- Email: [email protected]
CGP 35348 and CGP 55845A block the baclofen-induced depression of dorsal root evoked potentials in lumbar motoneurons of the neonatal rat
|[JB ELSEVIER
NeuroscienceLetters214 (1996) 103-106
CGP 35348 axtd CGP 55845A block the baclofen-induced depression of dorsal root evoked potentials in lumbar motoneurons of the neonatal rat L. V i n a y * , F. C l a r a c CNRS, UPR Neurobiologie et Mouvements, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Received 31 January 1996; revisedversionreceived 12 June 1996; accepted9 July 1996
Abstract
In vitro brainstem-spinal cord preparations isolated from neonatal (0-5 days old) rats were used to investigate the GABA8 receptormediated modulation of the dorsal root evoked potentials in lumbar motoneurons recorded intracellularly. The GABABreceptor agonist, baclofen, at low concentra~ions (1-10 #M), caused a reduction of the amplitude of the monosynaptic excitatory postsynaptic potential (EPSP), in a concentratiort-dependent manner. The depression of EPSPs was likely exerted at a presynaptic level since it occurred without any significant ch~mge of the passive membrane properties of the motoneurons. The two GABAa receptor antagonists, CGP 35348 and CGP 55845A blocked the effects of baclofen. These two compounds may be useful tools to study the evolution of GABAB receptor-mediated presynaptic inhibition during ontogenesis. Keywords: GABAa receptors; Monosynaptic reflex; Presynaptic inhibition; Spinal cord; Neonatal rat; Baclofen; CGP 35348; CGP
55845A
Presynaptic inhibition of the muscle spindle afferent terminals in the spinal cord provide an important mechanism for the regulation c,f the stretch reflex ([5]; see Ref. [14] for review). Presynaptic inhibition is considered to be produced, at least partly, by GABA which activates two types of receptors (GAI3;AA and GABAB receptors) in the intraspinal terminals of muscle afferents. Little is known about these mechanisms during ontogenesis and their contribution to the development of motor control. The development of dorsal root projections to the lumbar spinal cord has been studied in the rat using physiological and morphological techniques 1110,17]. Dorsal root stimulation evokes responses in motoneurons 3-5 days prior to birth. These responses first consist of long latency excitatory postsynaptic potentials (EPSPs) conveyed by polysynaptic pathways which are present 1-2 days before the formation of short-late, ncy monosynaptic connections [17]. The stretch reflex appears 1-2 days prior to birth [9]. Activation of GABAB receptors by baclofen reduces the responses evoked by dorsal root stimulation in lumbar motoneurons of both young rats (postnatal day (P) 1216 [16]; P6-P10 [11,13]), and adult cats [3,4,8]. The baclo* Corresponding author. Tel.: +33 91164086; fax: +33 91775084; email: [email protected]
fen-induced depression is, at least partly, mediated at a presynaptic level since, with low concentrations of baclofen (<5/~M), the synaptic transmission is depressed without any significant change of both the passive membrane properties of the recorded motoneurons and the depolarizations induced by exogenously applied glutamate [ 16]. In addition to synaptic depression, baclofen, with higher concentrations, causes a hyperpolarization and a decrease of the input resistance only in some of the motoneurons. The synaptic depressant effect of baclofen is not blocked by the GABAB receptor antagonist phaclofen [16]. A classification of GABAB receptors, based on their sensitivity to different antagonists, has been proposed [2]. The aim of this study was to examine the effect of low concentrations of baclofen (<5/~M) on dorsal root evoked potentials in lumbar motoneurons during the very first days following birth (P0-P5) and to test whether other GABAB receptor antagonists such as CGP 35348 and CGP 55845A [1] are able to block the baclofen-induced depression. Experiments were performed on 23 Wistar rats. The animals were anaesthetized with ether, decerebrated at a precollicular and postmamillary level and eviscerated. They were pinned down to a petri dish continuously perfused with saline solution, containing the following substances (in mM) NaC1, 130; KC1, 4; CaCI2, 3.75; MgSO4,
