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Flumazenil does not affect intracortical motor excitability in humans: a transcranial magnetic stimulation study
Clinical Neurophysiology 115 (2004) 325–329 www.elsevier.com/locate/clinph
Flumazenil does not affect intracortical motor excitability in humans: a transcranial magnetic stimulation study H.Y. Junga, Y.H. Sohnb, A. Masonb, E. Considineb, M. Hallettb,* a Department of Rehabilitation Medicine, Inha University College of Medicine, Inchon, South Korea Human Motor Control Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5N226, 10 Center Drive, MSC1428, Bethesda, MD 20892-1428, USA
b
Accepted 17 September 2003
Abstract Objective: The motor cortex may be subject to tonic inhibitory drive. One inhibitory mechanism is supported by activity at benzodiazepine (BZP) receptors. In this study we investigate whether or not the BZP antagonist, flumazenil, increases cortical motor excitability in humans. Methods: Eight healthy subjects received a 1 mg intravenous (i.v.) loading dose of flumazenil followed by a 0.5 mg i.v. infusion over the next 30 min. Before, during, and 1 h after flumazenil infusion, we measured cortical motor excitability using transcranial magnetic stimulation (TMS). This included resting motor threshold (rMT), paired-pulse measurements of intracortical inhibition and facilitation (ICI and ICF), recruitment curve (RC), and silent period (SP). We also measured F response and compound muscle action potential (CMAP) with peripheral nerve stimulation. The study was carried out using a randomized, double-blind crossover design controlled with a saline infusion. Results: None of the measures of cortical or peripheral excitability were significantly affected by drug administration. Conclusions: Our findings suggest that flumazenil has no effect on cortical motor excitability in normal humans. Significance: There does not appear to be any tonic activity at benzodiazepine receptors in the normal resting human motor cortex. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Flumazenil; Benzodiazepine; g-Aminobutyric acid; Cortical excitability; Transcranial magnetic stimulation
1. Introduction Cortical excitability can be modulated by a change in activity of g-animobutyric acid (GABA). Agents which enhance GABA increase intracortical inhibition (Ziemann et al., 1996). Events such as limb deafferentation and stroke decrease intracortical inhibition (Gomez-Fernandez, 2000; Hallett et al., 1999), which was mainly induced by reduced GABA activity. Reduced GABA inhibition can facilitate long-term potentiation (LTP) (Hallett, 1999; Nudo et al., 1996) and this process may be related to use-dependent plasticity in healthy subjects (Bu¨tefisch et al., 2000) and in stroke patients (Liepert et al., 2000; Taub and Morris, 2001). This suggests that if GABA is also inhibited pharmacologically, it might be a new strategy to enhance physical therapy in stroke patients. However, since clinically there is no * Corresponding author. Tel.: þ 1-301-496-9526; fax: þ1-301-480-2286. E-mail address: [email protected] (M. Hallett).
GABA antagonist available, we chose flumazenil for this study. Flumazenil is a potent BZP antagonist and competitively interacts at central BZP receptors to antagonize or reverse the behavioral, neurologic, and electrophysiologic effects of BZP agonists (Cone and Stott, 1994; Hoffman and Warren, 1993) and inverse agonists (Rex and Brogden, 1988). A BZP agonist, lorazepam, which enhances GABA activity, increases intracortical inhibition when tested with a pairedpulse paradigm in TMS (Di Lazzaro et al., 2000) while flumazenil reverses the effect of diazepam-induced intracortical inhibition on the motor cortex (Palmieri et al., 1999). According to these findings, it might be proposed that flumazenil would reverse the effect of GABA in the brain. However, the relationship between GABA and flumazenil without BZPs, i.e. the effect of flumazenil itself, is still not well known. If there is normally tonic activity at the BZP receptor, then flumazenil should reduce brain inhibition.
1388-2457/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00335-3
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H.Y. Jung et al. / Clinical Neurophysiology 115 (2004) 325–329
The purpose of this study was to investigate the effects of flumazenil on cortical excitability in healthy human subjects. We wished to determine if a therapeutic dose of intravenous (i.v.) flumazenil administration alters cortical excitability as measured by TMS.
