRs are a larger family of ligand-gated ion of nAChRs, including α2–α10, β2–β4, have been identi- channels widely expressed in both the central and the fied so far and these subunits form many different sub- Page 1 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 types of nAChRs with pentameric structures consisting of was to evaluate the effects of nAChR activation on the homomers or heteromers [1]. Homomeric nAChRs are inhibitory synaptic transmission in deep dorsal horn neu- made up of α7, α8 or α9 subunits, while heteromeric rons. nAChRs comprise various combinations of α2–α6 with β2–β4 subunits, α9 with α10 subunits [2,3]. These sub- Results types of nAChRs have different pharmacological and bio- Effects of nicotine and nAChR agonists on spontaneous IPSCs in the lamina V neurons physical properties [1]. It has been shown that nAChRs are involved in a variety of physiological functions includ- Whole-cell patch-clamp recordings were performed from ing learning, reinforcement, development, aging and lamina V neurons of spinal cord slices prepared from nociception [4]. adult rats. Stable recordings could be obtained from slices maintained in vitro for more than 12 hours. Glutamatergic Although Davis et al. (1932) first reported that nicotine excitatory postsynaptic transmission was blocked by has analgesic effects [5], high dosages of nicotine were CNQX (20 µM) and APV (50 µM). All lamina V neurons required to produce antinociception and its effect was rel- tested exhibited spontaneous inhibitory postsynaptic cur- atively modest with a short duration [6-8]. Epibatidine, a rents (sIPSCs) when cells were held at -10 mV. In the pres- potent nAChR agonist isolated from the skin of an Ecua- ence of bicuculline (20 µM) and strychnine (2 µM), dorian frog, was about 100-fold more potent than mor- sIPSCs were completely abolished in all lamina V neurons phine in rodents [9-12]. Unfortunately, the dosage of tested (n = 3; data not shown), indicating these sIPSCs epibatidine to produce antinociception was near that to were mediated by GABA and/or glycine receptors. Per- cause seizure, death, and other side effects [12]. The intol- fusion of nicotine (100 µM) for 1 min resulted in a rapid erable toxic effects of epibatidine were due to its actions and significant increase in sIPSC frequency in all neurons on a broad range of nAChR subtypes. Therefore, the key to tested (Fig. 1A–C). The average sIPSC frequency in con- the development of safe and effective nicotinic agonists as trols was 2.1 ± 0.6 Hz (0.4 – 5.1 Hz, n = 9) and the fre- analgesics is to first understand which nAChR subtypes quency increased to 15.8 ± 2.3 Hz (4.2 – 28.1 Hz, n = 9, P are involved in modulating nociceptive transmission. < 0.05) following the application of 100 µM nicotine (Fig. 1C); the sIPSC frequency increased to 1330 ± 310% of the The spinal dorsal horn is the first site in the central nerv- control (n = 9, P < 0.05). The nicotine-induced increase in ous system where somatosensory information is proc- sIPSC frequency was completely blocked in the presence essed and integrated. Multiple subtypes of nAChRs are of nAChR antagonist mecamylamine (5 µM) adminis- expressed in the spinal dorsal horn and these receptors trated 5 min prior to the application of nicotine (n = 3; have been indicated to modulate sensory inputs from the Fig. 1D, E). After the washout of mecamylamine, a second periphery. Genzen and McGehee (2003) have demon- application of nicotine (100 µM) increased the sIPSC fre- strated that the activation of α7 nAChRs located at the quency in all neurons tested (Fig. 1D, E). central terminals of primary afferents enhances the gluta- matergic excitatory transmission in the spinal dorsal horn We tested RJR-2403, a selective α4β2 nAChR agonist, to [13]. Several subtypes of nAChRs have been shown to see if it also increased sIPSC frequency. Similar to nico- exert tonic or phasic control on the descending inhibitory tine, application of 100 µM RJR-2403 