0304-3940/96/$12.00 © 1996 ElsevierScienceIreland Ltd. All rights reserved PII S0304-3940(96) 1290zl-9
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L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
1.3; NaH2PO4, 0.58; NaHCO3, 25; glucose, 10, bubbled with a 95% 02_5% CO2 mixture, and adjusted to pH 7.4. A dorsal craniotomy and laminectomy were performed. The brainstem, spinal cord and lumbar roots were then removed and pinned down with the ventral side up in the recording chamber (Fig. 1A). The pia was removed to give access to the lumbar motoneurons which were recorded intracellularly using glass microelectrodes filled with 1.5 M K-acetate (40-70 Mfl resistance). Intracellular potentials were recorded using an Intra 767 amplifier (World Precision Instruments, USA) or an Axoclamp 2B (Axon Instruments, USA), connected through an Instrutech ITC16 MAC interface to a Macintosh Quadra 650 (software programs from Axon Instruments). Only neurons exhibiting a resting membrane potential of at least -60 mV were considered for analysis. The input resistance was monitored by applying a hyperpolarizing current pulse (from -0.2 to -0.5 nA; 200 ms) through the recording electrode 400 ms prior to dorsal root stimulation. The roots were stimulated with monopolar platinum electrodes placed in contact with the nerve and insulated with Vaseline. The stimulation voltage ranged from 2 to 6 V (corresponding to 1-2 x threshold intensity (T)) and the pulse duration was 0.3 ms. Dorsal roots were stimulated at a low frequency (every 30 s) to allow recovery from the prolonged synaptic depression that has been described in this preparation [1 l]. Motoneurons were identified by the antidromic action potentials they produced in response to ventral root stimulation (inset in Fig. 1A). The latencies of the antidromic and dorsal root responses were 4.4 ± 1.7 ms (mean _+ SD) and 10.5 ± 2.3 ms, respectively. In individual motoneurons, the delay between these responses was 6 ± 1.9 ms, similar to the delays of monosynaptic dorsal root potentials in neonatal rats [9,15]. Seventeen out of the 30 cells tested were identified as motoneurons, eight in L3 (out of l0 cells recorded in this segment), four in L4 (11) and five in L5 (9). No difference in the results below was observed between the identified motoneurons and the other cells. Drugs used were (+)-baclofen (1-10 /xM; RBI, Natick, USA) and mephenesin (1 m M ; Sigma, St. Louis, MO, USA). CGP 35348 (100-150 /xM) and CGP 55845A (100-200 nM) were generous gifts from Ciba-Geigy. Drugs applied in the perfusion medium reached the recording chamber after a delay of about 60 s. Since EPSPs evoked by dorsal root stimulation at a strength of 1.2-1.5 × T triggered an action potential in lumbar motoneurons, a stimulation intensity subthreshold for the orthodromic spike was used to investigate the effect of baclofen on the postsynaptic potentials (PSPs) amplitude (Fig. 1B; 1.1 × T). These responses were composed of many components with a mixture of EPSPs and inhibitory postsynaptic potentials (IPSPs) [15] which were reversed at resting membrane potential (from -65 to -70 mV; reversal of IPSPs a t - 5 0 t o - 6 0 mV). Bath application of baclofen (2/~M) decreased the amplitude of all components of the response attenuating the long-latency
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20 ms Fig. 1. Baclofen reduces the dorsal root evoked potentials in lumbar motoneurons. (A) Experimental protocol. The trace is the averaged (10 sweeps) antidromic response to stimulation (stim) of the ipsilateral L5 ventral root (VR). (B) Baclofen (2 #M) caused a reduction of the responses evoked by stimulation of the ipsilateral homonymous dorsal root (DR) in this L5 motoneuron. Traces are averages of five consecutive sweeps elicited at 30 s intervals, before (control) and 2, 5 and 7 mill after starting the perfusion of baclofen.
