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Role of serotonin1A receptors on the modulation of rat spinal mono-synaptic reflexes in vitro
Neuroscience Letters 334 (2002) 41–44 www.elsevier.com/locate/neulet
Role of serotonin1A receptors on the modulation of rat spinal mono-synaptic reflexes in vitro Gonzalo Hedo, Mar Ajubita, Jose A. Lopez-Garcia* Department of Physiology, Edificio de Medicina, University of Alcala, 28871 Madrid, Spain Received 25 July 2002; received in revised form 10 September 2002; accepted 12 September 2002
Abstract The present study aimed at determining the role of the serotonin1A (5-HT1A) receptor subtype on the modulation of the mono-synaptic reflex (MSR) elicited by dorsal root stimulation and recorded from ventral roots in the hemisected spinal cord obtained from rat pups. Serotonin and 5-carboxamidotryptamine (5-CT) depressed both the MSR and the cumulative depolarisation (CD) produced by repetitive dorsal root stimulation, whereas the specific 5-HT1A receptor agonist (R)-(1)-8hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) applied at 1 mM showed a selective depressant effect on the MSR. Superfusion of the 5-HT1A receptor antagonist (1)-N-tert-butyl-3(4(2-methoxyphenyl)-piperazin-1-yl)-2-phenylprpanamide ((1)WAY 100135) produced a complete blockade of 8-OH-DPAT effects. In addition (1)WAY 100135 blocked partially the effects of 5-HT and 5-CT on the MSR but not the effects of these compounds on the CD. The results are consistent with an intervention of 5-HT1A receptors in the modulation of non-nociceptive reflexes but do not support a prominent role for this receptor in the modulation of nociceptive reflexes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin; Serotonin1A receptors; Mono-synaptic reflex; Nociceptive reflex; Spinal pathways
Extensive experimental evidence obtained from in vivo and in vitro experiments has revealed that serotonin (5-HT) exerts complex modulatory actions on spinal segmental reflexes mediated by A- and C-fibres (see review in Ref. [15]). The mono-synaptic reflex (MSR) recorded from ventral roots in response to dorsal root stimulation has been intensively studied in the hemisected cord of rat pups. This reflex is depressed by bath-applied 5-HT and a range of 5-HT receptor agonists [5,9,12]. In addition, conditioning stimuli delivered to descending tracks at the thoracic level produce depression of the MSR which is mediated by a 5-HT receptor sensitive to ketanserin [4,18], a classic 5-HT2 receptor antagonist. This receptor was first thought to be an atypical 5-HT2 receptor, but further pharmacological analysis concluded that it was more likely a 5-HT1-like receptor similar to the 5-HT1Da [12,17]. Interestingly ketanserin does not block the depressant effect of bath applied 5-HT suggesting that a different receptor mediates the effects of exogenous 5HT [6]. Crick and colleagues [5] found that a combination * Corresponding author. Tel.: 134-91-885-5106; fax: 134-91885-4807 E-mail address: [email protected] (J.A. Lopez-Garcia).
of antagonists directed against 5-HT1, 5-HT2 and 5-HT3 receptors failed to block the effects of bath applied 5-HT and suggested that a novel receptor or a combination of receptors mediate the depressant effect of 5-HT. Later on Clarke and colleagues [2] using an in vivo preparation of adult rabbit and more selective antagonists concluded that spinal 5-HT1A receptors mediate tonic depression of reflex responses recorded from gastrocnemius motoneurones in response to electrical stimulation of the sural nerve. Strong depressant effects of specific 5-HT1A agonists have been reported in rats [1,5] but none of the antagonists tested has proven useful. In vitro experiments using the hemisected cord preparation from rat pups have shown that spiperone, a non-specific 5-HT1A receptor antagonist, blocks only partially the effect of (R)-(1)-8hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on the MSR and fails to block the effect of 5-HT [4,5]. Intravenous spiroxantrine and the more selective 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl) piperazine (NAN-190) administered to adult rats in vivo have also failed to block inhibitory responses to 8-OHDPAT in spinalised rats [10]. We have shown recently that 8-OH-DPAT produces a strong, concentration-dependent and selective depression
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 06 4- 9
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G. Hedo et al. / Neuroscience Letters 334 (2002) 41–44
Fig. 1. The graphs in (A–C) show the effects of 8-OH-DPAT (1 mM), 5-HT (50 mM) and 5-CT (0.1 mM) on the amplitude of the mono-synaptic reflex in the absence (filled squares) and presence of (1)WAY 100135 (1 m, hollow triangles). The black bar signal the time of superfusion of the agonist, the numbers in parenthesis are the numbers of observations and the asterisks denote significant differences between control and antagonist conditions (two way ANOVA, Bonferroni post-hoc tests P # 0:05). Graphs in (D–F) show the corresponding effects of the agonists on the integrated area of the cumulative depolarisation in the absence (filled squares) and presence of (1)WAY 100135 (hollow triangles) obtained from the same pool of experiments (no significant differences detected).