for 1 min markedly serotonergic transmission [14]. Multiple subtypes of increased sIPSC frequency in 13 out of 14 neurons nAChRs are found to be expressed on both inhibitory and recorded (Fig. 2Aa–c). The average sIPSC frequency in the excitatory interneurons in the spinal dorsal horn [15]. The control and following the application of RJR-2403 was 5.8 activation of presynaptic nAChRs facilitates GABAergic ± 1.0 Hz (0.5 – 11.6 Hz, n = 14) and 15.7 ± 1.9 Hz (3.7 – and glycinergic inhibitory synaptic transmission in the 25.2 Hz, n = 14), respectively (Fig. 2Ac). The sIPSC fre- superficial dorsal horn [16-19]. Although the roles of pre- quency following the applications of RJR-2403 signifi- synaptic nAChRs were extensively studied in the superfi- cantly increased to 573 ± 189% of the control (n = 14, P < cial dorsal horn, it is unclear whether nAChRs also 0.05; Fig. 2C). While RJR-2403 alone produced a signifi- mediate sensory modulation in the deep dorsal horn of cant increase in sIPSC frequency, the effects of RJR-2403 the spinal cord in adult animals. A variety of sensory was completely blocked in the presence of dihydro-beta- inputs, including nociceptive and non-nociceptive inputs, erythroidine (DhβE, 1 µM), an α4β2 nAChR antagonist are transmitted into deep dorsal horn [20]. Deep dorsal (97 ± 2% of control, n = 3). Perfusion of choline (10 mM), horn neurons, especially those in the lamina V region, can a selective α7 nAChR agonist, for 1 min also increased the generate long-lasting afterdischarges in response to nocic- sIPSC frequency in 11 out of the 13 neurons examined eptive inputs and this hyperactivity has important impli- (Fig. 2Ba–c). The average sIPSC frequency in the control cations in pathological pain states [21]. Inhibitory and following the application of choline was 7.4 ± 1.2 Hz modulation in this region is critical in preventing the cen- (2.9 – 16.9 Hz, n = 13) and 15.2 ± 2.3 Hz (5.2 – 27.6 Hz, tral hyperactivity and hyperalgesia. The aim of this study n = 13), respectively (Fig. 2Bc). The sIPSC frequency fol- Page 2 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 lowing the applications of choline significantly increased to 221 ± 22% of control (n = 13, P < 0.05; Fig. 2C). While choline alone produced a significant increase in sIPSC fre- quency, choline did not produce any significant increase in sIPSC frequency in the presence of methyllycaconitine (MLA, 50 nM), an α7 nAChR antagonist (98 ± 2% of con- trol, n = 4). Effects of nicotine and nAChR agonists on mIPSCs in lamina V neurons We examined the effects of nicotine or nAChR agonists on mIPSC frequency in the presence of tetrodotoxin (TTX, 0.5 µM) to determine whether nAChRs might be localized at the presynaptic terminals of GABAergic and/or glyciner- gic inhibitory interneurons. Application of 0.5 µM TTX itself blocked the action potential-driven synaptic trans- mission and decreased the amplitude of IPSCs from 149.9 ± 125.6 pA to 30.0 ± 18.4 pA (n = 4). Under this condi- tion, bath application of nicotine (100 µM) largely increased mIPSC frequency in all neurons recorded, but there was no effect on mIPSC amplitude (n = 10; Fig. 3A). The average mIPSC frequency in the control and follow- ing the applications of nicotine was 1.7 ± 0.4 Hz (0.4 – 5.3 Hz, n = 10) and 15.0 ± 2.3 Hz (1.4 – 25.2 Hz, n = 10), respectively. The mIPSC frequency following the applica- tions of nicotine significantly increased to 1043 ± 153% of control (n = 10, P < 0.05, Fig. 3D). On the other hand, perfusion of 100 µM RJR-2403 (n = 6) or 10 mM choline (n = 7) did not affect mIPSC frequency and amplitude (Fig. 3B, 3C). The average mIPSC frequency following the application of RJR-2403 and choline was 95 ± 3% of con- trol (n = 6) and 98 ± 2% of control (n = 7), respectively (Fig. 3D). E Figure 1 ffects of nicotine on spontaneous IPSCs in lamina V neurons Effects of nicotine on spontaneous IPSCs in lamina V Whole-cell currents directly evoked by nicotine or nAChR neurons. A, A