responses before the early EPSPs (Fig. 1B). Short- and long-latency responses are likely transmitted by monoand polysynaptic pathways, respectively [17]. The activity of polysynaptic pathways is greatly reduced by perfusing a saline with either a high concentration of divalent cations (7.5 mM Ca+++ 8 mM Mg++; [7]) or mephenesin [6,11,17]. We therefore used these modified solutions to investigate the effect of baclofen on the monosynaptic response (Fig. 2 with high concentration of Ca +÷ and Mg +÷ (n = 4 neurons recorded in this condition) and Fig. 3 with mephenesin (n = 8)). In both conditions, baclofen caused a reduction of the amplitude of the EPSPs (measured from onset to peak) in a concentration-dependent manner (Fig. 2A1,B (open circles)). EPSPs almost disappeared with baclofen at a concentration of 10 #M (95% reduction). Since polysynaptic responses may still be evoked in the presence of either high concentrations of Ca ++ and Mg ++ or mephenesin (see Ref. [17]) and the minimum delay from the mono- to the polysynaptic reflex has been estimated to be 4 ms [10], we have measured the amplitude of the EPSP at 3.5 ms from the onset (Fig. 2A2 (vertical dotted line)). With increasing concentrations of baclofen of 1, 3, 5 and 10/~M the monosynaptic EPSP decreased to 68, 35, 23 and 10% compared with control, respectively (Fig. 2A1,B (filled circles)), while the input resistance (17 Mfl) and the membrane potential (from -68 to -69 mV) were not affected (see also Fig. 3B,C). Fig. 2C represents the baclofen concentration-dependent reduction of the monosynaptic EPSP for all the cells tested (n = 13). There was no significant change of the membrane potential ( < 1 mV) and input resistance (<5%). These results suggest that the depression of the dorsal root evoked EPSPs, at
L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
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Baclofen concentration 0aM) Fig. 2. Baclofen reduces the monosynaptic EPSP in a concentration-dependent manner. (A1,A2) Responses evoked by dorsal root stimulation in an L5 neuron with various concentrations of baclofen in the bath (modified saline with a high concentration of divalent cations (7.5 mM Ca +÷ + 8 mM Mg ÷ +). Traces are averages of 10 consecutive sweeps. (A2) Enlarged portion of (A1) with a higher sweep velocity. The horizontal dashed line indicates the resting membrane potential level (around - 68 mV); the vertical dashed line shows the time at which the amplitude of the monosynaptic EPSP was measured (3.5 ms from the EPSP onset). (B) Time course of the amplitude of the dorsal root evoked EPSP (from onset to peak, open circles; from onset to 3.5 ms, filled circles) before, during and after (washout) the application of baclofen at the increasing concentrations indicated. Each dot is the mean ( + SEM) amplitude of five consecutive dorsal root evoked EPSPs. Amplitudes are expressed as percentages of the mean amplitude obtained before the application of baclofen (dashed line at 100%; 4.5 mV for the filled circles, 20.9 mV for the open circles; n = 12). Note that the difference was highly significant (P < 0.001) for both amplitudes with baclofen concentrations as low as 1 ttM, as compared to control. (C) Percentage of reduction ( + SEM) of the monosynaptic EPSP (measured at 3.5-4 ms from the onset) following application of baclofen at the various concentrations indicated below each column. The number of cells tested is indicated above each column.
low concentrations of baclofen, may be exerted, at least partly, at a presynaptic level. The baclofen-induced depression of the monosynaptic EPSP was blocked by adding the GABAs receptor antagonist CGP 35348 (100 #M) or CGP 55845A (200 nM) to the baclofen (2 #M) containing solution (Fig. 3A-D). Washing out the antagonist w]aile keeping baclofen in the perfusion caused a reduction of the amplitude of EPSPs within 20-30 min, which shows that the increase of amplitude in the presence of GABAB :receptor antagonists is not due to a desensitization of receptors (Fig. 3B,C). Washout of CGP 35348 takes shorter time than washout of CGP 55845 (see Ref. [12]). Both antagoni~sts had no significant effect on the membrane potential (Fig. 3B) and the input resistance (Fig. 3C). The amplitude of the monosynaptic component of dorsal root evoked potentials was not significantly different with GABAB receptor antagonists in the bath as compared to control (e:ither normal solution (n = 8) or baclofen containing solution (n = 9); see, however, the slight increase in the presence of CGP 55845A in Fig. 3A). The present study conJirrns previous observations of the depression of the dorsal root evoked potentials by baclofen
in 6-16 day old rats [11,13,16], and extends them to the very first days following birth (P0-P5). With low concentrations of baclofen, it is likely that the depression of EPSPs is exerted at a presynaptic level [16]. However, a postsynaptic action, even without any significant change of the passive membrane properties of the motoneurons, cannot be excluded. Presynaptic GABAB receptors may inhibit the synaptic transmission by depressing voltage-gated calcium channels and thus decreasing the calcium influx that triggers transmitter release (see Ref. [12] for review). These results further demonstrate that CGP 35348 and CGP 55845A antagonize the baclofen-induced reduction of dorsal root evoked EPSPs. The presynaptic GABAs receptors involved in the reduction of glutamate release from primary afferents in neonatal rat are therefore baclofen sensitive, phaclofen insensitive [16] but sensitive to CGP 35348 and CGP 55845A (see the classification of GABAB receptors proposed on the basis of release studies [2]). These two GABAa receptor antagonists may be useful tools to study the evolution of presynaptic inhibition during ontogenesis and its contribution to the development of motor control.