2. Subjects and methods 2.1. Subjects Eight right-handed healthy subjects (2 females and 6 males, mean age 27.8 ^ 16.2 years, weighing 70 – 80 kg) were studied. The investigational procedure was explained to all subjects and all gave their written informed consent before the study. All procedures were approved by the Institutional Review Board. 2.2. Experimental procedures All subjects refrained from alcohol, caffeine-containing beverages, and food from the midnight before the investigation. The entire study took place in the morning and in the same room. Each subject participated in two experimental sessions (flumazenil or placebo) separated by at least one week. The experiment consisted of 3 parts. In the first part, an i.v. catheter was inserted in an antecubital vein in the dominant arm to administer 500 ml of 5% glucose solution and all were tested with the baseline TMS studies. In the second part, TMS studies were done in the steady state of an infusion of flumazenil or placebo. Flumazenil (Anexate, Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) or placebo was given as an i.v. bolus over 3 min (1 mg mixed in 10 ml of 5% glucose solution) followed by a 0.5 mg continuous i.v. infusion of flumazenil or placebo (mixed in 50 ml of 5% glucose solution) using a constant rate infusion pump for about 30 min. After finishing the second part, the i.v. infusion of flumazenil or placebo was discontinued and subjects only kept an i.v. line with 500 ml of 5% glucose solution to maintain the same condition as in the first experiment. One hour after testing the second part, a third set of TMS studies was carried out. Flumazenil and placebo were given in a double-blind, randomized, and crossover design. 2.3. Transcranial magnetic stimulation In all subjects, motor evoked potentials (MEPs) were recorded from the left first dorsal interosseus (FDI) muscles using Ag-AgCl surface electrodes in a belly-tendon montage. EMG amplitude was amplified using a conventional EMG machine (Counterpoint; Dantec Electronics, Skovlunde, Denmark) with bandpass between 20 and 2000 Hz. Auditory EMG feedback was given to ensure complete, voluntary relaxation of the target muscles. The EMG signal was digitalized at a frequency of 5 kHz and fed into a laboratory computer for further off-line analysis.
Focal TMS was applied through a figure-of-8 shaped magnetic coil (each loop 70 mm in diameter) connected to two MAGSTIM 200 stimulators via a Bistim module (Magstim, Whitland, Dyfed, UK). The magnetic coil was placed over the scalp overlying the right motor cortex at the optimal site for left FDI activation. The coil was held tangentially to the skull with the handle pointing back and lateally at a 458 away from the midline. Thus, the current induced in the brain neural tissue was directed approximately perpendicularly toward the position of the central sulcus. This is thought to be the best position for activating the pyramidal cells transsynaptically (Brasil-Neto et al., 1992). We used TMS to evaluate the effects of flumazenil on different measures of cortical excitability, including rMT (resting motor threshold), ICI (intracortical inhibition) and ICF (intracortical facilitation), SP (silent period), RC (recruitment curve) and F response. rMT was determined to the nearest 1% of the maximum stimulator output, and is defined as the minimal stimulus intensity required to produce MEPs of . 50 mV in at least 5 of 10 consecutive trials. MEP size was determined by averaging the peak-to-peak amplitudes over 10 single trials, each at stimulus intensities of 30– 50% of maximum stimulator output above the individual rMT. For the paired pulse paradigm, the stimulus intensity of the first, conditioning stimulus was set at 70% of rMT. The test stimulus was applied with 140% rMT. Single test pulses and paired stimuli with interstimulus interval (ISIs) of 2 and 15 ms were delivered 5 s apart in a random order; 20 trials were recorded for a single test and paired pulses at each ISI. The 2 ms interval was chosen to assess intracortical inhibition, and the 15 ms interval was chosen to assess intracortical facilitation. SP was measured in 15 trials at a stimulus intensity of 140% active MT in moderately active FDI. TMS was set to provide stimuli only when the EMG activity of FDI was maintained with 10– 20% of maximal voluntary contraction. The amount of voluntary muscle activation was controlled with auditory feedback. SP duration was defined as the time from the beginning of the magnetic stimulus to the first return of voluntary EMG activity. Peak-to-peak amplitudes for testing RC were measured in the resting FDI muscle at stimulation intensities of 120, 140, and 160% rMT. TMS stimuli were delivered randomly between 5 and 7 s apart, with 20 stimuli for each stimulus intensity beginning with the lowest intensity, i.e. 120% RMT. MEP amplitudes were related to the compound muscle action potential (CMAP) following supramaximal stimulation of the ulnar nerve at the wrist and expressed as a percent of CMAP. The ulnar nerve at the wrist was stimulated electrically and maximum peak-to-peak M-wave amplitude was measured. With this supramaximal stimulation, 20 stimulations in the relaxed FDI muscle were performed, and F responses were recorded and averaged.