continuous recording of sIPSCs in the con- agonists in lamina V neurons trol and following the application of nicotine (Nic, 100 µM). We determined whether nicotine, RJR-2403 and choline B, A histogram shows the time course of the changes in could evoke whole-cell currents in lamina V neurons. In sIPSC frequency following the application of nicotine; time this set of experiments, cells were held at -60 mV and bin is 10 s. C, The graph shows the individual result from 9 recordings were conducted in the presence of 20 µM lamina V neurons. D. Effects of mecamylamine (Mec) on nic- CNQX, 50 µM APV, 20 µM bicuculline and 10 µM PMBA otine-induced increase in sIPSC frequency. The consecutive (3-[2'-Phosphonomethyl[1,1'-biphenyl]-3-yl]alanine). traces on the left are sIPSCs in the control (upper panel) and following the application of nicotine (lower panel) in the Under this condition, both excitatory and inhibitory post- presence of mecamylamine (5 µM). The consecutive traces synaptic currents were completely disappeared. The bath on the right are sIPSCs in the control (upper panel) and fol- application of nicotine (100 µM) for 1 min evoked an lowing the application of nicotine (lower panel) after the inward current in all neurons tested (Fig. 4A). The average washout of mecamylamine. Note that the bath application of peak amplitude of the inward currents evoked by nicotine nicotine did not affect the sIPSCs in the presence of was 95 ± 19 pA (n = 8; Fig. 4D). The bath application of mecamylamine, but it markedly increased sIPSC frequency RJR-2403 (100 µM) for 1 min also evoked large inward after the washout of mecamylamine. E, Two histograms currents in 6 out of 8 neurons examined (Fig. 4B). The show time courses of changes in sIPSC frequency following average peak amplitude of the inward currents induced by the application of nicotine in the presence of mecamylamine RJR-2403 was 119 ± 42 pA (n = 6; Fig. 4D). In contrast to (left) and after the washout of mecamylamine (right); time bin nicotine and RJR-2403, choline (10 mM) did not elicit is 10 s. any detectable currents (n = 6; Fig. 4C). Page 3 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 E Figure 2 ffects of nicotinic receptor agonists on sIPSCs in lamina V neurons Effects of nicotinic receptor agonists on sIPSCs in lamina V neurons. A, (a) A continuous recording of sIPSCs in the control and following the application of the selective α4β2 nAChR agonist RJR-2403 (100 µM). (b), A histogram shows the time course of changes in sIPSC frequency following the application of RJR-2403; time bin is 10 s. (c), The graph shows the individual result from 14 lamina V neurons. B, The experiment was similar to that shown in (A) except that the selective α7 nAChR agonist choline (10 mM) was tested. Similar results were obtained in 11 out of 13 neurons. C, A histogram shows rel- ative sIPSC frequency following the application of nicotine (n = 9), RJR-2403 (n = 14), or choline (n = 13). sIPSC frequency before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean ± SEM; *P < 0.05. Page 4 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 E Figure 3 ffects of nicotinic receptor agonists on mIPSCs in lamina V neurons Effects of nicotinic receptor agonists on mIPSCs in lamina V neurons. A, The consecutive traces of mIPSCs are in the control (left) and following the application of nicotine (100 µM, right). Two histograms on the bottom panel show the time course of changes in mIPSC frequency (left) and amplitude (right) during the application of nicotine; time bin is 10 s. B, C, Experiments were similar to that shown in (A) except that 100 µM RJR-2403 or 10 mM choline was tested. D, A histogram shows relative mIPSC frequency following the applications of nicotine (n = 11), RJR-2403 (n = 6), or choline (n = 7). mIPSC fre- quency before the applications of testing drugs is used as control and is scaled at 100%. Data represent Mean ± SEM; *P < 0.05; n.s., not significant. neurotransmitters, including