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Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro

Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal... Background: 5-hydroxytryptamine (5-HT) is one of the major neurotransmitters widely distributed in the CNS. Several 5-HT receptor subtypes have been identified in the spinal dorsal horn which act on both pre- and postsynaptic sites of excitatory and inhibitory neurons. However, the receptor subtypes and sites of actions as well as underlying mechanism are not clarified rigorously. Several electrophysiological studies have been performed to investigate the effects of 5-HT on excitatory transmission in substantia gelatinosa (SG) of the spinal cord. In the present study, to understand the effects of 5-HT on the inhibitory synaptic transmission and to identify receptor subtypes, the blind whole cell recordings were performed from SG neurons of rat spinal cord slices. Results: Bath applied 5-HT (50 μM) increased the frequency but not amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) in 58% of neurons, and both amplitude and frequency in 23% of neurons. The frequencies of GABAergic and glycinergic mIPSCs were both enhanced. TTX (0.5 μM) had no effect on the increasing frequency, while the enhancement of amplitude of IPSCs was eliminated. Evoked-IPSCs (eIPSCs) induced by focal stimulation near the recording neurons in the presence of CNQX and APV were enhanced in amplitude by 2+ 5-HT. In the presence of Ba (1 mM), a potassium channel blocker, 5-HT had no effect on both frequency and amplitude. A 5-HT receptor agonist, TCB-2 mimicked the 5-HT effect, and ketanserin, an antagonist of 5-HT 2A 2A receptor, inhibited the effect of 5-HT partially and TCB-2 almost completely. A 5-HT receptor agonist WAY 161503 2C mimicked the 5-HT effect and this effect was blocked by a 5-HT receptor antagonist, N-desmethylclozapine. The 2C amplitudes of sIPSCs were unaffected by 5-HT or 5-HT agonists. A 5-HT receptor agonist mCPBG enhanced 2A 2C 3 both amplitude and frequency of sIPSCs. This effect was blocked by a 5-HT receptor antagonist ICS-205,930. The perfusion of 5-HT receptor agonist had no effect on sIPSCs. 2B Conclusions: Our results demonstrated that 5-HT modulated the inhibitory transmission in SG by the activation of 5-HT and 5-HT receptors subtypes located predominantly at inhibitory interneuron terminals, and 5-HT 2A 2C 3 receptors located at inhibitory interneuron terminals and soma-dendrites, consequently enhanced both frequency and amplitude of IPSCs. Keywords: IPSC, 5-HT receptor, Substantia gelatinosa, Presynaptic release * Correspondence: yoshimum@kumamoto-hsu.ac.jp Graduate School of Health Sciences, Kumamoto Health Science University, Kumamoto 861-5598, Japan Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Full list of author information is available at the end of the article © 2012 Xie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Xie et al. Molecular Pain 2012, 8:58 Page 2 of 12 http://www.molecularpain.com/content/8/1/58 Background 5-HT activates different subtypes of receptors on the The descending inhibitory system composed mainly of inhibitory neurons in the spinal dorsal horn, resulting in the periaqueductal gray and consecutive reticular forma- the modulation of the nociceptive transmission. Previous tion is a structure modulating the nociceptive transmis- electrophysiological studies [13,14,46] show possible sion from periphery to the central nervous system mechanisms underlying the 5-HT effects in the superfi- (CNS). 5-HT is one of the main neurotransmitters of the cial dorsal horn. First, 5-HT directly activates postsynap- descending system [1,2] which terminates preferentially tic 5-HT receptor and induces an outward current, 1A on superficial laminae (Laminae I and II), especially the inhibiting excitatory neurons and subsequently produ- substantial gelatinosa (SG, lamina II). The SG is com- cing the analgesic effect [13]. Second, 5-HT induces an posed of interneurons and plays as a local circuit for inward current in the small population of SG neurons processing nociceptive transmission. The 5-HT system through the activation of postsynaptic 5-HT receptors originates from the rostral ventromedial medulla (RVM) on inhibitory interneurons [13,47]. Third, 5-HT inhibits including the nucleus raphe magnus, projects to the glutamate release from C afferent fibers by activating spinal cord through the dorsolateral funiculus and mod- presynaptic 5-HT -like receptors and shows an inhibi- 1A ulates the nociceptive transmission by interacting with tory effect on nociception [14]. In this study, not only 5-HT receptor subtypes. Exact mechanisms and receptor inhibitory but also excitatory effects on glutamatergic subtypes modulating nociceptive transmission are, how- transmission are reported, 5-HT transiently inhibits a ever, still obscure [3-6]. frequency of mEPSCs and then enhances. Fourth, 5-HT The receptor of 5-HT has been classified into seven acts on inhibitory interneurons and enhances the release distinct classes (5-HT -5-HT ), some of these are further of GABA and/or glycine. The receptor subtypes and sites 1 7 divided into subtypes, through pharmacological and mo- of actions as well as underlying mechanism are, however, lecular biological studies [7-9]. The 5-HT receptors are not clarified rigorously. In the present study, using the G protein coupled, with exception of the 5-HT receptor blind whole cell recording technique, the effects of 5-HT which is a ligand gated ion channel [10-12]. Some of the on the synaptic transmission were studied in SG to iden- subtypes are found in the spinal cord, existing at pre- tify the receptor subtypes responsible for the enhance- synaptic or postsynaptic loci of excitatory or inhibitory ment of the inhibitory transmitter release. SG neurons [13-17]. The autoradiographic studies show that 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT Results 1A 1B 1D 2A 2C 3 and 5-HT receptors are distributed in the superficial Effects of 5-HT on sIPSCs and mIPSCs in the spinal laminae of the spinal cord [6,18-23]. RT-PCR study substantia gelatinosa shows that in DRG all subtypes of 5-HT receptors could The membrane potential was hold at 0 mV to observe be detected except for 5-HT , 5-HT and 5-HT [24]. the effects of 5-HT on sIPSCs in SG. Perfusion of 5-HT 1E 2B 5B Although there is controversy regarding the contribution of (50 μM) for 60 s resulted in two different effects in the 5-HT receptor subtypes on the sensory transmission, there total of 168 neurons tested. In 58% (98/168) neurons, are at least four families of 5-HT receptors (5-HT ,5-HT , significant increase in a frequency of sIPSCs from 1 2 5-HT and 5-HT ) have been shown to modulate the 4.4 ± 1.8 Hz to 12.9 ± 2.6 Hz (paired t-test, P < 0.01) by 3 7 nociceptive transmission [25,26]. Behavioral examina- 5-HT was observed without a change in a amplitude of tions show that stimulation of RVM or intrathecal ad- 12.6 ± 1.1 to 13.1 ± 1.2 pA (P > 0.05, Figure 1A). The same ministration of agonists of 5-HT or 5-HT receptor results were depicted in inter-event intervals and cumula- 2 3 mediates antinociception on such as formalin test tive histograms of the sIPSCs amplitudes (Figure 1B, C). [27-30]; paw pressure test [31,32] and hot plate tests [33]. The averages of the relative frequency and amplitude These effects are blocked by intrathecal administration of were 285% and 104% of the control, respectively 5-HT or 5-HT receptor antagonist. There are also (Figure 1D). While in 23% (36/168) of neurons, 5-HT sig- 2 3 reports showing an pronociceptive responses of 5-HT nificantly increased both frequency and amplitude of [11,13,34]. sIPSCs from 5.2 ± 1.0 Hz and 11.2 ± 1.3 pA, respectively, γ - aminobutyric acid (GABA) and glycine are major to 16.7 ± 2.3 Hz (P < 0.001) and 20.4 ± 1.8 pA (n = 36, inhibitory neurotransmitters in the spinal cord [35-37]. P < 0.01 Figure 1E) and also on the cumulative distribu- Inhibitory synaptic transmission mediated by GABA and tions (Figure 1F, G). The averages of the relative frequency glycine plays an important role in the modulation and and amplitude were 324% and 182% of the control, re- integration of nociceptive sensory transmission [38-40]. spectively, (Figure 1H). GABA-like and glycine-like immunoreactive neurons Next, the effects of 5-HT on mIPSCs were examined. In exist in the spinal dorsal horn, with fibers and terminals the presence of 0.5 μM TTX, the effect of 50 μM5-HT densely distributed in the SG. GABA and glycine coex- simply increased the frequency but not the amplitude of isting neurons are also observed in the SG [41-45]. mIPSCs (Figure 2A). mIPSCs changed from 1.8 ± 0.3 Hz Xie et al. Molecular Pain 2012, 8:58 Page 3 of 12 http://www.molecularpain.com/content/8/1/58 AB E F 5-HT 5-HT Control Control 5-HT 5-HT 50 pA 50 pA 60 s 1.0 60 s 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 2.5 00.51.0 1.5 2.0 0 0.2 0.4 0.60.81.0 Inter-event Inter-event 50 pA 20 pA interval (s) interval (s) 200 ms 100 ms C D G H 4 4 *** 1.0 ** 1.0 0.8 3 0.8 3 0.6 0.6 ** 2 2 0.4 0.4 0.2 0.2 1 1 0.0 0.0 020 40 0 20 40 60 80 100 60 80 100 0 0 Amp. (pA) Amp. (pA) Freq. Amp. Freq. Amp. Figure 1 Effects of 5-HT on sIPSCs recorded at the holding potential of 0 mV. (A) Bath applied 5-HT (50 μM) increased a frequency but not amplitude