H.Y. Jung et al. / Clinical Neurophysiology 115 (2004) 325–329
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Table 1 Motor excitability measures Measure
Flumenazil Baseline
Motor threshold (% of maximal output) Resting Paired simulations ICI (%) ICF (%) Recruitment curves 120% RMT 140% RMT 160% RMT Cortical silent period (ms) Peripheral nerve stimulation F-wave (% CMAP) CMAP (mV)
Placebo During
After 1 h
Baseline
During
After 1 h
42.3 ^ 3.5
42.5 ^ 3.4
42.8 ^ 3.3
42.6 ^ 3.4
42.3 ^ 3.0
42.0 ^ 2.9
68.0 ^ 17.9 100.0 ^ 15.1
66.0 ^ 13.8 118.0 ^ 17.8
64.0 ^ 16.9 113.0 ^ 15.0
67.0 ^ 18.0 110.0 ^ 16.7
69.0 ^ 19.1 107.0 ^ 13.7
68.0 ^ 10.5 112.0 ^ 14.4
6.0 ^ 1.5 10.8 ^ 2.6 14.7 ^ 3.3 167.0 ^ 11.5
7.0 ^ 2.0 13.9 ^ 3.7 16.5 ^ 5.0 159.0 ^ 10.0
7.3 ^ 2.7 14.8 ^ 3.1 17.9 ^ 4.9 163.0 ^ 8.7
6.9 ^ 1.9 12.4 ^ 3.2 15.5 ^ 3.7 170.0 ^ 13.9
7.6 ^ 1.5 14.6 ^ 3.0 16.4 ^ 4.6 177.0 ^ 17.4
7.4 ^ 2.3 14.3 ^ 3.9 16.1 ^ 4.0 177.0 ^ 13.6
6.7 ^ 1.2 4.9 ^ 0.4
5.7 ^ 1.2 4.9 ^ 0.5
6.7 ^ 1.1 4.9 ^ 0.4
6.1 ^ 1.0 4.9 ^ 0.3
5.8 ^ 1.2 5.0 ^ 0.3
5.9 ^ 1.0 5.2 ^ 0.4
All variables are expressed as mean ^ SE. Significance level indicates P , 0:05, analyzed by repeated measures analysis of variance. ICI, intracortical inhibition; ICF, intracortical facilitation; RMT, resting motor threshold; CMAP, compound muscle action potential.
2.4. Statistical analysis
3. Results
Data are given as mean ^ standard error of mean (SEM). Statistical analysis was performed with an analysis of variance for repeated measurements. The measures obtained were compared with the baseline data in post-hoc comparisons using Tukey’s test. Differences were regarded as significant when P , 0:05.
All subjects tolerated the experiment without any serious adverse effects or complications, except for subjective mild dizziness in two subjects and light-headedness in one subject, all with the flumazenil infusions. Flumazenil failed to produce any significant changes in motor cortical excitability measured by rMT, ICI, ICF, RC, and cortical
Fig. 1. Resting motor threshold (rMT: left side) and silent period (SP: right side) are shown as averaged data for the two groups (B, flumazenil; X, placebo) and expressed as the percent of maximum stimulator output and duration (ms) of the silence, respectively (y axis). x Axis represents the time intervals before, during and after intravenous infusion of flumazenil.
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Fig. 2. The effect of flumazenil (W) and placebo (X) on the recruitment curves of MEP amplitudes at different time with the intravenous infusion of flumazenil, recorded in FDI. MEP values are expressed as percent of maximum compound muscle action potential amplitude at 120% rMT (A), 140% rMT (B) and 160% rMT (C) stimulation.