serotonin, norepinephrine, Discussion The present study demonstrated in lamina V neurons of glutamate, GABA and glycine [22-24]. In the present the adult rat spinal cord that nicotine increased sIPSC fre- study, the activation of nAChRs enhanced the GABA and/ quency when glutamatergic excitatory transmission was or glycine release onto lamina V neurons. A similar blocked in the presence of CNQX and APV and that nico- enhancement of the inhibitory synaptic transmission by tine also increased mIPSC frequency when action poten- the activation of nAChRs has been reported in the super- tial-driven synaptic transmission was not permitted in the ficial layers of the spinal dorsal horn [16,18,19]. In neo- presence of TTX. Interestingly, however, neither the α4β2 natal rats, α4β2 nAChR subtype has been suggested to be nor α7 nAChR agonists increased mIPSC frequency expressed at presynaptic terminals and these receptors although both of them increased sIPSC frequency in lam- mediate significantly increases in the glycinergic [16] and ina V neurons. Together with the findings of our previous GABAergic inhibitory synaptic transmission in the super- study conducted on superficial laminas of the spinal cord ficial lamina of the spinal cord dorsal horn [19]. Interest- of adult rats [18], we have provided electrophysiological ingly, the expression of nAChR subunits in the spinal cord evidence showing that inhibitory synaptic activity in both changes during development [25]. Consistent with the superficial and deep laminas of the spinal cord dorsal development changes of nAChR subunits, our previous horn are modulated by different nAChR subtypes. study demonstrated that a non-α4β2 and non-α7 subtype nAChR mediated an enhancement of both the GABAergic Nicotinic receptors are abundant in different CNS regions, and glycinergic mIPSC frequency in the superficial lami- where they are shown to regulate the release of various nas of the adult rat spinal dorsal horn [18]. In the deeper Page 5 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 RJR-2403- and choline-induced increase in sIPSC fre- quency in our results suggests that α4β2 and α7 nAChRs are not expressed at presynaptic terminals. These receptors are likely to be expressed at the preterminals or other parts of GABAergic and/or glycinergic neurons whose axons innervate lamina V neurons. Because nicotine could still increase IPSC frequency in the presence of TTX, it suggests that a non-α4β2, non-α7 subtype of nAChR is located at the presynaptic terminals of GABAergic and/or glycinergic neurons that innervate lamina V neurons (Fig. 5). The Wh in lamina V neur Figure 4 ole-cell currents ev ons oked by nicotine and nAChR agonists Whole-cell currents evoked by nicotine and nAChR agonists in lamina V neurons. A, Whole-cell currents evoked by bath application of nicotine (100 µM) in a lamina V neuron. B, Whole-cell currents evoked by RJR-2403 (100 µM) in a different lamina V neuron. C, Choline (10 mM) did not evoke any membrane current in a neuron. C, The histo- gram shows the average amplitude of the whole-cell inward currents induced by nicotine (n = 8), RJR-2403 (n = 6), and choline (n = 6). laminas (lamina III–V) of neonatal rats, a previous study showed that presynaptic α4β2 nAChRs mediate the facili- tation of GABA release [19], a result similar to those shown in superficial laminas of neonatal rats [16,19]. However, the present study revealed that the nAChR- mediated modulation of inhibitory synaptic transmission Schematic diagram of nAChR-me s Figure 5 ory synaptic transmission in the dorsal horn of adu diated modulcation lt rats of sen- in the adult stage is more complicated than that in the Schematic diagram of nAChR-mediated modulcation neonatal stage. We showed that nicotine largely increased of sensory synaptic transmission in the dorsal horn of both sIPSC frequency and mIPSC frequency in the lamina adult rats. In adult rats, α4β2 nAChRs are expressed on the V neurons in adult