of sIPSCs in 58% of SG neurons. Lower traces show sIPSCs before (left) and after (right) the application of 5-HT in the expanded time scale. (B) cumulative sIPSCs of the inter-event interval (**P < 0.01, K-S test) and amplitude (C, P > 0.05, K-S test) distributions recorded in control and in the presence of 5-HT. (D) shows the relative frequency and amplitude compared with the pre-application levels (n = 98). (E) 5-HT (50 μM) increased the frequency and amplitudes of sIPSCs in 23% of SG neurons. Lower typical traces of sIPSCs observed before, during application of 5-HT. (F) Cumulative probability of the inter-event interval and amplitude (G) of sIPSCs for the neuron in (E). Both distributions of amplitude and frequency during 5-HT were significantly different from the control (***P < 0.001 and **P < 0.01, respectively, K-S test). (H) The bar graph normalized results of the frequency and amplitude with the control (n = 36). in frequency and 14.2 ± 2.1 pA in amplitude to potassium channels; 5-HT blocked the potassium channels 5.2 ± 0.7 Hz (P < 0.01) and 14.7 ± 2.2 pA, respectively at the inhibitory interneuron terminals and in some cases (P > 0.05, n = 5). The same results were depicted in inter- soma-dendrites and subsequently enhance the release of event intervals of the mIPSCs and cumulative histograms inhibitory neurotransmitters. of the mIPSCs amplitudes, (Figure 2B, C). The averages of the frequency and amplitude of mIPSCs to the pre- Effects of 5-HT on GABAergic and Glycinergic mIPSCs application levels of 5-HT were 289% and 104%, respect- Spontaneously occurring mIPSCs were recorded from ively (Figure 2D). SG neurons in the presence of TTX (0.5 μM) and The effects of 5-HT on mIPSCs of SG neurons were CNQX (10 μM). GABAergic and glycinergic interneur- 2+ further studied in the presence of TTX (0.5 μM) and Ba ons are distributed extensively in the superficial dorsal (1 mM), a potassium channel blocker. The frequency of horn. The effects of 5-HT on the mIPSCs mediated by mIPSCs in SG neurons was increased markedly by super- GABA or glycine were studied. fusion of slices with 1 mM BaCl .50 μM5-HT did not In the presence of 2 μM strychnine, 5-HT (50 μM) sig- affect the frequency of mIPSCs further more in this condi- nificantly increased the remaining mIPSCs frequency tion. The IPSCs were from 21.9 ± 3.6 Hz and 22.5 ± 3.7 pA from 5.2 ± 1.7 Hz to 16.9 ± 2.1 Hz (n = 7, P < 0.01), with- to 21.7 ± 4.2 Hz (P > 0.05) and 22.4 ± 4.0 pA (P > 0.05, n = 7, out affecting of current amplitude from 13.4 ± 2.5 pA to Figure 3A), respectively. The same results were depicted in 15.3 ± 2.7 pA (n = 7, P > 0.05). These mIPSCs were inter-event intervals and cumulative histograms of the blocked by additional administrations of 10 μM bicucul- 2+ mIPSCs amplitudes (Figure 3B, C). In the presence of Ba , line, confirming that facilitated mIPSCs were GABAergic the averages of frequency and amplitude to the pre- (Figure 4A). The averages of the relative frequency application levels of 5-HT were 103% and 104%, respect- and amplitude were 328% and 114% of the control, ively (Figure 3D). These results suggested that the effects respectively (Figure 4C). Likewise, in the presence of 10 of 5-HT on inhibitory transmission were concerned with μM bicuculline, 5-HT also increased the remaining Cumulative prob. Relative value Cumulative prob. Cumulative prob. Relative value Cumulative prob. Xie et al. Molecular Pain 2012, 8:58 Page 4 of 12 http://www.molecularpain.com/content/8/1/58 5-HT TTX TTX TTX+5-HT 1.0 20 pA 0.8 60 s 0.6 0.4 0.2 0.0 20 pA 00.5 1.0 1.5 2.0 2.5 500 ms Inter-event interval (s) C D 1.0 ** 0.8 0.6 0.4 0.2 0.0 010 20 30 40 Amp. (pA) Freq. Amp. Figure 2 Effects of 5-HT on mIPSCs. (A) Effect of 5-HT (50 μM) on mIPSCs in the presence of TTX (0.5 μM). Frequency, but not amplitude of mIPSCs increased following 5-HT application. Lower typical traces of mIPSCs were taken before (control) and after the onset of 5-HT application in the presence of TTX. (B, C) Cumulative probability of the inter-event interval and amplitude of mIPSCs were obtained from the neuron (A). (D) The change in frequency during 5-HT was significantly different from control (**P < 0.01, K-S test), the amplitude during 5-HT was not different from the control (K-S test, P > 0.05, n = 5). 2+ 2+ 2+ A washout Ba Ba +5-HT 5-HT (Ba ) 50 pA 100 ms 2+ B Ba C D 2+ Ba +5-HT 2+ washout (Ba ) 5-HT 1.5 1.0 1.0 0.8 0.8 1 0.6 0.6 0.4 0.4 0.5 0.2 0.2 0.0 0.0 0 00.1 0.2 0.3 0.4 020 40 60 80 100 Freq. Amp. Amp. (pA) Inter-event interval (s) 2+ Figure 3 Effects of 5-HT on mIPSCs in the presence of Ba .(A) Typical traces of mIPSCs observed before (control), during, and after the 2+ application of 5-HT (50 μM) in the presence of Ba (1 mM). (B) Cumulative mIPSCs of the inter-event interval and (C) amplitude distributions compiled from the trace indicated in (A). Both the frequency and amplitude during 5-HT was not different from the control. (D) The bar graph shows the relative of the mIPSCs frequency and amplitude (paired t-test, P > 0.05, n = 7). Cumulative prob. Cumulative prob. Cumulative prob. Cumulative prob. Relative value Relative value Xie et al. Molecular Pain 2012, 8:58 Page 5 of 12 http://www.molecularpain.com/content/8/1/58 A B Bic+5-HT+St St+5-HT+Bic Bic Bic+5-HT St St+5-HT 20 pA 100 pA 100 ms 200 ms C D ** 4 3 0 0 Freq. Amp. Freq. Amp. E F G GABAergic eIPSC Glycinergic eIPSC 5-HT 1.5 5-HT Control Control 0.5 20 pA 20 pA GABAergic Glycinergic 20 ms 20 ms Figure 4 Effects of 5-HT on mIPSCs and eIPSCs of GABAergic and glycinergic interneuron. (A) 5-HT (50 μM) increased the frequency of GABAergic mIPSCs in the presence of strychnine (St, 2 μM). Subsequent application of bicuculline (Bic, 10 μM) completely eliminated the remaining mIPSCs. (B) 5-HT (50 μM) also increased the frequency of glycinergic mIPSCs in the presence of bicuculline (10 μM). The facilitated mIPSCs were eliminated by the application of strychnine (2 μM). The bar graph shows the relative frequency and amplitude for GABAergic (C, n = 7) and glycinergic (D, n = 5) mIPSCs, respectively. (paired t-test, ** P < 0.01 and * P < 0.05). (E) 5-HT (50 μM) increased the amplitude of GABAergic (n = 8) eIPSCs with the control in the presence of strychnine (2 μM) together with CNQX (10 μM) and APV (50 μM). (F) 5-HT (50 μM) also increased the amplitude of glycinergic (n = 7) eIPSCs with the control in the presence bicuculline (10 μM) together with CNQX and APV. (G) The bar graph shows the relative amplitude for GABAergic and glycinergic eIPSCs (paired t-test, * P < 0.05 and * P < 0.05). mIPSCs in frequency from 3.8 ± 1.1 Hz to 7.7 ± 1.3 Hz were 168% and 154% of the control, respectively (P < 0.05), without affecting of current amplitude from (Figure 4G). 29.3 ± 4.4 pA to 32.2 ± 4.7 pA (n = 5, P > 0.05). This effect was blocked by simultaneous perfusion of 2 μM strych- Identification of 5-HT receptor subtypes in enhancement nine and 10 μM bicuculline (Figure 4B). The averages of of the inhibitory transmitter release the relative frequency and amplitude were 203% and Selective 5-HT receptor agonists and antagonists were 110% of the control, respectively (Figure 4D). Thus, the tested to identify which subtypes of 5-HT receptors were results demonstrated that 5-HT increased the frequen- responsible for the enhancement of the release of inhibi- cies of both GABAergic and glycinegic mIPSCs. Next, tory neurotransmitters. 5-HT , 5-HT , 5-HT and 5- 1A 2 3 the effects of 5-HT on evoked GABAergic and glyciner- HT subtypes are shown to expressed in the superficial gic eIPSCs were studied in the presence of CNQX (10 dorsal horn neurons and terminals. 5-HT and 5-HT 1 7 μM) and APV (50 μM). The results showed that the receptors are coupled to Gi/o, suggesting inhibitory amplitudes of both GABAergic (n = 8) and glycinergic effects. In fact, the 5-HT-induced membrane hyperpolar- (n = 7) eIPSCs were enhanced. The amplitudes of eIPSCs ization or outward current in SG is mediated by 5-HT 1A were from 54.4 ± 12.4 pA and 91.4 ± 7.4 pA to 91.4 ± 14.5 [13]. Based on these observations, we evaluated the pA (P < 0.05) and 140.8 ± 9.8 pA (P < 0.05), respectively effects of agonists and antagonists for 5-HT , 5-HT and 2 3 (Figure 4E, F). The averages of the relative amplitude 5-HT receptor subtypes, in particular 5-HT , 5-HT 7 2A 2C Relative value of GABAergic mIPSCs Relative value of glycinergic mIPSCs Relative amp. of eIPSCs Xie et al. Molecular Pain 2012, 8:58 Page 6 of 12 http://www.molecularpain.com/content/8/1/58 are expressed in the superficial spinal dorsal horn and markedly reduced the effects of the 5-HT agonist. The 2A behavioral studies show that intrathecal administration averages of the relative frequency and amplitude were of 5-HT receptor agonist exhibit analgesic effects 104% and 102%, respectively, of their pre-application [48,49]. It is, however, still obscure which subtypes of levels (Figures 5C, D). The 5-HT receptor agonist, 2C 5-HT receptors are involved. We firstly tested each WAY 161503 (30 μM) also mimicked the 5-HT effect, agonist for all subtypes of 5-HT receptors, and then enhanced the frequency but not amplitude of sIPSCs corresponding antagonists were added to the perfusion from 5.6 ± 1.0 Hz to 13.7 ± 1.9 Hz (P < 0.01) and 17.9 ± 0.8 to confirm the responsible subtypes with certainty. The pA to 19.1 ± 0.7 pA (Figure 6A, n = 7, P > 0.05), respect- agonists for 5-HT receptor (TCB-2) and 5-HT (BW ively. The averages of the relative frequency and ampli- 2A 2B 723C86) as well as 5-HT receptors (WAY 161503) tude were 245% and 106% of control, respectively 2C were given to the neurons whose sIPSCs were enhanced (Figure 6C, D). In the presence of 5-HT receptor 2C by prior application of 5-HT (Figures 5, 6, 7). The 5- selective antagonist, N-desmethylclozapine (10 μM), HT receptor agonist, TCB-2 (10 μM) mimicked the perfusion of WAY 161503 had no effect (Figure 6B). 