SP, as well as spinal and peripheral excitability (F response and CMAP), compared to the baseline test and the test done 1 h after cessation of flumazenil infusion (Table 1 and Figs. 1– 3).
4. Discussion Our study investigating the human motor cortex excitability under the influence of the BZP antagonist, flumazenil, shows that administration of a therapeutic i.v. dose of this drug in healthy subjects did not significantly modify cortical motor excitability. This suggests that flumazenil had no intrinsic effect on the cortex at such a dose or it had a very weak effect that could not be detected by TMS.
Following i.v. administration of single doses of 1 mg of flumazenil to healthy subjects, average peak plasma concentrations were 7– 8 mg/l, 5 min after completion of the bolus injection (Klotz et al., 1986; Roncari et al., 1986). The distribution half-life of flumazenil following single i.v. doses administered to healthy subjects was about 5 min. Peak plasma concentration rapidly declined, becoming undetectable after 4– 6 h; the bioavailability of flumazenil is also very low because of presystemic metabolism of 70 –80% (Cone and Stott, 1994). Most studies, however, were done with oral administration (Roncari et al., 1986) or only a single i.v. infusion (Neave et al., 2000; Schopf et al., 1984; Smolnik et al., 1998). The pharmacokinetics may contribute to the different results of previous studies. For our TMS study, a continuous i.v. infusion method was used because we required a flatter plasma concentration-time profile around the peak concentration, and the same or similar plasma drug concentration must be supplied for a cortical excitability study of about 30 –40 min. The intrinsic effect of flumazenil is still controversial and previous results should be interpreted cautiously. Flumazenil has been reported to have weak BZP antagonist effects (Schopf et al., 1984); no BZP effect (Breimer et al., 1991; File et al., 1982); and partial agonist properties (Neave et al., 2000). These differences are certainly due in part to the different methods and different dosages of drug administration. TMS of the cortex allows evaluation of the excitability of motor corticospinal pathways in experimental or pathologic conditions. Specifically, this technique can be used to investigate cortical motor excitability. Flumazenil’s lack of effect on all TMS measures (i.e. rMT, ICI, ICF, RC and SP) suggests that in healthy subjects, the pharmacological action of a therapeutic i.v. dose of flumazenil does not affect neuronal excitability mediated through GABAergic or glutamergic mechanisms or alternatively, there is no tonic activity at BZP receptors in the motor cortex. One possibility is that even though flumazenil competitively interacts with BZP in the motor cortex, GABAA/BZP receptors may not be directly affected by flumazenil. This is supported by the fact that BZP binding does not compete with GABA (Schofield et al., 1987), but BZPs bind to an additional site on the GABAA receptor-ion channel complex (Mohler and Okada, 1977; Schofield et al., 1987). Therefore, flumazenil may not directly modulate GABA receptors without BZPs also being present. In addition, if BZP receptors are not intrinsically very active in healthy subjects, then flumazenil can affect GABA. The second possibility is that even though reduced GABAergic activity was induced by flumazenil in the motor cortex at this therapeutic dose, it may be too small to be detected by TMS measures. Since we cannot exclude that this was a dose-related phenomenon, further studies may be needed.
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Fig. 3. Intracortical inhibition (left side) and intracortical facilitation (right side) are averaged across inhibitory interstimulus intervals (ISI 2 ms) and facilitatory intervals (15 ms), respectively (B, flumazenil; X, placebo). The x axis represents the time intervals before, during and after intravenous infusion of flumazenil. MEP size is expressed in percent as the ratio of conditioned motor evoked potential (MEP) versus control MEP amplitude (y axis).
Acknowledgements The authors thank Aaron H. Burstein, Ph.D., for his assistance in the study design and Devera G. Schoenberg, M.Sc., for skillful editing of the manuscript.