of the MSR whereas 5-HT and several other agonists depress the responses to repetitive nociceptive stimulation as well as the MSR [9]. In order to test whether this effect of 8-OH-DPAT is mediated by 5-HT1A receptors, here we test the effects of a selective 5-HT1A antagonist, (1)-N-tertbutyl-3(4(2-methoxyphenyl)-piperazin-1-yl)-2-phenylprpanamide ((1)WAY 100135), on the depressant actions of 8OH-DPAT, 5-HT and 5-CT. The present experiments were performed under similar experimental conditions to those described in [9]. Hemisected spinal cords were prepared from 8–12 days old Wistar rats under urethane anaesthesia (2 g/kg, 1.p.). A dorsal laminectomy was performed and the cord rapidly excised and placed in ice-cooled artificial cerebrospinal fluid (ACSF) for hemisection. A hemisected cord was placed in a recording chamber and continuously superfused with oxygenated (95% O2; 5% CO2) ACSF containing (in mM): NaCl (128); KCl (1.9); KH2PO4 (1.2); MgSO4 (1.3); CaCl2 (2.4); NaHCO3 (26); glucose (10) (pH 7.4). A lumbar dorsal root and the corresponding ventral root
were placed in tight fitting suction electrodes. Responses to electrical stimulation of the dorsal root were recorded from the ventral root via a DC coupled amplifier, digitised and stored for off-line computer-aided analysis. Trains of 20 high intensity electrical stimuli (200 ms; 300 mA; 1 Hz) were delivered to the dorsal root every 10 min. Responses to this stimulation schedule were recorded in control, after 8 min of agonist superfusion and during prolonged wash out periods. The agonists tested and the standard concentrations used were serotonin creatinine sulphate (5-HT; 50 mM), 8OH-DPAT (1 mM) and 5-carboxamidotryptamine maleate (5-CT; 0.1 mM) (all from Sigma). The standard concentrations used were selected to produce a strong depressant action of the MSR according to previous data [9]. The antagonist (1) WAY 100135 (1 mM) (donated by Wyeth Research, UK) was superfused for 90 min in isolation and sustained during superfusion of the agonists. Measurements of maximal amplitude from baseline were taken to quantify the mono-synaptic reflex (in a window of 4–9 ms from first stimulus artefact of each train). The cumu-
G. Hedo et al. / Neuroscience Letters 334 (2002) 41–44
lative depolarisation originated by repetitive stimulation was quantified as integrated area (area between baseline and waveform with a cut-off time of 24 s; see Refs. [8,16]). Statistical analysis were performed on raw data with Student’s t-test or with Mann–Whitney U-test as appropriate. The effect of the antagonist was analysed with two-way ANOVA followed by Bonferroni post-hoc test. All experimental procedures were performed according to European Union and Spanish Government regulations and were supervised and approved by the University Animal Care Facility. Under these experimental conditions, the mean amplitude of the MSR obtained in control experiments (n ¼ 19) was 4.4 ^ 0.5 (range 1.4–9.5 mV) and the mean integrated area of the cumulative depolarisation (CD) was 11.6 ^ 1 mV s (range 5.6–14 mV s). Bath application of (1)WAY 100135 at a concentration of 1 mM had no effect on the CD but produced a slow depression of the MSR peaking at 30 min of superfusion (to 48 ^ 14 % of control; t-test P # 0:01). The MSR returned slowly to baseline values during sustained superfusion of the antagonist (at 90 min of superfusion the MSR was not different from control; t-test P . 