2A enhancing effects of 5-HT on sIPSCs, increasing the The frequency and amplitude were 107% (Figure 6C, frequency without affecting the amplitude (Figure 5A). P > 0.05) and 104% of control (Figure 6D, P > 0.05), re- The frequency and amplitude of sIPSCs were, respect- spectively. Figure 7A showed that bath applied 5-HT ively, from 7.7 ± 4.0 Hz to 18.6 ± 5.6 Hz (n = 7, P < 0.01) increased the frequency but not amplitudes of sIPSCs and 19.6 ± 3.2 pA to 20.1 ± 3.4 pA (P > 0.05). The from 4.6 ± 0.6 Hz to 11.4 ± 2.0 Hz (P < 0.01, 248% of con- averages of the relative frequency and amplitude were trol, n = 7), and 18.9 ± 1.3 pA to 20.2 ± 1.5 pA (P > 0.05, 242% and 103% of control, respectively (Figure 5C, D). 106% of control), while an agonist of 5-HT receptor, 2B Perfusion of 5-HT receptor selective antagonist, BW 723 C86 (10 μM) induced no change in both fre- 2A ketanserin (10 μM), itself had no detectable effect on quency (to 5.9 ± 0.8 Hz, 115% of control, P > 0.05, the frequency and amplitude of sIPSCs (Figure 5B), Figure 7C) and amplitude (to 19.8 ± 1.6 pA, 104% of AB TCB-2 Ketanserin TCB-2 50 pA 60 s 50 pA 100 ms C D 1.5 ** 0.5 0 0 TCB-2 Ketanserin Ketanserin TCB-2 Ketanserin Ketanserin +TCB-2 +TCB-2 Figure 5 Effects of 5-HT2A receptor agonist and antagonist on sIPSCs. (A) Representative recording showing the effects of 5-HT2A receptor agonist TCB-2 (10 μM) mimicked the enhancing effects of 5-HT (50 μM) on sIPSCs, increasing the frequency, but not amplitude. Lower traces show sIPSCs before (left) and after (right) the application of TCB-2 in the expanded time scale. (B) Shows 5-HT2A receptor antagonist ketanserin (10 μM) on the effects of TCB-2 on sIPSCs recorded from the same neuron (A). Lower traces shows sIPSCs taken before control (ketanserin) and the after TCB-2 application in the presence of ketanserin. The bar graph shows the relative frequency (C) and amplitude (D) for TCB-2, ketanserin and TCB-2 + ketanserin, respectively (paired t-test, ** P < 0.01, n = 7). Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 7 of 12 http://www.molecularpain.com/content/8/1/58 A B WAY 161503 WAY 161503 N-desmethylciozapine 50 pA 60 s 50 pA 50 ms CD ** 1.5 0.5 WAY N-desme- WAY 161503 + WAY N-desme- WAY 161503 + thylciozapine thylciozapine N-desme- 161503 N-desme- thylciozapine thylciozapine Figure 6 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the 2C application of WAY 161503 (30 μM). Lower traces show sIPSCs before (left) and after (right) the application of WAY 161503 in the expanded time scale. (B) In the same (A) neuron, sample trace of sIPSCs recorded before (N-desmethylciozapine 10 μM), during and after the application of WAY 161503 in the presence of N-desmethylciozapine. Lower traces show sIPSCs before (left) and after (right) the application of N-desmethylciozapine. The bar graph shows the relative frequency (C) and amplitude (D) for WAY 161503, N-desmethylciozapine and WAY 161503 + N-desmethylciozapine, respectively (paired t-test, ** P < 0.01, n = 7). control, n = 7, P > 0.05, Figure 7D) of sIPSCs recorded agonist induced no change in both frequency (to from the same neuron. The results demonstrated that 5.7 ± 0.6 Hz, 107% of control, P > 0.05) and amplitude (to 5-HT and 5-HT receptors might locate in the pre- 20.2 ± 1.4 pA, 105% of control, n = 7, P>0.05 data not 2A 2C synaptic terminals of inhibitory interneurons and respon- shown). sible for the analgesic role of 5-HT. The 5-HT receptors are also showed to exist in the superficial dorsal horn and Discussion correlates with the analgesic role of 5-HT. Thus we fur- In the present study, using blind whole cell recordings ther studied a role of 5-HT receptor agonist mCPBG from adult rat spinal cord slices, the effects of 5-HT on (30 μM). The agonist mimicked the effects of 5-HT to inhibitory transmission were studied in SG to identify increase both frequency and amplitude of sIPSCs from the receptor subtypes responsible for enhancement of 6.8 ± 2.9 Hz and 22.9 ± 1.5 pA to 19.2 ± 7.6 Hz (292% of release of GABA or glycine. The results showed that control, p < 0.01) and 37.3 ± 1.9 pA (165% of control, 5-HT modulated the sensory transmission in SG by the p < 0.05, n = 7), respectively (Figure 8A). Both effects activation of 5-HT , 5-HT and/or 5-HT receptors, were blocked completely by a 5-HT receptor antagonist 3 2A 2C 3 possibly by reducing the potassium conductance as has ICS-205,930 (10 μM) (Figure 8B). The averages of the fre- been reported in the variety of CNS neurons. quency and amplitude were 105% and 103% of their pre- application levels, respectively (Figure 8C, D). In the pres- Substantial numbers of 5-HT receptor subtypes pre- ence of TTX (0.5 μM), mCPBG enhanced the frequency dominate in the superficial laminae of the spinal dorsal but not amplitude (data not shown). The results suggested horn, including 5-HT , 5-HT , 5-HT and 5-HT 1A 2 3 7 that 5-HT receptors were expressed at both on presynap- [19,49-53]. Among 5-HT receptor subtypes, 5-HT and 3 1A tic terminals and soma-dendritic sites of inhibitory inter- 5-HT receptors are coupled to Gi/o, suggesting an inhibi- neurons. In addition, we also tested the effect of 5-HT / tory effects [13,14,54,55]. In contrast 5-HT receptor is 1A 2 5-HT receptors agonist (8-OH-DPAT 10 μM). The coupled to Gq/11, and 5-HT receptor is directly linked to 7 3 Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 8 of 12 http://www.molecularpain.com/content/8/1/58 A B 5-HT BW 723C86 50 pA 60 s 50 pA 100 ms C D ** 1.5 0.5 0 0 5-HT BW 723C86 5-HT BW 723C86 Figure 7 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the 2B application of 5-HT (50 μM). Lower traces show sIPSCs before (left) and after (right) the application of 5-HT in the expanded time scale. (B) shows the same neuron (A) sample trace of sIPSCs recorded before, during and after the application of 5-HT receptor agonist BW 723 C86 (10 μM). 2B Lower races show sIPSCs before (left) and after (right) application of BW 723 C86. The bar graph shows the relative frequency (C), and amplitude (D) for 5-HT and BW 723 C86, respectively (paired t-test, ** P < 0.01, n = 7). nonselective cationic channels, suggesting an excitatory mediated by GABA [22,59-61], but a few reports con- effects [13,47,49]. cern with glycine. The present our study showed that Consistent with this, an agonist for 5-HT and 5-HT , not only GABAergic but also glycinergic transmission 1A 7 8-OH-DPAT produced an outward current and did not were augmented by 5-HT. The inconsistent results show any significant effect on the inhibitory transmission might be correlated with the location of the GABAergic (data not shown), indicating the 5-HT and 5-HT and glycinergic neurons in the spinal dorsal horn. The 1A 7 receptors might not be responsible for the enhancement previous studies show that glycinergic transmission are of GABA and glycine releases in the superficial dorsal more prominent in laminae III-IV, whereas GABAergic horn. We showed that the activation of 5-HT receptors transmission seem to predominate in lamina II [45,62]. enhanced both frequency and amplitude, and TTX In contrast to GABAergic neurons, the cell bodies of eliminated the effect on the amplitude, indicating that glycinergic neurons are preferentially located in lamina the 5-HT receptors were expressed at both terminals III and deeper laminae [40,42]. The behavioral studies and soma-dendritic trees of inhibitory interneurons. It show that intrathecal administration of 5-HT receptors could not, however, exclude that 5-HT and 5-HT agonists generally induce the inhibitory effects on the 2A 2C receptors were also expressed on both presynaptic term- nociception, which can be blocked by the corresponding inals and soma dendrites; application of the agonists we antagonists [27,29-31,33,63]. Accordingly, these results used for 5-HT and 5-HT would not be high enough suggest that 5-HT and 5-HT receptors located in the 2A 2C 2 3 to initiate spike firing at somas, because of difference in spinal dorsal horn are involved in the antinociception. density of receptors or efficacy of the agonists. Pile of evidence shows that 5-HT exhibits both inhibi- GABA and glycine are primary inhibitory transmitters tory and excitatory effects with complex mechanisms, in the spinal dorsal horn and play a critical role in and mechanisms are not fully understood up to now as modulating nociceptive transmission [37,40,56-58]. At there are too many subtypes of 5-HT receptor. The the spinal level, plenty of reports demonstrate that the complexity in the effects of 5-HT might be concerned analgesic effects via 5-HT and 5-HT receptors are with 5-HT activating distinct subtypes of 5-HT receptor 2 3 Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 9 of 12 http://www.molecularpain.com/content/8/1/58 mCPBG mCPBG ICS-205,930 50 pA 60 s 50 pA 