L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
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Fig. 3. CGP 55845A and CGP 35348 block the baclofen-induced depression of the monosynaptic EPSP. (A) EPSPs evoked by dorsal root stimulation in an IA neuron under control conditions and in the presence of baclofen (2/~M), either alone or together with CGP 55845A (200 nM). Traces are averages of five consecutive sweeps. (B) Time course of the mean ( + SEM) (filled triangles; n = 5 consecutive sweeps) amplitude of EPSPs and membrane potential (small squares). The response was significantly reduced (P < 0.001) under baclofen as compared to control. (C) Time course of the input resistance (open circles) and amplitude of EPSPs (filled triangles; both expressed as percentages of the control mean values) in another 1_.4motoneuron during application of baclofen (2 t~M), either alone or together with CGP 55845A (200 nM). Error bars are in most cases smaller than the symbol size. Note in (B) and (C) that, although there are spontaneous changes of the membrane potential and input resistance, there is no significant drift accompanying the decrease or increase of EPSP amplitude. (D) Averaged (10 traces) EPSPs evoked by dorsal root stimulation in an L4 motoneuron under control conditions, in the presence of 2 pM baclofen, and after 100 #M CGP 35348 has been added to the baclofen containing solution. Bathing solutions in (A,B,D) contained 1 mM mephenesin.
The authors wish to thank Ciba-Geigy for their gift of CGP 35348 and CGP 55845A and Dr. J.-Y. Barthe and Dr. J.-R. Cazalets for critical reading of the manuscript. [1] Bittiger, H., Froestl, W., Mickel, S.J. and Olpe, H.-R., GABAB receptor antagonists: from synthesis to therapeutic applications, Trends Phannacot. Sci., 14 (1993) 391-394. [2] Bonanno, G. and Raiteri, M., Multiple GABAB receptors, Trends Pharmacol. Sci., 14 (1993) 259-261. [3] Curtis, D.R. and Lacey, G., GABA-B receptur-mediated spinal inhibition, NeoroReport, 5 (1994) 540-542. [4] Curtis, D.R., Lodge, D., Bomstein, J.C. and Peet, M.J., Selective effects of baclofen on spinal synaptic transmission in the cat, Exp. Brain Res., 42 (1981) 158-170. [5] Frank, K. and Fuortes, M.G.F., Presynaptic and postsynaptic inhibition of monosynaptic reflexes, Fed. Proc., 16 (1957) 39-40. [6] Fulton, B.P, and Walton, K., Electrophysiological properties of neonatal rat motoneurones studied in vitro, J. Physiol., 370 (1986) 651-678. [7] Jahr, C.E. and Yoshioka, K., Ia afferent excitation of motoneurones in the in vitro new-born rat spinal cord is selectively antagonized by kynurenate, J. Physiol., 370 (1986) 515-530. [8] Jimdnez, I., Rudomin, P. and Enriquez, M., Differential effects of (+)-baciofen on Ia and descending monosynaptic EPSPs, Exp. Brain Res., 85 (1991) 103-113. [9] Kudo, N. and Yamada, T., Development of the monosynaptic
[10]
[11]
[12]
[13]
[14] [15]
[16]
[17]
stretch reflex in the rat: an in vitro study, J. Physiol., 369 (1985) 127-144. Kudo, N. and Yamada, T., Morphological and physiological studies of development of the monosynaptic reflex pathway in the rat lumbar spinal cord, J. Physiol., 389 (1987) 441-459. Lee-Toy, A. and Pinco, M., In vitro studies of prolonged synaptic depression in the neonatal rat spinal cord, J. Physiol., 447 (1992) 149-169. Misgeld, U., Bijak, M. and Jarolimek, W., A physiological role for GABAB receptors and the effects of baclofen in the mammalian central nervous system, Frog. Neurobiol., 46 (1995) 423-462. Pinco, M. and Lev-Tov, A., Synaptlc excitation of a-motoneurons by dorsal root afferents in the neonatal rat spinal cord, J. Neurophysiol., 70 (1993) 406-417. Rudomin, P., Quevedo, J. and Eguibar, J.R., Presynaptic modulation of spinal reflexes, CmT. Opin. Neurobiol., 3 (1993) 997-1004. Seebach, B.S. and Ziskind-Conhaim, L., Formation of transient inappropriate sensorimotor synapses in developing rat spinal cords, J. Neurosei., 14 (1994) 4520-4528. Wang, M.Y. and Dun, N.J., Plmelofen-insensitive presynaptic inhibitory action of (+)-baelofen in neonatal rat motoneurones in vitro, Br. J. Pharmacol., 99 (1990) 413-421. Ziskind-Conhaim, L., NMDA receptors mediate poly- and monosynaptic potentials in motoneurons of rat embryos, J. Neurosci., 10 (1990) 125-135.