References Brasil-Neto JP, Cohen LG, Panizza M, Nilsson J, Roth BJ, Hallett M. Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity. J Clin Neurophysiol 1992;9:132 –6. Breimer LT, Hennis PJ, Burm AG, Danhof M, Bovill JG, Spierdijk J, Vletter AA. Pharmacokinetics and EEG effects of flumazenil in volunteers. Clin Pharmacokinet 1991;20:491–6. Bu¨tefisch CM, Davis BC, Wise SP, Sawaki L, Kopylev L, Classen J, Cohen LG. Mechanisms of use-dependent plasticity in the human motor cortex. Proc Natl Acad Sci USA 2000;97:3661–5. Cone AM, Stott SA. Flumazenil. Br J Hosp Med 1994;51:346 –8. Di Lazzaro V, Oliviero A, Meglio M, Cioni B, Tamburrini G, Tonali P, Rothwell JC. Direct demonstration of the effect of lorazepam on the excitability of the human motor cortex. Clin Neurophysiol 2000;111: 794–9. File SE, Lister RG, Nutt DJ. Intrinsic actions of benzodiazepine antagonists. Neurosci Lett 1982;32:165–8. Gomez-Fernandez L. Cortical plasticity and restoration of neurologic functions: an update on this topic. Rev Neurol 2000;31:749 –56. Hallett M. Motor cortex plasticity. Electroenceph clin Neurophysiol Suppl 1999;50:85 –91. Hallett M, Chen R, Ziemann U, Cohen LG. Reorganization in motor cortex in amputees and in normal volunteers after ischemic limb deafferentation. Electroenceph clin Neurophysiol Suppl 1999;51: 183–7. Hoffman EJ, Warren EW. Flumazenil: a benzodiazepine antagonist. Clin Pharm 1993;12:641–56.
Klotz U, Ziegler G, Rosenkranz B, Mikus G. Does the benzodiazepine antagonist Ro 15-1788 antagonize the action of ethanol? Br J Clin Pharmacol 1986;22:513 –20. Liepert J, Graef S, Uhde I, Leidner O, Weiller C. Training-induced changes of motor cortex representations in stroke patients. Acta Neurol Scand 2000;101:321–6. Mohler H, Okada T. Properties of 3H-diazepam binding to benzodiazepine receptors in rat cerebral cortex. Life Sci 1977;20:2101–10. Neave N, Reid C, Scholey AB, Thompson JM, Moss M, Ayre G, Wesnes K, Girdler NM. Dose-dependent effects of flumazenil on cognition, mood, and cardio- respiratory physiology in healthy volunteers. Br Dent J 2000;189:668–74. Nudo RJ, Wise BM, Sifuentes F, Milliken GW. Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 1996;272:1791– 4. Palmieri MG, Iani C, Scalise A, Desiato MT, Loberti M, Telera S, Caramia MD. The effect of benzodiazepines and flumazenil on motor cortical excitability in the human brain. Brain Res 1999;815:192 –9. Rex N, Brogden KLG. Flumazenil: a preliminary review of its benzodiazepine antagonist properties, intrinsic and therapeutic use. Drugs 1988;35:448 –67. Roncari G, Ziegler WH, Guentert TW. Pharmacokinetics of the new benzodiazepine antagonist Ro 15-1788 in man following intravenous and oral administration. Br J Clin Pharmacol 1986;22:421 –8. Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, Seeburg PH, Barnard EA. Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 1987;328:221–7. Schopf J, Laurian S, Le PK, Gaillard JM. Intrinsic activity of the benzodiazepine antagonist Ro 15-1788 in man: an electrophysiological investigation. Pharmacopsychiatry 1984;17:79–83. Smolnik R, Pietrowsky R, Fehm HL, Born J. Enhanced selective attention after low-dose administration of the benzodiazepine antagonist flumazenil. J Clin Psychopharmacol 1998;18:241–7. Taub E, Morris DM. Constraint-induced movement therapy to enhance recovery after stroke. Curr Atheroscler Rep 2001;3:279 –86. Ziemann U, Lonnecker S, Steinhoff BJ, Paulus W. The effect of lorazepam on the motor cortical excitability in man. Exp Brain Res 1996;109:127–35.