100 ms C D ** 2 1 mCPBG ICS-205,930 mCPBG + mCPBG ICS-205,930 mCPBG + ICS-205,930 ICS-205,930 Figure 8 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the application of mCPBG (30 μM). Lower traces show sIPSCs before (left) and after (right) the application of mCPBG in the expanded time scale. (B) In the same neuron from (A), sample trace of sIPSCs recorded before ICS-205,930 (10 μM), during and after the application of mCPBG in the presence of ICS-205,930. Lower traces show sIPSCs before (left) and after (right) the application of mCPBG in the presence of ICS-205,930 in the expanded time scale. The bar graph shows the relative frequency (C) and amplitude (D) for mCPBG, ICS-205,930 and mCPBG + ICS-205,930, respectively (paired t-test, ** P < 0.01 and *P < 0.05, n = 7). or affecting different types of neurons. Recent reports localized in the superficial spinal dorsal horn [17,18]. show that SG is composed at least of four types neurons, However, our electrophysiological studies did not show islet, small islet, vertical and radial neurons. The islet that the 5-HT receptor modulated the inhibitory trans- neurons are inhibitory interneurons and the small islet mission in SG. This result was confirmed by perfusion neurons include both excitatory and inhibitory inter- of 8-OH-OPAT (10 μM), a mixed 5-HT /5-HT recep- 1A 7 neurons [13,64,65]. It is reported that 5-HT depolarizes tors agonist had no effect on sIPSCs (Data not shown). a small population of SG neurons which is morphologic- ally classified into islet cell type [13]. Conclusions The previous electrophysiological studies show that 5-HT The present study demonstrated that 5-HT could en- exerts different postsynaptic effects on different types of SG hance the release of GABA and glycine by activating the neurons. Depolarization is induced in 6.8% neurons 5-HT , 5-HT and/or 5-HT receptors expressed on 2A 2C 3 mimicked by 5-HT receptor agonist mCPBG and inhibitory interneurons to inhibit sensory transmission. block by 5-HT receptor antagonist ICS-205,930 [13]. The 5-HT and 5-HT receptors predominantly exist 3 2A 2C In DRG neurons, the activation of 5-HT receptors also at presynaptic terminals, while the 5-HT receptor might induces a rapid depolarization. Also slice studies show that exist at both cell bodies and terminals. It is reported that 5-HT potentiates the GABA or glycine-induced Cl -current 5-HT receptors also located on soma-dendritic trees 2A in the rat sacral dorsal commissural nucleus and superficial [69]. But our result did not coincide with the report. spinal dorsal horn [60,66-68]. The contradiction might be concerned with the distribu- In recent studies, behavioral nociceptive tests show tion density of the receptors. that the 5-HT receptor play an antinociceptive role at In short, the present study provided more evidences to the level of the spinal cord [26,54,55]. Immunocyto- explain the various mechanisms of 5-HT on modulating chemical studies found that 5-HT receptors are nociceptive transmission in SG of the spinal dorsal horn. Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 10 of 12 http://www.molecularpain.com/content/8/1/58 Methods a personal computer using the pCLAMP data acquisition All the experimental procedures involving the use of ani- program (version 8.2, Molecular Devices, CA, USA). The mals were approved by the Ethics Committee on Animal recordings were made under the voltage-clamp mode at Experiments, Kyushu University, and were in accordance holding membrane potentials of 0 mV to isolate IPSCs. with the UK Animals (Scientific Procedures) Act 1986 At this potential the glutamate-mediated excitatory post- and associated guidelines. All efforts were made to synaptic currents (EPSCs) were negligible, because of a minimize the number of animals used for the studies. reversal potential of EPSCs. In fact, no remaining synap- tic currents were observed in the presence of antagonists Spinal cord slice preparation for GABA and glycine receptors [70]. Frequencies and Methods for obtaining adult rat spinal cord slices and amplitudes of spontaneous IPSCs (sIPSCs) and miniature for bind patch-clamp recordings from SG neurons were IPSCs (mIPSCs) in the presence of TTX (0.5 μM) were identical to those described elsewhere [70,71]2. Briefly, measured automatically with MiniAnalysis software male adult Sprague–Dawley rats (6–7 weeks) were (Synaptosoft, Decatur, GA). The frequency of IPSCs was deeply anesthetized with urethane (1.2g/Kg, ip), and further confirmed by their shapes with eyes. then thoracolumbar laminectomy was performed. The lumbosacral spinal cord was removed and placed in a Drug application preoxygenated cold Krebs solution containing (in mM): Drugs dissolved in Krebs solution were applied to the sur- NaCl 117, KCl 3.6, CaCl 2.5, MgCl 1.2, NaH PO 1.2, 2 2 2 4 face of the spinal cord by exchanging solutions via a NaHCO 25 and glucose 11 at 1-3°C. The pia-arachnoid three-way stopcock without any change in both perfusion membrane was removed after cutting all the ventral and rate and temperature. The time necessary for the solution dorsal roots. The spinal cord was mounted on a vibra- to flow from the stopcock to the surface of the spinal cord tome and then a 500 μm thick transverse or parasagittal was approximately 5 s and the solution in the recording slice was cut. The slice was placed on a nylon mesh in chamber was completely exchanged with a drug contain- the recording chamber and then perfused at a rate of ing solution within 15 s. The drugs used were 5-HT 15–20 ml/min with Krebs solution saturated with 95% hydrogen maleate (Sigma, St. Louis, MO, USA), tetrodo- O and 5% CO , at 36 ± 1°C. 2 2 toxin (TTX) (Wako, Osaka, Japan), 6-cyano-7-nitroqui- noxaline-2,3-dione (CNQX) (Sigma), DL-2-amino-5- Patch-clamp recordings from substantia gelatinosa phosphonovaleric acid (APV) (Sigma), BaCl (Sigma), neurons strychnine (Sigma), bicuculline (Sigma), 4-bromo-3,6- Blind whole-cell voltage-clamp recordings were made dimethoxybenzocyclobuten-1-yl) methylamine hydrobro- from SG neurons with patch pipettes filled with a solu- mide (TBC-2) (Tocris Cookson, Bristol, UK), ketanserin tion containing (mM): Cs SO 110, tetraethylammonium 2 4 (Sigma), α-methyl-5-(2-thienylmethoxy)-H-indole-3-etha- (TEA) 5, CaCl 0.5, MgCl 2, EGTA 5, HEPES 5 and 2 2 namine hydrochloride (BW723C86) (Sigma), 8,9-dchloro- ATP-Mg 5 (PH 7.2). Cs SO and TEA were main chemi- 2 4 2,3,4 4a-tetrahydro-1H-pyrazino[1,2-a] quinoxalin-5(6H)- cals which could inhibit the postsynaptic effects of 5-HT one hydrochloride (WAY161503) (Tocris), 8-chloro-11-(1- on the K channels, and enabled us to investigate the piperazinyl)-5H-dibenzo[b,e][1,4]diazipine (N-desmethyl- presynaptic effects on IPSCs by 5-HT. Recorded neurons clozpine) (Tocris), 1-(m-chlorophenyl)-biguanide (mCPBG) were identified as SG by their locations and morphologic (Sigma), 3-tropanylindole-3-carboxylate methiodide (ICS- features. SG was easily identifiably as a relatively translu- 205,930) (Sigma), (±)-8-hydroxy-2-dipropylaminotetralin cent band across the dorsal horn. In some instances, hydrobromide (8-OH-DPAT) (Sigma). neurobiotin was injected in the recorded neurons through electrodes. After completing experiments, the Statistical analysis recorded neuron were stained and their morphological All the data were expressed as the mean ± S.E.M. Statis- features were compared with those reported previously tical significance was determined as P < 0.05 using the [13,64,65,72]. Monosynaptic IPSCs were evoked in the paired t-test. Cumulative probability plots were con- presence of a non-NMDA-receptor antagonist CNQX, structed for sIPSC amplitude and frequency and were and an NMDA-receptor antagonist APV, at a frequency compared, under different experimental conditions, of 0.2 Hz by a focal monopolar silver electrode (50 μm using the Kolmogorov-Smirnov test. In all cases, n refers diameter), insulated except for the tip, located within 150 to the number of neurons studied. μm of the recorded neurons. Signals were acquired with a patch clamp amplifier (Axopatch 200B, Molecular Abbreviations 5-HT, 5-hydroxytryptamine; CNS, central nervous system; SG, substantia Devices, Union City, CA, USA). The data were digitized gelatinosa; sIPSCs, spontaneous inhibitory postsynaptic currents; with an analog-to-digital converter (digidata 1321A, Mo- eIPSCs, evoked IPSCs; RVM, rostral ventromedial medulla; GABA, lecular Devices, CA, USA), and stored and analyzed with γ-aminobutyric acid; TEA, tetraethylammonium; EPSCs, excitatory Xie et al. Molecular Pain 2012, 8:58 Page 11 of 12 http://www.molecularpain.com/content/8/1/58 postsynaptic currents; TTX, tetrodotoxin; CNQX, 6-cyano-7-nitroquinoxaline- 13. Abe K, Kato G, Katafuchi T, Tamae A, Furue H, Yoshimura M: Responses to 2,3-dione; APV, DL-2-amino-5-phosphonovaleric acid; TBC-2, 4-bromo-3,6- 5-HT in morphologically identified neurons in the rat substantia dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide; BW723C86, gelatinosa in vitro. Neuroscience 2009, 159:316–324. α-methyl-5-(2-thienylmethoxy)-H-indole-3-ethanamine hydrochloride; 14. 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Identification of 5-HT receptor subtypes enhancing inhibitory transmission in the rat spinal dorsal horn in vitro

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Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology; Neurosciences; Neurology