NeuroscienceLetters214 (1996) 103-106
CGP 35348 axtd CGP 55845A block the baclofen-induced depression of dorsal root evoked potentials in lumbar motoneurons of the neonatal rat L. V i n a y * , F. C l a r a c CNRS, UPR Neurobiologie et Mouvements, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
Received 31 January 1996; revisedversionreceived 12 June 1996; accepted9 July 1996
Abstract
In vitro brainstem-spinal cord preparations isolated from neonatal (0-5 days old) rats were used to investigate the GABA8 receptormediated modulation of the dorsal root evoked potentials in lumbar motoneurons recorded intracellularly. The GABABreceptor agonist, baclofen, at low concentra~ions (1-10 #M), caused a reduction of the amplitude of the monosynaptic excitatory postsynaptic potential (EPSP), in a concentratiort-dependent manner. The depression of EPSPs was likely exerted at a presynaptic level since it occurred without any significant ch~mge of the passive membrane properties of the motoneurons. The two GABAa receptor antagonists, CGP 35348 and CGP 55845A blocked the effects of baclofen. These two compounds may be useful tools to study the evolution of GABAB receptor-mediated presynaptic inhibition during ontogenesis. Keywords: GABAa receptors; Monosynaptic reflex; Presynaptic inhibition; Spinal cord; Neonatal rat; Baclofen; CGP 35348; CGP
55845A
Presynaptic inhibition of the muscle spindle afferent terminals in the spinal cord provide an important mechanism for the regulation c,f the stretch reflex ([5]; see Ref. [14] for review). Presynaptic inhibition is considered to be produced, at least partly, by GABA which activates two types of receptors (GAI3;AA and GABAB receptors) in the intraspinal terminals of muscle afferents. Little is known about these mechanisms during ontogenesis and their contribution to the development of motor control. The development of dorsal root projections to the lumbar spinal cord has been studied in the rat using physiological and morphological techniques 1110,17]. Dorsal root stimulation evokes responses in motoneurons 3-5 days prior to birth. These responses first consist of long latency excitatory postsynaptic potentials (EPSPs) conveyed by polysynaptic pathways which are present 1-2 days before the formation of short-late, ncy monosynaptic connections [17]. The stretch reflex appears 1-2 days prior to birth [9]. Activation of GABAB receptors by baclofen reduces the responses evoked by dorsal root stimulation in lumbar motoneurons of both young rats (postnatal day (P) 1216 [16]; P6-P10 [11,13]), and adult cats [3,4,8]. The baclo* Corresponding author. Tel.: +33 91164086; fax: +33 91775084; email: [email protected]
fen-induced depression is, at least partly, mediated at a presynaptic level since, with low concentrations of baclofen (<5/~M), the synaptic transmission is depressed without any significant change of both the passive membrane properties of the recorded motoneurons and the depolarizations induced by exogenously applied glutamate [ 16]. In addition to synaptic depression, baclofen, with higher concentrations, causes a hyperpolarization and a decrease of the input resistance only in some of the motoneurons. The synaptic depressant effect of baclofen is not blocked by the GABAB receptor antagonist phaclofen [16]. A classification of GABAB receptors, based on their sensitivity to different antagonists, has been proposed [2]. The aim of this study was to examine the effect of low concentrations of baclofen (<5/~M) on dorsal root evoked potentials in lumbar motoneurons during the very first days following birth (P0-P5) and to test whether other GABAB receptor antagonists such as CGP 35348 and CGP 55845A [1] are able to block the baclofen-induced depression. Experiments were performed on 23 Wistar rats. The animals were anaesthetized with ether, decerebrated at a precollicular and postmamillary level and eviscerated. They were pinned down to a petri dish continuously perfused with saline solution, containing the following substances (in mM) NaC1, 130; KC1, 4; CaCI2, 3.75; MgSO4,