rats. On the other hand, RJR-2403, a soma of inhibitory interneurons located in the lamina V potent activator of α4β2 nAChR, and choline, a selective region. These nAChRs may be also expressed on pretermi- agonist for α7 nAChR, significantly increased the sIPSC nals but not at presynaptic sites of the lamina V inhibitory frequency, but did not change mIPSC frequency. It has neurons. Lamina V neurons are also synapsed by inhibitory neurons expressing α7 nAChRs at their preterminals and/or been demonstrated that nAChRs are expressed at two cel- on their somas and these inhibitory neurons are likely to be lular locations in the central nervous system [26]. One is located in other lamina regions. The modulation of inhibitory presynaptic sites or synaptic boutons where nAChR activa- activity in lamina V by both α4β2 nAChRs and α7 nAChRs tion modulates transmitter release in a TTX-insensitive depends on membrane depolarization and action potentials. manner. The other is preterminals at terminal axon There is a non-α4β2/non-α7 subtype of nAChRs that are branches where nAChR activation affects transmitter expressed at the presynpatic terminals of inhibitory neurons release by depolarizing axonal membranes to fire action in lamina V region. The modulation of inhibitory transmission potentials. A recent immunohistochemical study revealed in lamina V by non-α4β2/non-α7 subtype of nAChRs is inde- the immunoreactivity of nAChRs in lamina V neurons of pendent of membrane depolarization and action potentials. the spinal dorsal horn at both presynaptic and pretermi- The distribution of nAChR subtypes in the spinal cord lamina nal sites [27]. At the preterminal sites, nAChR-mediated II region of adult rats [18] is also presented in this diagram for a comparison. regulation of transmitter releases is TTX-sensitive and can be blocked in the presence of TTX. The block by TTX of the Page 6 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 presence of a non-α4β2, non-α7 subtype of nAChR in spi- have an antinociceptive effect [37]. Several mechanisms nal cord dorsal horns are supported by previous studies have been proposed to contribute to nAChR-mediated using α4β2 nAChR knock-out mouse [28], in situ hybrid- analgesic effects, including the desensitization of nAChRs ization [29] and the combination of patch-clamp record- on nociceptive primary afferent fibers, the increase of ings with single-cell RT-PCR [15]. All these previous noradrenaline and serotonin release within the spinal studies pointed to the potential presence of other func- cord, the activation of the descending inhibitory pathways tional nAChR subtypes in addition to α4β2 and α7 recep- [14,38], and the increases of GABA and glycine release tors. However, the subunit compositions of the non-α4β2 from inhibitory interneurons in the superficial spinal cord and non-α7 subtype of nAChRs remain to be identified. dorsal horn [39]. Our study suggest that α4β2, α7 nAChRs, and another undefined subtype of nAchRs are We have shown that both nicotine and the α4β2 nAChR involved in regulating GABA and/or glycine release in the agonist RJR-2403 directly evoked whole-cell inward cur- deep lamina of the spinal cord dorsal horn in adult ani- rents in the majority of lamina V neurons. On the other mals. hand, the α7 nAChR agonist choline did not evoke any detectable inward currents in lamina V neurons. Since In this study, the application of nicotine or nicotinic ago- strychnine has been noted to also be an effective antago- nists significantly facilitated GABAergic and/or glycinergic nist at α7 nAChRs [30,31], the effect of choline was exam- inhibitory synaptic transmission in the deep dorsal horn ined in the presence of PMBA, a glycine receptor of the spinal cord. This raises a possibility that acetylcho- antagonist that has no effect on α7 nAChRs [32,33]. The line released endogenously may modulcate inhibitory lack of choline-evoked whole-cell currents raise a possibil- synaptic transmission