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Abstract

Background: 5-hydroxytryptamine (5-HT) is one of the major neurotransmitters widely distributed in the CNS. Several 5-HT receptor subtypes have been identified in the spinal dorsal horn which act on both pre- and postsynaptic sites of excitatory and inhibitory neurons. However, the receptor subtypes and sites of actions as well as underlying mechanism are not clarified rigorously. Several electrophysiological studies have been performed to investigate the effects of 5-HT on excitatory transmission in substantia gelatinosa (SG) of the spinal cord. In the present study, to understand the effects of 5-HT on the inhibitory synaptic transmission and to identify receptor subtypes, the blind whole cell recordings were performed from SG neurons of rat spinal cord slices. Results: Bath applied 5-HT (50 μM) increased the frequency but not amplitudes of spontaneous inhibitory postsynaptic currents (sIPSCs) in 58% of neurons, and both amplitude and frequency in 23% of neurons. The frequencies of GABAergic and glycinergic mIPSCs were both enhanced. TTX (0.5 μM) had no effect on the increasing frequency, while the enhancement of amplitude of IPSCs was eliminated. Evoked-IPSCs (eIPSCs) induced by focal stimulation near the recording neurons in the presence of CNQX and APV were enhanced in amplitude by 2+ 5-HT. In the presence of Ba (1 mM), a potassium channel blocker, 5-HT had no effect on both frequency and amplitude. A 5-HT receptor agonist, TCB-2 mimicked the 5-HT effect, and ketanserin, an antagonist of 5-HT 2A 2A receptor, inhibited the effect of 5-HT partially and TCB-2 almost completely. A 5-HT receptor agonist WAY 161503 2C mimicked the 5-HT effect and this effect was blocked by a 5-HT receptor antagonist, N-desmethylclozapine. The 2C amplitudes of sIPSCs were unaffected by 5-HT or 5-HT agonists. A 5-HT receptor agonist mCPBG enhanced 2A 2C 3 both amplitude and frequency of sIPSCs. This effect was blocked by a 5-HT receptor antagonist ICS-205,930. The perfusion of 5-HT receptor agonist had no effect on sIPSCs. 2B Conclusions: Our results demonstrated that 5-HT modulated the inhibitory transmission in SG by the activation of 5-HT and 5-HT receptors subtypes located predominantly at inhibitory interneuron terminals, and 5-HT 2A 2C 3 receptors located at inhibitory interneuron terminals and soma-dendrites, consequently enhanced both frequency and amplitude of IPSCs. Keywords: IPSC, 5-HT receptor, Substantia gelatinosa, Presynaptic release * Correspondence: yoshimum@kumamoto-hsu.ac.jp Graduate School of Health Sciences, Kumamoto Health Science University, Kumamoto 861-5598, Japan Department of Integrative Physiology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan Full list of author information is available at the end of the article © 2012 Xie et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Xie et al. Molecular Pain 2012, 8:58 Page 2 of 12 http://www.molecularpain.com/content/8/1/58 Background 5-HT activates different subtypes of receptors on the The descending inhibitory system composed mainly of inhibitory neurons in the spinal dorsal horn, resulting in the periaqueductal gray and consecutive reticular forma- the modulation of the nociceptive transmission. Previous tion is a structure modulating the nociceptive transmis- electrophysiological studies [13,14,46] show possible sion from periphery to the central nervous system mechanisms underlying the 5-HT effects in the superfi- (CNS). 5-HT is one of the main neurotransmitters of the cial dorsal horn. First, 5-HT directly activates postsynap- descending system [1,2] which terminates preferentially tic 5-HT receptor and induces an outward current, 1A on superficial laminae (Laminae I and II), especially the inhibiting excitatory neurons and subsequently produ- substantial gelatinosa (SG, lamina II). The SG is com- cing the analgesic effect [13]. Second, 5-HT induces an posed of interneurons and plays as a local circuit for inward current in the small population of SG neurons processing nociceptive transmission. The 5-HT system through the activation of postsynaptic 5-HT receptors originates from the rostral ventromedial medulla (RVM) on inhibitory interneurons [13,47]. Third, 5-HT inhibits including the nucleus raphe magnus, projects to the glutamate release from C afferent fibers by activating spinal cord through the dorsolateral funiculus and mod- presynaptic 5-HT -like receptors and shows an inhibi- 1A ulates the nociceptive transmission by interacting with tory effect on nociception [14]. In this study, not only 5-HT receptor subtypes. Exact mechanisms and receptor inhibitory but also excitatory effects on glutamatergic subtypes modulating nociceptive transmission are, how- transmission are reported, 5-HT transiently inhibits a ever, still obscure [3-6]. frequency of mEPSCs and then enhances. Fourth, 5-HT The receptor of 5-HT has been classified into seven acts on inhibitory interneurons and enhances the release distinct classes (5-HT -5-HT ), some of these are further of GABA and/or glycine. The receptor subtypes and sites 1 7 divided into subtypes, through pharmacological and mo- of actions as well as underlying mechanism are, however, lecular biological studies [7-9]. The 5-HT receptors are not clarified rigorously. In the present study, using the G protein coupled, with exception of the 5-HT receptor blind whole cell recording technique, the effects of 5-HT which is a ligand gated ion channel [10-12]. Some of the on the synaptic transmission were studied in SG to iden- subtypes are found in the spinal cord, existing at pre- tify the receptor subtypes responsible for the enhance- synaptic or postsynaptic loci of excitatory or inhibitory ment of the inhibitory transmitter release. SG neurons [13-17]. The autoradiographic studies show that 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT Results 1A 1B 1D 2A 2C 3 and 5-HT receptors are distributed in the superficial Effects of 5-HT on sIPSCs and mIPSCs in the spinal laminae of the spinal cord [6,18-23]. RT-PCR study substantia gelatinosa shows that in DRG all subtypes of 5-HT receptors could The membrane potential was hold at 0 mV to observe be detected except for 5-HT , 5-HT and 5-HT [24]. the effects of 5-HT on sIPSCs in SG. Perfusion of 5-HT 1E 2B 5B Although there is controversy regarding the contribution of (50 μM) for 60 s resulted in two different effects in the 5-HT receptor subtypes on the sensory transmission, there total of 168 neurons tested. In 58% (98/168) neurons, are at least four families of 5-HT receptors (5-HT ,5-HT , significant increase in a frequency of sIPSCs from 1 2 5-HT and 5-HT ) have been shown to modulate the 4.4 ± 1.8 Hz to 12.9 ± 2.6 Hz (paired t-test, P < 0.01) by 3 7 nociceptive transmission [25,26]. Behavioral examina- 5-HT was observed without a change in a amplitude of tions show that stimulation of RVM or intrathecal ad- 12.6 ± 1.1 to 13.1 ± 1.2 pA (P > 0.05, Figure 1A). The same ministration of agonists of 5-HT or 5-HT receptor results were depicted in inter-event intervals and cumula- 2 3 mediates antinociception on such as formalin test tive histograms of the sIPSCs amplitudes (Figure 1B, C). [27-30]; paw pressure test [31,32] and hot plate tests [33]. The averages of the relative frequency and amplitude These effects are blocked by intrathecal administration of were 285% and 104% of the control, respectively 5-HT or 5-HT receptor antagonist. There are also (Figure 1D). While in 23% (36/168) of neurons, 5-HT sig- 2 3 reports showing an pronociceptive responses of 5-HT nificantly increased both frequency and amplitude of [11,13,34]. sIPSCs from 5.2 ± 1.0 Hz and 11.2 ± 1.3 pA, respectively, γ - aminobutyric acid (GABA) and glycine are major to 16.7 ± 2.3 Hz (P < 0.001) and 20.4 ± 1.8 pA (n = 36, inhibitory neurotransmitters in the spinal cord [35-37]. P < 0.01 Figure 1E) and also on the cumulative distribu- Inhibitory synaptic transmission mediated by GABA and tions (Figure 1F, G). The averages of the relative frequency glycine plays an important role in the modulation and and amplitude were 324% and 182% of the control, re- integration of nociceptive sensory transmission [38-40]. spectively, (Figure 1H). GABA-like and glycine-like immunoreactive neurons Next, the effects of 5-HT on mIPSCs were examined. In exist in the spinal dorsal horn, with fibers and terminals the presence of 0.5 μM TTX, the effect of 50 μM5-HT densely distributed in the SG. GABA and glycine coex- simply increased the frequency but not the amplitude of isting neurons are also observed in the SG [41-45]. mIPSCs (Figure 2A). mIPSCs changed from 1.8 ± 0.3 Hz Xie et al. Molecular Pain 2012, 8:58 Page 3 of 12 http://www.molecularpain.com/content/8/1/58 AB E F 5-HT 5-HT Control Control 5-HT 5-HT 50 pA 50 pA 60 s 1.0 60 s 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 2.5 00.51.0 1.5 2.0 0 0.2 0.4 0.60.81.0 Inter-event Inter-event 50 pA 20 pA interval (s) interval (s) 200 ms 100 ms C D G H 4 4 *** 1.0 ** 1.0 0.8 3 0.8 3 0.6 0.6 ** 2 2 0.4 0.4 0.2 0.2 1 1 0.0 0.0 020 40 0 20 40 60 80 100 60 80 100 0 0 Amp. (pA) Amp. (pA) Freq. Amp. Freq. Amp. Figure 1 Effects of 5-HT on sIPSCs recorded at the holding potential of 0 mV. (A) Bath applied 5-HT (50 μM) increased a frequency but not amplitude