0304-3940/96/$12.00 © 1996 ElsevierScienceIreland Ltd. All rights reserved PII S0304-3940(96) 1290zl-9
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L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
1.3; NaH2PO4, 0.58; NaHCO3, 25; glucose, 10, bubbled with a 95% 02_5% CO2 mixture, and adjusted to pH 7.4. A dorsal craniotomy and laminectomy were performed. The brainstem, spinal cord and lumbar roots were then removed and pinned down with the ventral side up in the recording chamber (Fig. 1A). The pia was removed to give access to the lumbar motoneurons which were recorded intracellularly using glass microelectrodes filled with 1.5 M K-acetate (40-70 Mfl resistance). Intracellular potentials were recorded using an Intra 767 amplifier (World Precision Instruments, USA) or an Axoclamp 2B (Axon Instruments, USA), connected through an Instrutech ITC16 MAC interface to a Macintosh Quadra 650 (software programs from Axon Instruments). Only neurons exhibiting a resting membrane potential of at least -60 mV were considered for analysis. The input resistance was monitored by applying a hyperpolarizing current pulse (from -0.2 to -0.5 nA; 200 ms) through the recording electrode 400 ms prior to dorsal root stimulation. The roots were stimulated with monopolar platinum electrodes placed in contact with the nerve and insulated with Vaseline. The stimulation voltage ranged from 2 to 6 V (corresponding to 1-2 x threshold intensity (T)) and the pulse duration was 0.3 ms. Dorsal roots were stimulated at a low frequency (every 30 s) to allow recovery from the prolonged synaptic depression that has been described in this preparation [1 l]. Motoneurons were identified by the antidromic action potentials they produced in response to ventral root stimulation (inset in Fig. 1A). The latencies of the antidromic and dorsal root responses were 4.4 ± 1.7 ms (mean _+ SD) and 10.5 ± 2.3 ms, respectively. In individual motoneurons, the delay between these responses was 6 ± 1.9 ms, similar to the delays of monosynaptic dorsal root potentials in neonatal rats [9,15]. Seventeen out of the 30 cells tested were identified as motoneurons, eight in L3 (out of l0 cells recorded in this segment), four in L4 (11) and five in L5 (9). No difference in the results below was observed between the identified motoneurons and the other cells. Drugs used were (+)-baclofen (1-10 /xM; RBI, Natick, USA) and mephenesin (1 m M ; Sigma, St. Louis, MO, USA). CGP 35348 (100-150 /xM) and CGP 55845A (100-200 nM) were generous gifts from Ciba-Geigy. Drugs applied in the perfusion medium reached the recording chamber after a delay of about 60 s. Since EPSPs evoked by dorsal root stimulation at a strength of 1.2-1.5 × T triggered an action potential in lumbar motoneurons, a stimulation intensity subthreshold for the orthodromic spike was used to investigate the effect of baclofen on the postsynaptic potentials (PSPs) amplitude (Fig. 1B; 1.1 × T). These responses were composed of many components with a mixture of EPSPs and inhibitory postsynaptic potentials (IPSPs) [15] which were reversed at resting membrane potential (from -65 to -70 mV; reversal of IPSPs a t - 5 0 t o - 6 0 mV). Bath application of baclofen (2/~M) decreased the amplitude of all components of the response attenuating the long-latency
L~ m V A
~
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20 ms Fig. 1. Baclofen reduces the dorsal root evoked potentials in lumbar motoneurons. (A) Experimental protocol. The trace is the averaged (10 sweeps) antidromic response to stimulation (stim) of the ipsilateral L5 ventral root (VR). (B) Baclofen (2 #M) caused a reduction of the responses evoked by stimulation of the ipsilateral homonymous dorsal root (DR) in this L5 motoneuron. Traces are averages of five consecutive sweeps elicited at 30 s intervals, before (control) and 2, 5 and 7 mill after starting the perfusion of baclofen.