in a similar fashion. Recently, ity that α7 nAChRs are not expressed on lamina V neu- Rashid et al. (2006) suggested that endogenous acetylcho- rons. If this is the case, then choline-induced increases of line tonically stimulated the GABA and glycine release via sIPSC frequency might be mediated by α7 nAChRs that α4β2 subtype of nAChRs in the superficial dorsal horn in are expressed on the inhibitory interneurons whose cell mice [40]. Acetylcholine may be released from the bodies are located in other lamina regions in the spinal interneurons in the dorsal horn since the cell bodies of cord. In contrast to α7 nAChRs, our results suggest that cholinergic interneurons have been found in lamina III–V α4β2 nAChR expressing inhibitory interneurons are [41]. It appears that in the deep dorsal horn there are no located in lamina V. Consistently, several reports have descending cholinergic systems in the rat [20,42]. Thus, indicated the presence of α4β2 nAChR in the deep dorsal cholinergic interneurons in the dorsal horn [43,44] may horn and α7 nAChRs in other lamina of the spinal cord play an important role in modulating inhibitory synaptic [15,33-35]. Bradia et al. (2002) has reported that α-bun- transmission. garotoxin-sensitive α7 nAChRs are located in the para- sympathetic preganglionic neurons surrounding the In the dorsal horn, GABAergic and glicinergic inhibitory central canal of the spinal cord (lamina X) [33]. A low synapses undergo developmental changes [45-47]. In the level of α7 transcripts were also detected by in situ hybrid- present study, we did not separate inhibitory activity ization in the area around the central canal [35]. Moreo- between those of GABAergic synapses and those of glycin- ver, the single-cell RT-PCR study revealed a more ergic synapses. It would be interesting to further study widespread expression of α7 nAChR subunits in mouse whether nAchR subtype expression on GABAergic and gly- spinal dorsal horn neurons [15]. These findings support cinergic neurons is different in the spinal cord dorsal the idea that α7 nAChR-expressing inhibitory interneu- horn. rons innervate lamina V neurons from other lamina regions in the spinal cord. Conclusion We have demonstrated that several nAChR subtypes are The role of nAChRs in modulating pain transmission has expressed on the presynaptic terminals, preterminals, and been reported by a number of studies. Using α4β2 knock- neuronal cell bodies within lamina V and that they are out mice, Marubio et al. (1999) showed a reduced antino- involved in the facilitation of inhibitory synaptic trans- ciceptive effect in a behavior study [28]. In a neuropathic mission. Therefore, the activation of nAChRs in the deep mouse model, epibatidine, a potent agonist of nAChRs dorsal horn of the spinal cord may be capable of inhibit- showed strong analgesic effects. However, the effects of ing nociceptive signaling in physiological and pathologi- epibatidine were not completely prevented by the α4β2 cal pain sensations. nAChR antagonist dihydro-β-erythroidine [36]. These studies suggested that in addition to α4β2 nAChR, other Methods nAChRs were involved in nAChR-mediated analgesic All the experimental procedures involving the use of ani- effects. Consistent with this idea, the intrathecal injection mals were approved by the Ethics Committee on Animal of choline, an α7 nAChR agonist, has been reported to Experiments, Wakayama Medical University, and were in Page 7 of 10 (page number not for citation purposes) Molecular Pain 2007, 3:26 http://www.molecularpain.com/content/3/1/26 accordance with the UK Animals (Scientific Procedures) USA), choline (Sigma-Aldrich), mecamylamine (Sigma- Act 1986 and all associated guidelines. Aldrich), 3-[2'-Phosphonomethyl[1,1'-biphenyl]-3- yl]alanine (PMBA, Sigma RBI), bicuculline (Sigma- Spinal cord slice preparation Aldrich), strychnine (Sigma-Aldrich), 6-cyano-7-nitroqui- The method used to prepare adult rat spinal cord slices noxaline-2,3-dion (CNQX, Tocris), D(-)-2-Amino-5- has been described previously [48]. In brief, male adult phosphonopentanoic acid (D-APV, Tocris), and tetrodo- Sprague-Dawley rats (6–8 weeks of age, 200–300 g) were toxin (TTX, Tocris). deeply anaesthetized with isoflurane through a nose cone inhalation, and then lumbosacral laminectomy was per- Statistical analysis formed. The lumbosacral spinal cord (L1-S3) was All numerical data were expressed as the mean ± S.E.M. removed and placed in pre-oxygenated Krebs solution at Statistical significance was determined as P < 0.05 using 1–3°C. Immediately after the removal of the spinal cord, paired Student's t-test. For electrophysiological data, n the rats were killed by exsanguination. The pia-arachnoid refers to the number of neurons recorded. membrane was removed after cutting all the ventral and dorsal roots near the root entry zone. The spinal cord was Abbreviations mounted on a vibratome and then a 600 µm-thick trans- nACh, nicotinic acetylcholine receptor; verse slice was cut. The slice was placed on nylon mesh in the recording chamber, which had a volume of 0.5 ml, IPSC, inhibitory postsynaptic current; and then was perfused at a rate of 10–15 ml/min with Krebs solution saturated with 95% O and 5% CO , and RJR2-403, N-methyl-4-(3-pyridinyl)-3-butene-1-amine; 2 2 maintained at room temperature. A platinum grid was placed on the top of the slice to prevent slice movement. TTX, tetrodotoxin; The Krebs solution contained (in mM) 117 NaCl, 3.6 KCl, 2.5 CaCl , 1.2 MgCl , 1.2 NaH PO , 25 NaHCO and 11 GABA, gamma-aminobutyric acid; 2 2 2 4 3 glucose. TEA, tetraethylammonium; Patch-clamp recordings from lamina V neurons Blind whole-cell patch-clamp recordings were made from EGTA, ethyleneglycol bis(2-aminoethylether)tetraacetic lamina V neurons with patch-pipette electrodes having a acid; resistance of 5–10 MΩ [48]. The patch-pipette solution was composed of (in mM) 110 Cs SO , 5 Tetraethylam- HEPES, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesul- 2 4 monium (TEA), 0.5 CaCl , 2 MgCl , 5 EGTA, 5 HEPES, 5 fonic acid); 2 2 ATP-Mg, pH 7.2. Signals were acquired with a patch- clamp amplifier (Axopatch 200B; Axon Instruments, Fos- ATP-Mg, adenosine triphosphate-magnesium; ter City, CA, USA). The data were digitized with an A/D converter (Digidata 1200, Axon Instruments) and stored CNQX,6-cyano-7-nitroquinoxaline-2,3-dion; and analyzed with a personal computer using the pCLAMP data acquisition program (Version 8.2, Axon PMBA,3-[2'-Phosphonomethyl[1,1'-biphenyl]-3- Instruments). Lamina V neurons were viable for up to 24 yl]alanine; h in slices perfused with a pre-oxygenated Krebs solution. All the recordings described in this study were made D-APV, D(-)-2-Amino-5-phosphonopentanoic acid; within 12 h. Whole-cell patch-clamp recordings were sta- ble for up to 4 h. All of the neurons had membrane poten- Competing interests tials more negative than -50 mV. Unless otherwise noted, The author(s) declare that they have no competing inter- all the recordings in this study were performed in the pres- ests. ence of CNQX (20 µM) and APV (50 µM). Acknowledgements This work was supported by The General Insurance Association of Japan, Drug Applications The Japanese Health Sciences Foundation, and Grants-in-Aid for Scientific Drugs were dissolved in Krebs solution and then were Research from the Ministry of Education, Science, Sports and Culture of applied by perfusion via a three-way stopcock without any Japan to T.N. change in the perfusion rate or the temperature. The time necessary for the solution to flow from the stopcock to the References surface of the spinal cord slice was approximately 20 s. 1. Changeux JP, Edelstein SJ: Allosteric receptors after 30 years. The drugs used in this study were nicotine (Sigma-Aldrich, Neuron 1998, 21:959-980. 2. 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Molecular Pain – Springer Journals

Published: Sep 25, 2007

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