of sIPSCs in 58% of SG neurons. Lower traces show sIPSCs before (left) and after (right) the application of 5-HT in the expanded time scale. (B) cumulative sIPSCs of the inter-event interval (**P < 0.01, K-S test) and amplitude (C, P > 0.05, K-S test) distributions recorded in control and in the presence of 5-HT. (D) shows the relative frequency and amplitude compared with the pre-application levels (n = 98). (E) 5-HT (50 μM) increased the frequency and amplitudes of sIPSCs in 23% of SG neurons. Lower typical traces of sIPSCs observed before, during application of 5-HT. (F) Cumulative probability of the inter-event interval and amplitude (G) of sIPSCs for the neuron in (E). Both distributions of amplitude and frequency during 5-HT were significantly different from the control (***P < 0.001 and **P < 0.01, respectively, K-S test). (H) The bar graph normalized results of the frequency and amplitude with the control (n = 36). in frequency and 14.2 ± 2.1 pA in amplitude to potassium channels; 5-HT blocked the potassium channels 5.2 ± 0.7 Hz (P < 0.01) and 14.7 ± 2.2 pA, respectively at the inhibitory interneuron terminals and in some cases (P > 0.05, n = 5). The same results were depicted in inter- soma-dendrites and subsequently enhance the release of event intervals of the mIPSCs and cumulative histograms inhibitory neurotransmitters. of the mIPSCs amplitudes, (Figure 2B, C). The averages of the frequency and amplitude of mIPSCs to the pre- Effects of 5-HT on GABAergic and Glycinergic mIPSCs application levels of 5-HT were 289% and 104%, respect- Spontaneously occurring mIPSCs were recorded from ively (Figure 2D). SG neurons in the presence of TTX (0.5 μM) and The effects of 5-HT on mIPSCs of SG neurons were CNQX (10 μM). GABAergic and glycinergic interneur- 2+ further studied in the presence of TTX (0.5 μM) and Ba ons are distributed extensively in the superficial dorsal (1 mM), a potassium channel blocker. The frequency of horn. The effects of 5-HT on the mIPSCs mediated by mIPSCs in SG neurons was increased markedly by super- GABA or glycine were studied. fusion of slices with 1 mM BaCl .50 μM5-HT did not In the presence of 2 μM strychnine, 5-HT (50 μM) sig- affect the frequency of mIPSCs further more in this condi- nificantly increased the remaining mIPSCs frequency tion. The IPSCs were from 21.9 ± 3.6 Hz and 22.5 ± 3.7 pA from 5.2 ± 1.7 Hz to 16.9 ± 2.1 Hz (n = 7, P < 0.01), with- to 21.7 ± 4.2 Hz (P > 0.05) and 22.4 ± 4.0 pA (P > 0.05, n = 7, out affecting of current amplitude from 13.4 ± 2.5 pA to Figure 3A), respectively. The same results were depicted in 15.3 ± 2.7 pA (n = 7, P > 0.05). These mIPSCs were inter-event intervals and cumulative histograms of the blocked by additional administrations of 10 μM bicucul- 2+ mIPSCs amplitudes (Figure 3B, C). In the presence of Ba , line, confirming that facilitated mIPSCs were GABAergic the averages of frequency and amplitude to the pre- (Figure 4A). The averages of the relative frequency application levels of 5-HT were 103% and 104%, respect- and amplitude were 328% and 114% of the control, ively (Figure 3D). These results suggested that the effects respectively (Figure 4C). Likewise, in the presence of 10 of 5-HT on inhibitory transmission were concerned with μM bicuculline, 5-HT also increased the remaining Cumulative prob. Relative value Cumulative prob. Cumulative prob. Relative value Cumulative prob. Xie et al. Molecular Pain 2012, 8:58 Page 4 of 12 http://www.molecularpain.com/content/8/1/58 5-HT TTX TTX TTX+5-HT 1.0 20 pA 0.8 60 s 0.6 0.4 0.2 0.0 20 pA 00.5 1.0 1.5 2.0 2.5 500 ms Inter-event interval (s) C D 1.0 ** 0.8 0.6 0.4 0.2 0.0 010 20 30 40 Amp. (pA) Freq. Amp. Figure 2 Effects of 5-HT on mIPSCs. (A) Effect of 5-HT (50 μM) on mIPSCs in the presence of TTX (0.5 μM). Frequency, but not amplitude of mIPSCs increased following 5-HT application. Lower typical traces of mIPSCs were taken before (control) and after the onset of 5-HT application in the presence of TTX. (B, C) Cumulative probability of the inter-event interval and amplitude of mIPSCs were obtained from the neuron (A). (D) The change in frequency during 5-HT was significantly different from control (**P < 0.01, K-S test), the amplitude during 5-HT was not different from the control (K-S test, P > 0.05, n = 5). 2+ 2+ 2+ A washout Ba Ba +5-HT 5-HT (Ba ) 50 pA 100 ms 2+ B Ba C D 2+ Ba +5-HT 2+ washout (Ba ) 5-HT 1.5 1.0 1.0 0.8 0.8 1 0.6 0.6 0.4 0.4 0.5 0.2 0.2 0.0 0.0 0 00.1 0.2 0.3 0.4 020 40 60 80 100 Freq. Amp. Amp. (pA) Inter-event interval (s) 2+ Figure 3 Effects of 5-HT on mIPSCs in the presence of Ba .(A) Typical traces of mIPSCs observed before (control), during, and after the 2+ application of 5-HT (50 μM) in the presence of Ba (1 mM). (B) Cumulative mIPSCs of the inter-event interval and (C) amplitude distributions compiled from the trace indicated in (A). Both the frequency and amplitude during 5-HT was not different from the control. (D) The bar graph shows the relative of the mIPSCs frequency and amplitude (paired t-test, P > 0.05, n = 7). Cumulative prob. Cumulative prob. Cumulative prob. Cumulative prob. Relative value Relative value Xie et al. Molecular Pain 2012, 8:58 Page 5 of 12 http://www.molecularpain.com/content/8/1/58 A B Bic+5-HT+St St+5-HT+Bic Bic Bic+5-HT St St+5-HT 20 pA 100 pA 100 ms 200 ms C D ** 4 3 0 0 Freq. Amp. Freq. Amp. E F G GABAergic eIPSC Glycinergic eIPSC 5-HT 1.5 5-HT Control Control 0.5 20 pA 20 pA GABAergic Glycinergic 20 ms 20 ms Figure 4 Effects of 5-HT on mIPSCs and eIPSCs of GABAergic and glycinergic interneuron. (A) 5-HT (50 μM) increased the frequency of GABAergic mIPSCs in the presence of strychnine (St, 2 μM). Subsequent application of bicuculline (Bic, 10 μM) completely eliminated the remaining mIPSCs. (B) 5-HT (50 μM) also increased the frequency of glycinergic mIPSCs in the presence of bicuculline (10 μM). The facilitated mIPSCs were eliminated by the application of strychnine (2 μM). The bar graph shows the relative frequency and amplitude for GABAergic (C, n = 7) and glycinergic (D, n = 5) mIPSCs, respectively. (paired t-test, ** P < 0.01 and * P < 0.05). (E) 5-HT (50 μM) increased the amplitude of GABAergic (n = 8) eIPSCs with the control in the presence of strychnine (2 μM) together with CNQX (10 μM) and APV (50 μM). (F) 5-HT (50 μM) also increased the amplitude of glycinergic (n = 7) eIPSCs with the control in the presence bicuculline (10 μM) together with CNQX and APV. (G) The bar graph shows the relative amplitude for GABAergic and glycinergic eIPSCs (paired t-test, * P < 0.05 and * P < 0.05). mIPSCs in frequency from 3.8 ± 1.1 Hz to 7.7 ± 1.3 Hz were 168% and 154% of the control, respectively (P < 0.05), without affecting of current amplitude from (Figure 4G). 29.3 ± 4.4 pA to 32.2 ± 4.7 pA (n = 5, P > 0.05). This effect was blocked by simultaneous perfusion of 2 μM strych- Identification of 5-HT receptor subtypes in enhancement nine and 10 μM bicuculline (Figure 4B). The averages of of the inhibitory transmitter release the relative frequency and amplitude were 203% and Selective 5-HT receptor agonists and antagonists were 110% of the control, respectively (Figure 4D). Thus, the tested to identify which subtypes of 5-HT receptors were results demonstrated that 5-HT increased the frequen- responsible for the enhancement of the release of inhibi- cies of both GABAergic and glycinegic mIPSCs. Next, tory neurotransmitters. 5-HT , 5-HT , 5-HT and 5- 1A 2 3 the effects of 5-HT on evoked GABAergic and glyciner- HT subtypes are shown to expressed in the superficial gic eIPSCs were studied in the presence of CNQX (10 dorsal horn neurons and terminals. 5-HT and 5-HT 1 7 μM) and APV (50 μM). The results showed that the receptors are coupled to Gi/o, suggesting inhibitory amplitudes of both GABAergic (n = 8) and glycinergic effects. In fact, the 5-HT-induced membrane hyperpolar- (n = 7) eIPSCs were enhanced. The amplitudes of eIPSCs ization or outward current in SG is mediated by 5-HT 1A were from 54.4 ± 12.4 pA and 91.4 ± 7.4 pA to 91.4 ± 14.5 [13]. Based on these observations, we evaluated the pA (P < 0.05) and 140.8 ± 9.8 pA (P < 0.05), respectively effects of agonists and antagonists for 5-HT , 5-HT and 2 3 (Figure 4E, F). The averages of the relative amplitude 5-HT receptor subtypes, in particular 5-HT , 5-HT 7 2A 2C Relative value of GABAergic mIPSCs Relative value of glycinergic mIPSCs Relative amp. of eIPSCs Xie et al. Molecular Pain 2012, 8:58 Page 6 of 12 http://www.molecularpain.com/content/8/1/58 are expressed in the superficial spinal dorsal horn and markedly reduced the effects of the 5-HT agonist. The 2A behavioral studies show that intrathecal administration averages of the relative frequency and amplitude were of 5-HT receptor agonist exhibit analgesic effects 104% and 102%, respectively, of their pre-application [48,49]. It is, however, still obscure which subtypes of levels (Figures 5C, D). The 5-HT receptor agonist, 2C 5-HT receptors are involved. We firstly tested each WAY 161503 (30 μM) also mimicked the 5-HT effect, agonist for all subtypes of 5-HT receptors, and then enhanced the frequency but not amplitude of sIPSCs corresponding antagonists were added to the perfusion from 5.6 ± 1.0 Hz to 13.7 ± 1.9 Hz (P < 0.01) and 17.9 ± 0.8 to confirm the responsible subtypes with certainty. The pA to 19.1 ± 0.7 pA (Figure 6A, n = 7, P > 0.05), respect- agonists for 5-HT receptor (TCB-2) and 5-HT (BW ively. The averages of the relative frequency and ampli- 2A 2B 723C86) as well as 5-HT receptors (WAY 161503) tude were 245% and 106% of control, respectively 2C were given to the neurons whose sIPSCs were enhanced (Figure 6C, D). In the presence of 5-HT receptor 2C by prior application of 5-HT (Figures 5, 6, 7). The 5- selective antagonist, N-desmethylclozapine (10 μM), HT receptor agonist, TCB-2 (10 μM) mimicked the perfusion of WAY 161503 had no effect (Figure 6B). 