responses before the early EPSPs (Fig. 1B). Short- and long-latency responses are likely transmitted by monoand polysynaptic pathways, respectively [17]. The activity of polysynaptic pathways is greatly reduced by perfusing a saline with either a high concentration of divalent cations (7.5 mM Ca+++ 8 mM Mg++; [7]) or mephenesin [6,11,17]. We therefore used these modified solutions to investigate the effect of baclofen on the monosynaptic response (Fig. 2 with high concentration of Ca +÷ and Mg +÷ (n = 4 neurons recorded in this condition) and Fig. 3 with mephenesin (n = 8)). In both conditions, baclofen caused a reduction of the amplitude of the EPSPs (measured from onset to peak) in a concentration-dependent manner (Fig. 2A1,B (open circles)). EPSPs almost disappeared with baclofen at a concentration of 10 #M (95% reduction). Since polysynaptic responses may still be evoked in the presence of either high concentrations of Ca ++ and Mg ++ or mephenesin (see Ref. [17]) and the minimum delay from the mono- to the polysynaptic reflex has been estimated to be 4 ms [10], we have measured the amplitude of the EPSP at 3.5 ms from the onset (Fig. 2A2 (vertical dotted line)). With increasing concentrations of baclofen of 1, 3, 5 and 10/~M the monosynaptic EPSP decreased to 68, 35, 23 and 10% compared with control, respectively (Fig. 2A1,B (filled circles)), while the input resistance (17 Mfl) and the membrane potential (from -68 to -69 mV) were not affected (see also Fig. 3B,C). Fig. 2C represents the baclofen concentration-dependent reduction of the monosynaptic EPSP for all the cells tested (n = 13). There was no significant change of the membrane potential ( < 1 mV) and input resistance (<5%). These results suggest that the depression of the dorsal root evoked EPSPs, at
L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
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Baclofen concentration 0aM) Fig. 2. Baclofen reduces the monosynaptic EPSP in a concentration-dependent manner. (A1,A2) Responses evoked by dorsal root stimulation in an L5 neuron with various concentrations of baclofen in the bath (modified saline with a high concentration of divalent cations (7.5 mM Ca +÷ + 8 mM Mg ÷ +). Traces are averages of 10 consecutive sweeps. (A2) Enlarged portion of (A1) with a higher sweep velocity. The horizontal dashed line indicates the resting membrane potential level (around - 68 mV); the vertical dashed line shows the time at which the amplitude of the monosynaptic EPSP was measured (3.5 ms from the EPSP onset). (B) Time course of the amplitude of the dorsal root evoked EPSP (from onset to peak, open circles; from onset to 3.5 ms, filled circles) before, during and after (washout) the application of baclofen at the increasing concentrations indicated. Each dot is the mean ( + SEM) amplitude of five consecutive dorsal root evoked EPSPs. Amplitudes are expressed as percentages of the mean amplitude obtained before the application of baclofen (dashed line at 100%; 4.5 mV for the filled circles, 20.9 mV for the open circles; n = 12). Note that the difference was highly significant (P < 0.001) for both amplitudes with baclofen concentrations as low as 1 ttM, as compared to control. (C) Percentage of reduction ( + SEM) of the monosynaptic EPSP (measured at 3.5-4 ms from the onset) following application of baclofen at the various concentrations indicated below each column. The number of cells tested is indicated above each column.
low concentrations of baclofen, may be exerted, at least partly, at a presynaptic level. The baclofen-induced depression of the monosynaptic EPSP was blocked by adding the GABAs receptor antagonist CGP 35348 (100 #M) or CGP 55845A (200 nM) to the baclofen (2 #M) containing solution (Fig. 3A-D). Washing out the antagonist w]aile keeping baclofen in the perfusion caused a reduction of the amplitude of EPSPs within 20-30 min, which shows that the increase of amplitude in the presence of GABAB :receptor antagonists is not due to a desensitization of receptors (Fig. 3B,C). Washout of CGP 35348 takes shorter time than washout of CGP 55845 (see Ref. [12]). Both antagoni~sts had no significant effect on the membrane potential (Fig. 3B) and the input resistance (Fig. 3C). The amplitude of the monosynaptic component of dorsal root evoked potentials was not significantly different with GABAB receptor antagonists in the bath as compared to control (e:ither normal solution (n = 8) or baclofen containing solution (n = 9); see, however, the slight increase in the presence of CGP 55845A in Fig. 3A). The present study conJirrns previous observations of the depression of the dorsal root evoked potentials by baclofen
in 6-16 day old rats [11,13,16], and extends them to the very first days following birth (P0-P5). With low concentrations of baclofen, it is likely that the depression of EPSPs is exerted at a presynaptic level [16]. However, a postsynaptic action, even without any significant change of the passive membrane properties of the motoneurons, cannot be excluded. Presynaptic GABAB receptors may inhibit the synaptic transmission by depressing voltage-gated calcium channels and thus decreasing the calcium influx that triggers transmitter release (see Ref. [12] for review). These results further demonstrate that CGP 35348 and CGP 55845A antagonize the baclofen-induced reduction of dorsal root evoked EPSPs. The presynaptic GABAs receptors involved in the reduction of glutamate release from primary afferents in neonatal rat are therefore baclofen sensitive, phaclofen insensitive [16] but sensitive to CGP 35348 and CGP 55845A (see the classification of GABAB receptors proposed on the basis of release studies [2]). These two GABAa receptor antagonists may be useful tools to study the evolution of presynaptic inhibition during ontogenesis and its contribution to the development of motor control.