Flumazenil does not affect intracortical motor excitability in humans: a transcranial magnetic stimulation study H.Y. Junga, Y.H. Sohnb, A. Masonb, E. Considineb, M. Hallettb,* a Department of Rehabilitation Medicine, Inha University College of Medicine, Inchon, South Korea Human Motor Control Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Building 10, Room 5N226, 10 Center Drive, MSC1428, Bethesda, MD 20892-1428, USA
b
Accepted 17 September 2003
Abstract Objective: The motor cortex may be subject to tonic inhibitory drive. One inhibitory mechanism is supported by activity at benzodiazepine (BZP) receptors. In this study we investigate whether or not the BZP antagonist, flumazenil, increases cortical motor excitability in humans. Methods: Eight healthy subjects received a 1 mg intravenous (i.v.) loading dose of flumazenil followed by a 0.5 mg i.v. infusion over the next 30 min. Before, during, and 1 h after flumazenil infusion, we measured cortical motor excitability using transcranial magnetic stimulation (TMS). This included resting motor threshold (rMT), paired-pulse measurements of intracortical inhibition and facilitation (ICI and ICF), recruitment curve (RC), and silent period (SP). We also measured F response and compound muscle action potential (CMAP) with peripheral nerve stimulation. The study was carried out using a randomized, double-blind crossover design controlled with a saline infusion. Results: None of the measures of cortical or peripheral excitability were significantly affected by drug administration. Conclusions: Our findings suggest that flumazenil has no effect on cortical motor excitability in normal humans. Significance: There does not appear to be any tonic activity at benzodiazepine receptors in the normal resting human motor cortex. q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. Keywords: Flumazenil; Benzodiazepine; g-Aminobutyric acid; Cortical excitability; Transcranial magnetic stimulation
1. Introduction Cortical excitability can be modulated by a change in activity of g-animobutyric acid (GABA). Agents which enhance GABA increase intracortical inhibition (Ziemann et al., 1996). Events such as limb deafferentation and stroke decrease intracortical inhibition (Gomez-Fernandez, 2000; Hallett et al., 1999), which was mainly induced by reduced GABA activity. Reduced GABA inhibition can facilitate long-term potentiation (LTP) (Hallett, 1999; Nudo et al., 1996) and this process may be related to use-dependent plasticity in healthy subjects (Bu¨tefisch et al., 2000) and in stroke patients (Liepert et al., 2000; Taub and Morris, 2001). This suggests that if GABA is also inhibited pharmacologically, it might be a new strategy to enhance physical therapy in stroke patients. However, since clinically there is no * Corresponding author. Tel.: þ 1-301-496-9526; fax: þ1-301-480-2286. E-mail address: [email protected] (M. Hallett).
GABA antagonist available, we chose flumazenil for this study. Flumazenil is a potent BZP antagonist and competitively interacts at central BZP receptors to antagonize or reverse the behavioral, neurologic, and electrophysiologic effects of BZP agonists (Cone and Stott, 1994; Hoffman and Warren, 1993) and inverse agonists (Rex and Brogden, 1988). A BZP agonist, lorazepam, which enhances GABA activity, increases intracortical inhibition when tested with a pairedpulse paradigm in TMS (Di Lazzaro et al., 2000) while flumazenil reverses the effect of diazepam-induced intracortical inhibition on the motor cortex (Palmieri et al., 1999). According to these findings, it might be proposed that flumazenil would reverse the effect of GABA in the brain. However, the relationship between GABA and flumazenil without BZPs, i.e. the effect of flumazenil itself, is still not well known. If there is normally tonic activity at the BZP receptor, then flumazenil should reduce brain inhibition.
1388-2457/$30.00 q 2003 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S1388-2457(03)00335-3
326
H.Y. Jung et al. / Clinical Neurophysiology 115 (2004) 325–329
The purpose of this study was to investigate the effects of flumazenil on cortical excitability in healthy human subjects. We wished to determine if a therapeutic dose of intravenous (i.v.) flumazenil administration alters cortical excitability as measured by TMS.