2A enhancing effects of 5-HT on sIPSCs, increasing the The frequency and amplitude were 107% (Figure 6C, frequency without affecting the amplitude (Figure 5A). P > 0.05) and 104% of control (Figure 6D, P > 0.05), re- The frequency and amplitude of sIPSCs were, respect- spectively. Figure 7A showed that bath applied 5-HT ively, from 7.7 ± 4.0 Hz to 18.6 ± 5.6 Hz (n = 7, P < 0.01) increased the frequency but not amplitudes of sIPSCs and 19.6 ± 3.2 pA to 20.1 ± 3.4 pA (P > 0.05). The from 4.6 ± 0.6 Hz to 11.4 ± 2.0 Hz (P < 0.01, 248% of con- averages of the relative frequency and amplitude were trol, n = 7), and 18.9 ± 1.3 pA to 20.2 ± 1.5 pA (P > 0.05, 242% and 103% of control, respectively (Figure 5C, D). 106% of control), while an agonist of 5-HT receptor, 2B Perfusion of 5-HT receptor selective antagonist, BW 723 C86 (10 μM) induced no change in both fre- 2A ketanserin (10 μM), itself had no detectable effect on quency (to 5.9 ± 0.8 Hz, 115% of control, P > 0.05, the frequency and amplitude of sIPSCs (Figure 5B), Figure 7C) and amplitude (to 19.8 ± 1.6 pA, 104% of AB TCB-2 Ketanserin TCB-2 50 pA 60 s 50 pA 100 ms C D 1.5 ** 0.5 0 0 TCB-2 Ketanserin Ketanserin TCB-2 Ketanserin Ketanserin +TCB-2 +TCB-2 Figure 5 Effects of 5-HT2A receptor agonist and antagonist on sIPSCs. (A) Representative recording showing the effects of 5-HT2A receptor agonist TCB-2 (10 μM) mimicked the enhancing effects of 5-HT (50 μM) on sIPSCs, increasing the frequency, but not amplitude. Lower traces show sIPSCs before (left) and after (right) the application of TCB-2 in the expanded time scale. (B) Shows 5-HT2A receptor antagonist ketanserin (10 μM) on the effects of TCB-2 on sIPSCs recorded from the same neuron (A). Lower traces shows sIPSCs taken before control (ketanserin) and the after TCB-2 application in the presence of ketanserin. The bar graph shows the relative frequency (C) and amplitude (D) for TCB-2, ketanserin and TCB-2 + ketanserin, respectively (paired t-test, ** P < 0.01, n = 7). Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 7 of 12 http://www.molecularpain.com/content/8/1/58 A B WAY 161503 WAY 161503 N-desmethylciozapine 50 pA 60 s 50 pA 50 ms CD ** 1.5 0.5 WAY N-desme- WAY 161503 + WAY N-desme- WAY 161503 + thylciozapine thylciozapine N-desme- 161503 N-desme- thylciozapine thylciozapine Figure 6 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the 2C application of WAY 161503 (30 μM). Lower traces show sIPSCs before (left) and after (right) the application of WAY 161503 in the expanded time scale. (B) In the same (A) neuron, sample trace of sIPSCs recorded before (N-desmethylciozapine 10 μM), during and after the application of WAY 161503 in the presence of N-desmethylciozapine. Lower traces show sIPSCs before (left) and after (right) the application of N-desmethylciozapine. The bar graph shows the relative frequency (C) and amplitude (D) for WAY 161503, N-desmethylciozapine and WAY 161503 + N-desmethylciozapine, respectively (paired t-test, ** P < 0.01, n = 7). control, n = 7, P > 0.05, Figure 7D) of sIPSCs recorded agonist induced no change in both frequency (to from the same neuron. The results demonstrated that 5.7 ± 0.6 Hz, 107% of control, P > 0.05) and amplitude (to 5-HT and 5-HT receptors might locate in the pre- 20.2 ± 1.4 pA, 105% of control, n = 7, P>0.05 data not 2A 2C synaptic terminals of inhibitory interneurons and respon- shown). sible for the analgesic role of 5-HT. The 5-HT receptors are also showed to exist in the superficial dorsal horn and Discussion correlates with the analgesic role of 5-HT. Thus we fur- In the present study, using blind whole cell recordings ther studied a role of 5-HT receptor agonist mCPBG from adult rat spinal cord slices, the effects of 5-HT on (30 μM). The agonist mimicked the effects of 5-HT to inhibitory transmission were studied in SG to identify increase both frequency and amplitude of sIPSCs from the receptor subtypes responsible for enhancement of 6.8 ± 2.9 Hz and 22.9 ± 1.5 pA to 19.2 ± 7.6 Hz (292% of release of GABA or glycine. The results showed that control, p < 0.01) and 37.3 ± 1.9 pA (165% of control, 5-HT modulated the sensory transmission in SG by the p < 0.05, n = 7), respectively (Figure 8A). Both effects activation of 5-HT , 5-HT and/or 5-HT receptors, were blocked completely by a 5-HT receptor antagonist 3 2A 2C 3 possibly by reducing the potassium conductance as has ICS-205,930 (10 μM) (Figure 8B). The averages of the fre- been reported in the variety of CNS neurons. quency and amplitude were 105% and 103% of their pre- application levels, respectively (Figure 8C, D). In the pres- Substantial numbers of 5-HT receptor subtypes pre- ence of TTX (0.5 μM), mCPBG enhanced the frequency dominate in the superficial laminae of the spinal dorsal but not amplitude (data not shown). The results suggested horn, including 5-HT , 5-HT , 5-HT and 5-HT 1A 2 3 7 that 5-HT receptors were expressed at both on presynap- [19,49-53]. Among 5-HT receptor subtypes, 5-HT and 3 1A tic terminals and soma-dendritic sites of inhibitory inter- 5-HT receptors are coupled to Gi/o, suggesting an inhibi- neurons. In addition, we also tested the effect of 5-HT / tory effects [13,14,54,55]. In contrast 5-HT receptor is 1A 2 5-HT receptors agonist (8-OH-DPAT 10 μM). The coupled to Gq/11, and 5-HT receptor is directly linked to 7 3 Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 8 of 12 http://www.molecularpain.com/content/8/1/58 A B 5-HT BW 723C86 50 pA 60 s 50 pA 100 ms C D ** 1.5 0.5 0 0 5-HT BW 723C86 5-HT BW 723C86 Figure 7 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the 2B application of 5-HT (50 μM). Lower traces show sIPSCs before (left) and after (right) the application of 5-HT in the expanded time scale. (B) shows the same neuron (A) sample trace of sIPSCs recorded before, during and after the application of 5-HT receptor agonist BW 723 C86 (10 μM). 2B Lower races show sIPSCs before (left) and after (right) application of BW 723 C86. The bar graph shows the relative frequency (C), and amplitude (D) for 5-HT and BW 723 C86, respectively (paired t-test, ** P < 0.01, n = 7). nonselective cationic channels, suggesting an excitatory mediated by GABA [22,59-61], but a few reports con- effects [13,47,49]. cern with glycine. The present our study showed that Consistent with this, an agonist for 5-HT and 5-HT , not only GABAergic but also glycinergic transmission 1A 7 8-OH-DPAT produced an outward current and did not were augmented by 5-HT. The inconsistent results show any significant effect on the inhibitory transmission might be correlated with the location of the GABAergic (data not shown), indicating the 5-HT and 5-HT and glycinergic neurons in the spinal dorsal horn. The 1A 7 receptors might not be responsible for the enhancement previous studies show that glycinergic transmission are of GABA and glycine releases in the superficial dorsal more prominent in laminae III-IV, whereas GABAergic horn. We showed that the activation of 5-HT receptors transmission seem to predominate in lamina II [45,62]. enhanced both frequency and amplitude, and TTX In contrast to GABAergic neurons, the cell bodies of eliminated the effect on the amplitude, indicating that glycinergic neurons are preferentially located in lamina the 5-HT receptors were expressed at both terminals III and deeper laminae [40,42]. The behavioral studies and soma-dendritic trees of inhibitory interneurons. It show that intrathecal administration of 5-HT receptors could not, however, exclude that 5-HT and 5-HT agonists generally induce the inhibitory effects on the 2A 2C receptors were also expressed on both presynaptic term- nociception, which can be blocked by the corresponding inals and soma dendrites; application of the agonists we antagonists [27,29-31,33,63]. Accordingly, these results used for 5-HT and 5-HT would not be high enough suggest that 5-HT and 5-HT receptors located in the 2A 2C 2 3 to initiate spike firing at somas, because of difference in spinal dorsal horn are involved in the antinociception. density of receptors or efficacy of the agonists. Pile of evidence shows that 5-HT exhibits both inhibi- GABA and glycine are primary inhibitory transmitters tory and excitatory effects with complex mechanisms, in the spinal dorsal horn and play a critical role in and mechanisms are not fully understood up to now as modulating nociceptive transmission [37,40,56-58]. At there are too many subtypes of 5-HT receptor. The the spinal level, plenty of reports demonstrate that the complexity in the effects of 5-HT might be concerned analgesic effects via 5-HT and 5-HT receptors are with 5-HT activating distinct subtypes of 5-HT receptor 2 3 Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 9 of 12 http://www.molecularpain.com/content/8/1/58 mCPBG mCPBG ICS-205,930 50 pA 60 s 50 pA 