L. Vinay, F. Clarac / Neuroscience Letters 214 (1996) 103-106
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Fig. 3. CGP 55845A and CGP 35348 block the baclofen-induced depression of the monosynaptic EPSP. (A) EPSPs evoked by dorsal root stimulation in an IA neuron under control conditions and in the presence of baclofen (2/~M), either alone or together with CGP 55845A (200 nM). Traces are averages of five consecutive sweeps. (B) Time course of the mean ( + SEM) (filled triangles; n = 5 consecutive sweeps) amplitude of EPSPs and membrane potential (small squares). The response was significantly reduced (P < 0.001) under baclofen as compared to control. (C) Time course of the input resistance (open circles) and amplitude of EPSPs (filled triangles; both expressed as percentages of the control mean values) in another 1_.4motoneuron during application of baclofen (2 t~M), either alone or together with CGP 55845A (200 nM). Error bars are in most cases smaller than the symbol size. Note in (B) and (C) that, although there are spontaneous changes of the membrane potential and input resistance, there is no significant drift accompanying the decrease or increase of EPSP amplitude. (D) Averaged (10 traces) EPSPs evoked by dorsal root stimulation in an L4 motoneuron under control conditions, in the presence of 2 pM baclofen, and after 100 #M CGP 35348 has been added to the baclofen containing solution. Bathing solutions in (A,B,D) contained 1 mM mephenesin.
The authors wish to thank Ciba-Geigy for their gift of CGP 35348 and CGP 55845A and Dr. J.-Y. Barthe and Dr. J.-R. Cazalets for critical reading of the manuscript. [1] Bittiger, H., Froestl, W., Mickel, S.J. and Olpe, H.-R., GABAB receptor antagonists: from synthesis to therapeutic applications, Trends Phannacot. Sci., 14 (1993) 391-394. [2] Bonanno, G. and Raiteri, M., Multiple GABAB receptors, Trends Pharmacol. Sci., 14 (1993) 259-261. [3] Curtis, D.R. and Lacey, G., GABA-B receptur-mediated spinal inhibition, NeoroReport, 5 (1994) 540-542. [4] Curtis, D.R., Lodge, D., Bomstein, J.C. and Peet, M.J., Selective effects of baclofen on spinal synaptic transmission in the cat, Exp. Brain Res., 42 (1981) 158-170. [5] Frank, K. and Fuortes, M.G.F., Presynaptic and postsynaptic inhibition of monosynaptic reflexes, Fed. Proc., 16 (1957) 39-40. [6] Fulton, B.P, and Walton, K., Electrophysiological properties of neonatal rat motoneurones studied in vitro, J. Physiol., 370 (1986) 651-678. [7] Jahr, C.E. and Yoshioka, K., Ia afferent excitation of motoneurones in the in vitro new-born rat spinal cord is selectively antagonized by kynurenate, J. Physiol., 370 (1986) 515-530. [8] Jimdnez, I., Rudomin, P. and Enriquez, M., Differential effects of (+)-baciofen on Ia and descending monosynaptic EPSPs, Exp. Brain Res., 85 (1991) 103-113. [9] Kudo, N. and Yamada, T., Development of the monosynaptic
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[11]
[12]
[13]
[14] [15]
[16]
[17]
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