2. Subjects and methods 2.1. Subjects Eight right-handed healthy subjects (2 females and 6 males, mean age 27.8 ^ 16.2 years, weighing 70 – 80 kg) were studied. The investigational procedure was explained to all subjects and all gave their written informed consent before the study. All procedures were approved by the Institutional Review Board. 2.2. Experimental procedures All subjects refrained from alcohol, caffeine-containing beverages, and food from the midnight before the investigation. The entire study took place in the morning and in the same room. Each subject participated in two experimental sessions (flumazenil or placebo) separated by at least one week. The experiment consisted of 3 parts. In the first part, an i.v. catheter was inserted in an antecubital vein in the dominant arm to administer 500 ml of 5% glucose solution and all were tested with the baseline TMS studies. In the second part, TMS studies were done in the steady state of an infusion of flumazenil or placebo. Flumazenil (Anexate, Hoffmann-La Roche AG, Grenzach-Wyhlen, Germany) or placebo was given as an i.v. bolus over 3 min (1 mg mixed in 10 ml of 5% glucose solution) followed by a 0.5 mg continuous i.v. infusion of flumazenil or placebo (mixed in 50 ml of 5% glucose solution) using a constant rate infusion pump for about 30 min. After finishing the second part, the i.v. infusion of flumazenil or placebo was discontinued and subjects only kept an i.v. line with 500 ml of 5% glucose solution to maintain the same condition as in the first experiment. One hour after testing the second part, a third set of TMS studies was carried out. Flumazenil and placebo were given in a double-blind, randomized, and crossover design. 2.3. Transcranial magnetic stimulation In all subjects, motor evoked potentials (MEPs) were recorded from the left first dorsal interosseus (FDI) muscles using Ag-AgCl surface electrodes in a belly-tendon montage. EMG amplitude was amplified using a conventional EMG machine (Counterpoint; Dantec Electronics, Skovlunde, Denmark) with bandpass between 20 and 2000 Hz. Auditory EMG feedback was given to ensure complete, voluntary relaxation of the target muscles. The EMG signal was digitalized at a frequency of 5 kHz and fed into a laboratory computer for further off-line analysis.
Focal TMS was applied through a figure-of-8 shaped magnetic coil (each loop 70 mm in diameter) connected to two MAGSTIM 200 stimulators via a Bistim module (Magstim, Whitland, Dyfed, UK). The magnetic coil was placed over the scalp overlying the right motor cortex at the optimal site for left FDI activation. The coil was held tangentially to the skull with the handle pointing back and lateally at a 458 away from the midline. Thus, the current induced in the brain neural tissue was directed approximately perpendicularly toward the position of the central sulcus. This is thought to be the best position for activating the pyramidal cells transsynaptically (Brasil-Neto et al., 1992). We used TMS to evaluate the effects of flumazenil on different measures of cortical excitability, including rMT (resting motor threshold), ICI (intracortical inhibition) and ICF (intracortical facilitation), SP (silent period), RC (recruitment curve) and F response. rMT was determined to the nearest 1% of the maximum stimulator output, and is defined as the minimal stimulus intensity required to produce MEPs of . 50 mV in at least 5 of 10 consecutive trials. MEP size was determined by averaging the peak-to-peak amplitudes over 10 single trials, each at stimulus intensities of 30– 50% of maximum stimulator output above the individual rMT. For the paired pulse paradigm, the stimulus intensity of the first, conditioning stimulus was set at 70% of rMT. The test stimulus was applied with 140% rMT. Single test pulses and paired stimuli with interstimulus interval (ISIs) of 2 and 15 ms were delivered 5 s apart in a random order; 20 trials were recorded for a single test and paired pulses at each ISI. The 2 ms interval was chosen to assess intracortical inhibition, and the 15 ms interval was chosen to assess intracortical facilitation. SP was measured in 15 trials at a stimulus intensity of 140% active MT in moderately active FDI. TMS was set to provide stimuli only when the EMG activity of FDI was maintained with 10– 20% of maximal voluntary contraction. The amount of voluntary muscle activation was controlled with auditory feedback. SP duration was defined as the time from the beginning of the magnetic stimulus to the first return of voluntary EMG activity. Peak-to-peak amplitudes for testing RC were measured in the resting FDI muscle at stimulation intensities of 120, 140, and 160% rMT. TMS stimuli were delivered randomly between 5 and 7 s apart, with 20 stimuli for each stimulus intensity beginning with the lowest intensity, i.e. 120% RMT. MEP amplitudes were related to the compound muscle action potential (CMAP) following supramaximal stimulation of the ulnar nerve at the wrist and expressed as a percent of CMAP. The ulnar nerve at the wrist was stimulated electrically and maximum peak-to-peak M-wave amplitude was measured. With this supramaximal stimulation, 20 stimulations in the relaxed FDI muscle were performed, and F responses were recorded and averaged.