0:05, not significant). Fig. 1A–C shows a comparison between the effects of the agonists on the amplitude of the MSR in the presence and absence of (1)WAY 100135 (1 mM). Bath application of either 8-OH-DPAT (1 mM), 5-HT (50 mM) or 5-CT (0.1 mM) produced a depression of the MSR. Serotonin and 5CT showed a robust depressant action on the CD whereas 8OH-DPAT had a weak effect (to 78 ^ 7 % of control integrated area; t-test P # 0:05). Superfusion of 8-OH-DPAT produced a very strong and long-lasting depression of the MSR which was virtually abolished by (1)WAY 100135 (see Fig. 1A). Superfusion of 5-HT in control ACSF produced a large reduction of the MSR (to 16 ^ 4 of control) which was significantly attenuated in the presence of (1)WAY 100135 (to 50 ^ 4 % of control; Bonferroni post-hoc P # 0:05; see Figs. 1B and 2). The median time for full recovery was reduced from 40 min in control to 10 min in (1)WAY 100135 (see Fig. 1B; Mann–Whitney P # 0:001). Similarly (1)WAY 100135 reduced significantly the maximal effect and the duration of the depression caused by 5CT on the MSR (Fig. 1C). In contrast to the blocking action of (1)WAY 100135 on agonist-induced depression of the MSR, this compound did not modify the peak effect of the agonists on the CD nor the time course of recovery (Bonferroni post-hoc P . 0:05; see Fig. 1D–F). These results give strong experimental support to the hypothesis that 5-HT1A receptors mediate the inhibitory action of exogenously applied 5-HT on the MSR using a selective antagonist for this receptor. At the concentration used, (1)WAY 100135 is expected to saturate 5-HT1A receptors and should not bind to more than 20–30% of
43
other serotonergic, dopaminergic or adrenergic receptors [7,3]. Consistent with a functional elimination of 5-HT1A receptors, this compound blocked almost completely the depressant effect of 8-OH-DPAT on the MSR. In addition (1)WAY 100135 attenuated significantly the maximal effect and the duration of the depressions produced by 5HT and 5-CT. Two aspects of the results obtained are worth noting. Firstly, (1)WAY 100135 behaved as a partial agonist inhibiting the MSR although this effect weakened with time probably due to desensitisation of receptors. Mixed agonist-antagonist effects have been reported for 5-HT1A antagonists (see Ref. [11]). Secondly and more important, the strong blockade of 5-HT1A receptors provided by (1)WAY 100135 was insufficient to revert completely the
Fig. 2. Original recordings of the MSR showing the effect of 5-HT (50 mM) obtained from two different preparations: (A) in control ACSF and (B) in the presence of 1 mM (1)WAY 100135. These recordings were obtained at 10 min intervals before (a), during (b) and after superfusion of 50 mM 5-HT (c–d). Arrows indicate the time of stimulus artefact (hidden for clarity).