100 ms C D ** 2 1 mCPBG ICS-205,930 mCPBG + mCPBG ICS-205,930 mCPBG + ICS-205,930 ICS-205,930 Figure 8 Effects of 5-HT receptor agonist and antagonist on sIPSCs. (A) Sample trace of sIPSCs recorded before, during and after the application of mCPBG (30 μM). Lower traces show sIPSCs before (left) and after (right) the application of mCPBG in the expanded time scale. (B) In the same neuron from (A), sample trace of sIPSCs recorded before ICS-205,930 (10 μM), during and after the application of mCPBG in the presence of ICS-205,930. Lower traces show sIPSCs before (left) and after (right) the application of mCPBG in the presence of ICS-205,930 in the expanded time scale. The bar graph shows the relative frequency (C) and amplitude (D) for mCPBG, ICS-205,930 and mCPBG + ICS-205,930, respectively (paired t-test, ** P < 0.01 and *P < 0.05, n = 7). or affecting different types of neurons. Recent reports localized in the superficial spinal dorsal horn [17,18]. show that SG is composed at least of four types neurons, However, our electrophysiological studies did not show islet, small islet, vertical and radial neurons. The islet that the 5-HT receptor modulated the inhibitory trans- neurons are inhibitory interneurons and the small islet mission in SG. This result was confirmed by perfusion neurons include both excitatory and inhibitory inter- of 8-OH-OPAT (10 μM), a mixed 5-HT /5-HT recep- 1A 7 neurons [13,64,65]. It is reported that 5-HT depolarizes tors agonist had no effect on sIPSCs (Data not shown). a small population of SG neurons which is morphologic- ally classified into islet cell type [13]. Conclusions The previous electrophysiological studies show that 5-HT The present study demonstrated that 5-HT could en- exerts different postsynaptic effects on different types of SG hance the release of GABA and glycine by activating the neurons. Depolarization is induced in 6.8% neurons 5-HT , 5-HT and/or 5-HT receptors expressed on 2A 2C 3 mimicked by 5-HT receptor agonist mCPBG and inhibitory interneurons to inhibit sensory transmission. block by 5-HT receptor antagonist ICS-205,930 [13]. The 5-HT and 5-HT receptors predominantly exist 3 2A 2C In DRG neurons, the activation of 5-HT receptors also at presynaptic terminals, while the 5-HT receptor might induces a rapid depolarization. Also slice studies show that exist at both cell bodies and terminals. It is reported that 5-HT potentiates the GABA or glycine-induced Cl -current 5-HT receptors also located on soma-dendritic trees 2A in the rat sacral dorsal commissural nucleus and superficial [69]. But our result did not coincide with the report. spinal dorsal horn [60,66-68]. The contradiction might be concerned with the distribu- In recent studies, behavioral nociceptive tests show tion density of the receptors. that the 5-HT receptor play an antinociceptive role at In short, the present study provided more evidences to the level of the spinal cord [26,54,55]. Immunocyto- explain the various mechanisms of 5-HT on modulating chemical studies found that 5-HT receptors are nociceptive transmission in SG of the spinal dorsal horn. Relative freq. Relative amp. Xie et al. Molecular Pain 2012, 8:58 Page 10 of 12 http://www.molecularpain.com/content/8/1/58 Methods a personal computer using the pCLAMP data acquisition All the experimental procedures involving the use of ani- program (version 8.2, Molecular Devices, CA, USA). The mals were approved by the Ethics Committee on Animal recordings were made under the voltage-clamp mode at Experiments, Kyushu University, and were in accordance holding membrane potentials of 0 mV to isolate IPSCs. with the UK Animals (Scientific Procedures) Act 1986 At this potential the glutamate-mediated excitatory post- and associated guidelines. All efforts were made to synaptic currents (EPSCs) were negligible, because of a minimize the number of animals used for the studies. reversal potential of EPSCs. In fact, no remaining synap- tic currents were observed in the presence of antagonists Spinal cord slice preparation for GABA and glycine receptors [70]. Frequencies and Methods for obtaining adult rat spinal cord slices and amplitudes of spontaneous IPSCs (sIPSCs) and miniature for bind patch-clamp recordings from SG neurons were IPSCs (mIPSCs) in the presence of TTX (0.5 μM) were identical to those described elsewhere [70,71]2. Briefly, measured automatically with MiniAnalysis software male adult Sprague–Dawley rats (6–7 weeks) were (Synaptosoft, Decatur, GA). The frequency of IPSCs was deeply anesthetized with urethane (1.2g/Kg, ip), and further confirmed by their shapes with eyes. then thoracolumbar laminectomy was performed. The lumbosacral spinal cord was removed and placed in a Drug application preoxygenated cold Krebs solution containing (in mM): Drugs dissolved in Krebs solution were applied to the sur- NaCl 117, KCl 3.6, CaCl 2.5, MgCl 1.2, NaH PO 1.2, 2 2 2 4 face of the spinal cord by exchanging solutions via a NaHCO 25 and glucose 11 at 1-3°C. The pia-arachnoid three-way stopcock without any change in both perfusion membrane was removed after cutting all the ventral and rate and temperature. The time necessary for the solution dorsal roots. The spinal cord was mounted on a vibra- to flow from the stopcock to the surface of the spinal cord tome and then a 500 μm thick transverse or parasagittal was approximately 5 s and the solution in the recording slice was cut. The slice was placed on a nylon mesh in chamber was completely exchanged with a drug contain- the recording chamber and then perfused at a rate of ing solution within 15 s. The drugs used were 5-HT 15–20 ml/min with Krebs solution saturated with 95% hydrogen maleate (Sigma, St. Louis, MO, USA), tetrodo- O and 5% CO , at 36 ± 1°C. 2 2 toxin (TTX) (Wako, Osaka, Japan), 6-cyano-7-nitroqui- noxaline-2,3-dione (CNQX) (Sigma), DL-2-amino-5- Patch-clamp recordings from substantia gelatinosa phosphonovaleric acid (APV) (Sigma), BaCl (Sigma), neurons strychnine (Sigma), bicuculline (Sigma), 4-bromo-3,6- Blind whole-cell voltage-clamp recordings were made dimethoxybenzocyclobuten-1-yl) methylamine hydrobro- from SG neurons with patch pipettes filled with a solu- mide (TBC-2) (Tocris Cookson, Bristol, UK), ketanserin tion containing (mM): Cs SO 110, tetraethylammonium 2 4 (Sigma), α-methyl-5-(2-thienylmethoxy)-H-indole-3-etha- (TEA) 5, CaCl 0.5, MgCl 2, EGTA 5, HEPES 5 and 2 2 namine hydrochloride (BW723C86) (Sigma), 8,9-dchloro- ATP-Mg 5 (PH 7.2). Cs SO and TEA were main chemi- 2 4 2,3,4 4a-tetrahydro-1H-pyrazino[1,2-a] quinoxalin-5(6H)- cals which could inhibit the postsynaptic effects of 5-HT one hydrochloride (WAY161503) (Tocris), 8-chloro-11-(1- on the K channels, and enabled us to investigate the piperazinyl)-5H-dibenzo[b,e][1,4]diazipine (N-desmethyl- presynaptic effects on IPSCs by 5-HT. Recorded neurons clozpine) (Tocris), 1-(m-chlorophenyl)-biguanide (mCPBG) were identified as SG by their locations and morphologic (Sigma), 3-tropanylindole-3-carboxylate methiodide (ICS- features. SG was easily identifiably as a relatively translu- 205,930) (Sigma), (±)-8-hydroxy-2-dipropylaminotetralin cent band across the dorsal horn. In some instances, hydrobromide (8-OH-DPAT) (Sigma). neurobiotin was injected in the recorded neurons through electrodes. After completing experiments, the Statistical analysis recorded neuron were stained and their morphological All the data were expressed as the mean ± S.E.M. Statis- features were compared with those reported previously tical significance was determined as P < 0.05 using the [13,64,65,72]. Monosynaptic IPSCs were evoked in the paired t-test. Cumulative probability plots were con- presence of a non-NMDA-receptor antagonist CNQX, structed for sIPSC amplitude and frequency and were and an NMDA-receptor antagonist APV, at a frequency compared, under different experimental conditions, of 0.2 Hz by a focal monopolar silver electrode (50 μm using the Kolmogorov-Smirnov test. In all cases, n refers diameter), insulated except for the tip, located within 150 to the number of neurons studied. μm of the recorded neurons. Signals were acquired with a patch clamp amplifier (Axopatch 200B, Molecular Abbreviations 5-HT, 5-hydroxytryptamine; CNS, central nervous system; SG, substantia Devices, Union City, CA, USA). The data were digitized gelatinosa; sIPSCs, spontaneous inhibitory postsynaptic currents; with an analog-to-digital converter (digidata 1321A, Mo- eIPSCs, evoked IPSCs; RVM, rostral ventromedial medulla; GABA, lecular Devices, CA, USA), and stored and analyzed with γ-aminobutyric acid; TEA, tetraethylammonium; EPSCs, excitatory Xie et al. Molecular Pain 2012, 8:58 Page 11 of 12 http://www.molecularpain.com/content/8/1/58 postsynaptic currents; TTX, tetrodotoxin; CNQX, 6-cyano-7-nitroquinoxaline- 13. Abe K, Kato G, Katafuchi T, Tamae A, Furue H, Yoshimura M: Responses to 2,3-dione; APV, DL-2-amino-5-phosphonovaleric acid; TBC-2, 4-bromo-3,6- 5-HT in morphologically identified neurons in the rat substantia dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide; BW723C86, gelatinosa in vitro. Neuroscience 2009, 159:316–324. α-methyl-5-(2-thienylmethoxy)-H-indole-3-ethanamine hydrochloride; 14. 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Molecular Pain – Springer Journals

Published: Aug 20, 2012

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