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G. Hedo et al. / Neuroscience Letters 334 (2002) 41–44
inhibitory effect of 5-HT on the MSR. We interpret this later observation as an indication of the involvement of another 5-HT receptor in the mediation of this inhibitory effect of 5HT. This unidentified receptor is sensitive to 5-CT and therefore is likely to belong to the 5-HT1 or to the 5-HT1like receptor family. Previous attempts to antagonise depressant responses to serotonergic agonists on the mono synaptic reflex [5,10] may be due to the use of non-selective tools and to the complex regulation of this reflex. Nevertheless, descending serotonergic systems are known to start developing in early embryonic stages and to reach an adult pattern only by postnatal day 21 [14]. At the age of the animals used in the present study (8–12 days) this system is still susceptible of further maturation and therefore the results obtained here will need validation in older animals. The results presented also confirm our previous observation that 5-HT1A receptors have a very limited role on the modulation of nociceptive somato-motor transmission which is partly mediated by 5-HT1B receptors [9]. In fact 8-OH-DPAT failed to mimic the inhibitory effects of 5-HT and 5-CT on the cumulative depolarisation which is known to depend upon activation of thin afferent fibres [6]. In addition, (1)WAY 100135 failed to reverse the effects of 5-HT and 5-CT on the cumulative depolarisation and did not act as a partial agonist on this signal. We conclude that spinal 5-HT1A receptors have a significant role as mediators of serotonergic actions in the spinal cord of rat pups. The role of 5-HT1A receptors is related to the modulation of non-nociceptive reflexes involved in the regulation of muscle control. The activation of 5-HT1A receptors leads to depression of these reflexes and its blockade results in a significantly diminished response to exogenous 5-HT. In contrast no experimental support has been found for the involvement of this receptor subtype on the modulation of nociceptive reflexes. It is therefore possible that inconsistent analgesic and proalgesic effects attributed to 8-OH-DPAT (see Ref. [13]) are due to impoverished motor control. This study was funded by the Spanish Ministry of Science and Technology (SAF-2000-0199) and the Madrid Regional Government (Contrato Programa) to Dr Cervero. The Authors wish to thank Dr Cervero and Dr Laird for their help in revising the manuscript. [1] Bras, H., Jankowska, E., Noga, B. and Skoog, B., Comparison of effects of various types of NA and 5-HT agonists on transmission from group II muscle afferents in the cat, Eur. J. Neurosci., 2 (1990) 1029–1039. [2] Clarke, R.W., Harris, J. and Houghton, A.K., Spinal 5-HTreceptors and tonic modulation of transmission through a withdrawal reflex pathway in the decerebrated rabbit, Br. J. Pharmacol., 119 (1996) 1167–1176.
[3] Cliffe, I.A., Brightwell, C.I., Fletcher, A., Forster, E.A., Mansell, H.L., Reilly, Y., Routledge, C. and White, A.C., (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2phenylpropanamide [(S)-WAY-100135]: a selective antagonist at presynaptic and postsynaptic 5-HT1A receptors, J. Med. Chem., 36 (1993) 1509–1510. [4] Crick, H. and Wallis, D.I., Inhibition of reflex responses of neonate rat lumbar spinal cord by 5-hydroxytryptamine, Br. J. Pharmacol., 103 (1991) 1769–1775. [5] Crick, H., Manuel, N.A. and Wallis, D.I., A novel 5-HT receptor or a combination of 5-HT receptor subtypes may mediate depression of a spinal monosynaptic reflex in vitro, Neuropharmacology, 33 (1994) 897–904. [6] Elliott, P. and Wallis, D.I., Serotonin and L-norepinephrine as mediators of altered excitability in neonatal rat motoneurons studied in vitro, Neuroscience, 47 (1992) 533–544. [7] Fletcher, A., Bill, D.J., Bill, S.J., Cliffe, I.A., Dover, G.M., Forster, E.A., Haskins, J.T., Jones, D., Mansell, H.L. and Reilly, Y., WAY100135: a novel, selective antagonist at presynaptic and postsynaptic 5-HT1A receptors, Eur. J. Pharmacol., 237 (1993) 283–291. [8] Hedo, G., Laird, J.M. and Lopez-Garcia, J.A., Time-course of spinal sensitization following carrageenan-induced inflammation in the young rat: a comparative electrophysiological and behavioural study in vitro and in vivo, Neuroscience, 92 (1999) 309–318. [9] Hedo, G. and Lopez-Garcia, J.A., 5-HT1B but not 5-HT6 or 5HT7 receptors mediate depression of spinal nociceptive reflexes in vitro, Br. J. Pharmacol., 135 (2002) 935–942. [10] Honda, M. and Ono, H., Differential effects of (R)- and (S)-8hydroxy-2-(di-n-propylamino)tetralin on the monosynaptic spinal reflex in rats, Eur. J. Pharmacol., 373 (1999) 171–179. [11] Hoyer, D., Clarke, D.E., Fozard, J.R., Hartig, P.R., Martin, G.R., Mylecharane, E.J., Saxena, P.R. and Humphrey, P.P., International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin), Pharmacol. Rev., 46 (1994) 157–203. [12] Manuel, N.A., Wallis, D.I. and Crick, H., Ketanserin-sensitive depressant actions of 5-HT receptor agonists in the neonatal rat spinal cord, Br. J. Pharmacol., 116 (1995) 2647–2654. [13] Millan, M.J., The role of descending noradrenergic and serotonergic pathways in the modulation of nociception: focus on receptor multiplicity, In A. Dickenson and J.M. Besson (Eds.), The Pharmacology of Pain, Springer-Verlag, Berlin, 1997, pp. 385–446. [14] Rajaofetra, N., Sandillon, F., Geffard, M. and Privat, A., Preand post-natal ontogeny of serotonergic projections to the rat spinal cord, J. Neurosci. Res., 22 (1989) 305–321. [15] Schmidt, B.J. and Jordan, L.M., The role of serotonin in reflex modulation and locomotor rhythm production in the mammalian spinal cord, Brain Res. Bull., 53 (2000) 689–710. [16] Thompson, S.W., Dray, A. and Urban, L., Injury-induced plasticity of spinal reflex activity: NK1 neurokinin receptor activation and enhanced A- and C-fiber mediated responses in the rat spinal cord in vitro, J. Neurosci., 14 (1994) 3672–3687. [17] Wallis, D.I. and Wu, J., FAST and SLOW ipsilateral and contralateral spinal reflexes in the neonate rat are modulated by 5-HT, Gen. Pharmacol., 23 (1992) 1035–1044. [18] Yomono, H.S., Suzuki, H. and Yoshioka, K., Serotonergic fibers induce a long-lasting inhibition of monosynaptic reflex in the neonatal rat spinal cord, Neuroscience, 47 (1992) 521–531.
Role of serotonin1A receptors on the modulation of rat spinal mono-synaptic reflexes in vitro Gonzalo Hedo, Mar Ajubita, Jose A. Lopez-Garcia* Department of Physiology, Edificio de Medicina, University of Alcala, 28871 Madrid, Spain Received 25 July 2002; received in revised form 10 September 2002; accepted 12 September 2002
Abstract The present study aimed at determining the role of the serotonin1A (5-HT1A) receptor subtype on the modulation of the mono-synaptic reflex (MSR) elicited by dorsal root stimulation and recorded from ventral roots in the hemisected spinal cord obtained from rat pups. Serotonin and 5-carboxamidotryptamine (5-CT) depressed both the MSR and the cumulative depolarisation (CD) produced by repetitive dorsal root stimulation, whereas the specific 5-HT1A receptor agonist (R)-(1)-8hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) applied at 1 mM showed a selective depressant effect on the MSR. Superfusion of the 5-HT1A receptor antagonist (1)-N-tert-butyl-3(4(2-methoxyphenyl)-piperazin-1-yl)-2-phenylprpanamide ((1)WAY 100135) produced a complete blockade of 8-OH-DPAT effects. In addition (1)WAY 100135 blocked partially the effects of 5-HT and 5-CT on the MSR but not the effects of these compounds on the CD. The results are consistent with an intervention of 5-HT1A receptors in the modulation of non-nociceptive reflexes but do not support a prominent role for this receptor in the modulation of nociceptive reflexes. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Serotonin; Serotonin1A receptors; Mono-synaptic reflex; Nociceptive reflex; Spinal pathways
Extensive experimental evidence obtained from in vivo and in vitro experiments has revealed that serotonin (5-HT) exerts complex modulatory actions on spinal segmental reflexes mediated by A- and C-fibres (see review in Ref. [15]). The mono-synaptic reflex (MSR) recorded from ventral roots in response to dorsal root stimulation has been intensively studied in the hemisected cord of rat pups. This reflex is depressed by bath-applied 5-HT and a range of 5-HT receptor agonists [5,9,12]. In addition, conditioning stimuli delivered to descending tracks at the thoracic level produce depression of the MSR which is mediated by a 5-HT receptor sensitive to ketanserin [4,18], a classic 5-HT2 receptor antagonist. This receptor was first thought to be an atypical 5-HT2 receptor, but further pharmacological analysis concluded that it was more likely a 5-HT1-like receptor similar to the 5-HT1Da [12,17]. Interestingly ketanserin does not block the depressant effect of bath applied 5-HT suggesting that a different receptor mediates the effects of exogenous 5HT [6]. Crick and colleagues [5] found that a combination * Corresponding author. Tel.: 134-91-885-5106; fax: 134-91885-4807 E-mail address: [email protected] (J.A. Lopez-Garcia).
of antagonists directed against 5-HT1, 5-HT2 and 5-HT3 receptors failed to block the effects of bath applied 5-HT and suggested that a novel receptor or a combination of receptors mediate the depressant effect of 5-HT. Later on Clarke and colleagues [2] using an in vivo preparation of adult rabbit and more selective antagonists concluded that spinal 5-HT1A receptors mediate tonic depression of reflex responses recorded from gastrocnemius motoneurones in response to electrical stimulation of the sural nerve. Strong depressant effects of specific 5-HT1A agonists have been reported in rats [1,5] but none of the antagonists tested has proven useful. In vitro experiments using the hemisected cord preparation from rat pups have shown that spiperone, a non-specific 5-HT1A receptor antagonist, blocks only partially the effect of (R)-(1)-8hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) on the MSR and fails to block the effect of 5-HT [4,5]. Intravenous spiroxantrine and the more selective 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-(4-phthalimidobutyl) piperazine (NAN-190) administered to adult rats in vivo have also failed to block inhibitory responses to 8-OHDPAT in spinalised rats [10]. We have shown recently that 8-OH-DPAT produces a strong, concentration-dependent and selective depression
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 2) 01 06 4- 9
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G. Hedo et al. / Neuroscience Letters 334 (2002) 41–44
Fig. 1. The graphs in (A–C) show the effects of 8-OH-DPAT (1 mM), 5-HT (50 mM) and 5-CT (0.1 mM) on the amplitude of the mono-synaptic reflex in the absence (filled squares) and presence of (1)WAY 100135 (1 m, hollow triangles). The black bar signal the time of superfusion of the agonist, the numbers in parenthesis are the numbers of observations and the asterisks denote significant differences between control and antagonist conditions (two way ANOVA, Bonferroni post-hoc tests P # 0:05). Graphs in (D–F) show the corresponding effects of the agonists on the integrated area of the cumulative depolarisation in the absence (filled squares) and presence of (1)WAY 100135 (hollow triangles) obtained from the same pool of experiments (no significant differences detected).
of the MSR whereas 5-HT and several other agonists depress the responses to repetitive nociceptive stimulation as well as the MSR [9]. In order to test whether this effect of 8-OH-DPAT is mediated by 5-HT1A receptors, here we test the effects of a selective 5-HT1A antagonist, (1)-N-tertbutyl-3(4(2-methoxyphenyl)-piperazin-1-yl)-2-phenylprpanamide ((1)WAY 100135), on the depressant actions of 8OH-DPAT, 5-HT and 5-CT. The present experiments were performed under similar experimental conditions to those described in [9]. Hemisected spinal cords were prepared from 8–12 days old Wistar rats under urethane anaesthesia (2 g/kg, 1.p.). A dorsal laminectomy was performed and the cord rapidly excised and placed in ice-cooled artificial cerebrospinal fluid (ACSF) for hemisection. A hemisected cord was placed in a recording chamber and continuously superfused with oxygenated (95% O2; 5% CO2) ACSF containing (in mM): NaCl (128); KCl (1.9); KH2PO4 (1.2); MgSO4 (1.3); CaCl2 (2.4); NaHCO3 (26); glucose (10) (pH 7.4). A lumbar dorsal root and the corresponding ventral root
were placed in tight fitting suction electrodes. Responses to electrical stimulation of the dorsal root were recorded from the ventral root via a DC coupled amplifier, digitised and stored for off-line computer-aided analysis. Trains of 20 high intensity electrical stimuli (200 ms; 300 mA; 1 Hz) were delivered to the dorsal root every 10 min. Responses to this stimulation schedule were recorded in control, after 8 min of agonist superfusion and during prolonged wash out periods. The agonists tested and the standard concentrations used were serotonin creatinine sulphate (5-HT; 50 mM), 8OH-DPAT (1 mM) and 5-carboxamidotryptamine maleate (5-CT; 0.1 mM) (all from Sigma). The standard concentrations used were selected to produce a strong depressant action of the MSR according to previous data [9]. The antagonist (1) WAY 100135 (1 mM) (donated by Wyeth Research, UK) was superfused for 90 min in isolation and sustained during superfusion of the agonists. Measurements of maximal amplitude from baseline were taken to quantify the mono-synaptic reflex (in a window of 4–9 ms from first stimulus artefact of each train). The cumu-
G. Hedo et al. / Neuroscience Letters 334 (2002) 41–44
lative depolarisation originated by repetitive stimulation was quantified as integrated area (area between baseline and waveform with a cut-off time of 24 s; see Refs. [8,16]). Statistical analysis were performed on raw data with Student’s t-test or with Mann–Whitney U-test as appropriate. The effect of the antagonist was analysed with two-way ANOVA followed by Bonferroni post-hoc test. All experimental procedures were performed according to European Union and Spanish Government regulations and were supervised and approved by the University Animal Care Facility. Under these experimental conditions, the mean amplitude of the MSR obtained in control experiments (n ¼ 19) was 4.4 ^ 0.5 (range 1.4–9.5 mV) and the mean integrated area of the cumulative depolarisation (CD) was 11.6 ^ 1 mV s (range 5.6–14 mV s). Bath application of (1)WAY 100135 at a concentration of 1 mM had no effect on the CD but produced a slow depression of the MSR peaking at 30 min of superfusion (to 48 ^ 14 % of control; t-test P # 0:01). The MSR returned slowly to baseline values during sustained superfusion of the antagonist (at 90 min of superfusion the MSR was not different from control; t-test P . 0:05, not significant). Fig. 1A–C shows a comparison between the effects of the agonists on the amplitude of the MSR in the presence and absence of (1)WAY 100135 (1 mM). Bath application of either 8-OH-DPAT (1 mM), 5-HT (50 mM) or 5-CT (0.1 mM) produced a depression of the MSR. Serotonin and 5CT showed a robust depressant action on the CD whereas 8OH-DPAT had a weak effect (to 78 ^ 7 % of control integrated area; t-test P # 0:05). Superfusion of 8-OH-DPAT produced a very strong and long-lasting depression of the MSR which was virtually abolished by (1)WAY 100135 (see Fig. 1A). Superfusion of 5-HT in control ACSF produced a large reduction of the MSR (to 16 ^ 4 of control) which was significantly attenuated in the presence of (1)WAY 100135 (to 50 ^ 4 % of control; Bonferroni post-hoc P # 0:05; see Figs. 1B and 2). The median time for full recovery was reduced from 40 min in control to 10 min in (1)WAY 100135 (see Fig. 1B; Mann–Whitney P # 0:001). Similarly (1)WAY 100135 reduced significantly the maximal effect and the duration of the depression caused by 5CT on the MSR (Fig. 1C). In contrast to the blocking action of (1)WAY 100135 on agonist-induced depression of the MSR, this compound did not modify the peak effect of the agonists on the CD nor the time course of recovery (Bonferroni post-hoc P . 0:05; see Fig. 1D–F). These results give strong experimental support to the hypothesis that 5-HT1A receptors mediate the inhibitory action of exogenously applied 5-HT on the MSR using a selective antagonist for this receptor. At the concentration used, (1)WAY 100135 is expected to saturate 5-HT1A receptors and should not bind to more than 20–30% of
43
other serotonergic, dopaminergic or adrenergic receptors [7,3]. Consistent with a functional elimination of 5-HT1A receptors, this compound blocked almost completely the depressant effect of 8-OH-DPAT on the MSR. In addition (1)WAY 100135 attenuated significantly the maximal effect and the duration of the depressions produced by 5HT and 5-CT. Two aspects of the results obtained are worth noting. Firstly, (1)WAY 100135 behaved as a partial agonist inhibiting the MSR although this effect weakened with time probably due to desensitisation of receptors. Mixed agonist-antagonist effects have been reported for 5-HT1A antagonists (see Ref. [11]). Secondly and more important, the strong blockade of 5-HT1A receptors provided by (1)WAY 100135 was insufficient to revert completely the
Fig. 2. Original recordings of the MSR showing the effect of 5-HT (50 mM) obtained from two different preparations: (A) in control ACSF and (B) in the presence of 1 mM (1)WAY 100135. These recordings were obtained at 10 min intervals before (a), during (b) and after superfusion of 50 mM 5-HT (c–d). Arrows indicate the time of stimulus artefact (hidden for clarity).
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inhibitory effect of 5-HT on the MSR. We interpret this later observation as an indication of the involvement of another 5-HT receptor in the mediation of this inhibitory effect of 5HT. This unidentified receptor is sensitive to 5-CT and therefore is likely to belong to the 5-HT1 or to the 5-HT1like receptor family. Previous attempts to antagonise depressant responses to serotonergic agonists on the mono synaptic reflex [5,10] may be due to the use of non-selective tools and to the complex regulation of this reflex. Nevertheless, descending serotonergic systems are known to start developing in early embryonic stages and to reach an adult pattern only by postnatal day 21 [14]. At the age of the animals used in the present study (8–12 days) this system is still susceptible of further maturation and therefore the results obtained here will need validation in older animals. The results presented also confirm our previous observation that 5-HT1A receptors have a very limited role on the modulation of nociceptive somato-motor transmission which is partly mediated by 5-HT1B receptors [9]. In fact 8-OH-DPAT failed to mimic the inhibitory effects of 5-HT and 5-CT on the cumulative depolarisation which is known to depend upon activation of thin afferent fibres [6]. In addition, (1)WAY 100135 failed to reverse the effects of 5-HT and 5-CT on the cumulative depolarisation and did not act as a partial agonist on this signal. We conclude that spinal 5-HT1A receptors have a significant role as mediators of serotonergic actions in the spinal cord of rat pups. The role of 5-HT1A receptors is related to the modulation of non-nociceptive reflexes involved in the regulation of muscle control. The activation of 5-HT1A receptors leads to depression of these reflexes and its blockade results in a significantly diminished response to exogenous 5-HT. In contrast no experimental support has been found for the involvement of this receptor subtype on the modulation of nociceptive reflexes. It is therefore possible that inconsistent analgesic and proalgesic effects attributed to 8-OH-DPAT (see Ref. [13]) are due to impoverished motor control. This study was funded by the Spanish Ministry of Science and Technology (SAF-2000-0199) and the Madrid Regional Government (Contrato Programa) to Dr Cervero. The Authors wish to thank Dr Cervero and Dr Laird for their help in revising the manuscript. [1] Bras, H., Jankowska, E., Noga, B. and Skoog, B., Comparison of effects of various types of NA and 5-HT agonists on transmission from group II muscle afferents in the cat, Eur. J. Neurosci., 2 (1990) 1029–1039. [2] Clarke, R.W., Harris, J. and Houghton, A.K., Spinal 5-HTreceptors and tonic modulation of transmission through a withdrawal reflex pathway in the decerebrated rabbit, Br. J. Pharmacol., 119 (1996) 1167–1176.
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