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Table 1 Motor excitability measures Measure
Flumenazil Baseline
Motor threshold (% of maximal output) Resting Paired simulations ICI (%) ICF (%) Recruitment curves 120% RMT 140% RMT 160% RMT Cortical silent period (ms) Peripheral nerve stimulation F-wave (% CMAP) CMAP (mV)
Placebo During
After 1 h
Baseline
During
After 1 h
42.3 ^ 3.5
42.5 ^ 3.4
42.8 ^ 3.3
42.6 ^ 3.4
42.3 ^ 3.0
42.0 ^ 2.9
68.0 ^ 17.9 100.0 ^ 15.1
66.0 ^ 13.8 118.0 ^ 17.8
64.0 ^ 16.9 113.0 ^ 15.0
67.0 ^ 18.0 110.0 ^ 16.7
69.0 ^ 19.1 107.0 ^ 13.7
68.0 ^ 10.5 112.0 ^ 14.4
6.0 ^ 1.5 10.8 ^ 2.6 14.7 ^ 3.3 167.0 ^ 11.5
7.0 ^ 2.0 13.9 ^ 3.7 16.5 ^ 5.0 159.0 ^ 10.0
7.3 ^ 2.7 14.8 ^ 3.1 17.9 ^ 4.9 163.0 ^ 8.7
6.9 ^ 1.9 12.4 ^ 3.2 15.5 ^ 3.7 170.0 ^ 13.9
7.6 ^ 1.5 14.6 ^ 3.0 16.4 ^ 4.6 177.0 ^ 17.4
7.4 ^ 2.3 14.3 ^ 3.9 16.1 ^ 4.0 177.0 ^ 13.6
6.7 ^ 1.2 4.9 ^ 0.4
5.7 ^ 1.2 4.9 ^ 0.5
6.7 ^ 1.1 4.9 ^ 0.4
6.1 ^ 1.0 4.9 ^ 0.3
5.8 ^ 1.2 5.0 ^ 0.3
5.9 ^ 1.0 5.2 ^ 0.4
All variables are expressed as mean ^ SE. Significance level indicates P , 0:05, analyzed by repeated measures analysis of variance. ICI, intracortical inhibition; ICF, intracortical facilitation; RMT, resting motor threshold; CMAP, compound muscle action potential.
2.4. Statistical analysis
3. Results
Data are given as mean ^ standard error of mean (SEM). Statistical analysis was performed with an analysis of variance for repeated measurements. The measures obtained were compared with the baseline data in post-hoc comparisons using Tukey’s test. Differences were regarded as significant when P , 0:05.
All subjects tolerated the experiment without any serious adverse effects or complications, except for subjective mild dizziness in two subjects and light-headedness in one subject, all with the flumazenil infusions. Flumazenil failed to produce any significant changes in motor cortical excitability measured by rMT, ICI, ICF, RC, and cortical
Fig. 1. Resting motor threshold (rMT: left side) and silent period (SP: right side) are shown as averaged data for the two groups (B, flumazenil; X, placebo) and expressed as the percent of maximum stimulator output and duration (ms) of the silence, respectively (y axis). x Axis represents the time intervals before, during and after intravenous infusion of flumazenil.
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Fig. 2. The effect of flumazenil (W) and placebo (X) on the recruitment curves of MEP amplitudes at different time with the intravenous infusion of flumazenil, recorded in FDI. MEP values are expressed as percent of maximum compound muscle action potential amplitude at 120% rMT (A), 140% rMT (B) and 160% rMT (C) stimulation.
SP, as well as spinal and peripheral excitability (F response and CMAP), compared to the baseline test and the test done 1 h after cessation of flumazenil infusion (Table 1 and Figs. 1– 3).
4. Discussion Our study investigating the human motor cortex excitability under the influence of the BZP antagonist, flumazenil, shows that administration of a therapeutic i.v. dose of this drug in healthy subjects did not significantly modify cortical motor excitability. This suggests that flumazenil had no intrinsic effect on the cortex at such a dose or it had a very weak effect that could not be detected by TMS.
Following i.v. administration of single doses of 1 mg of flumazenil to healthy subjects, average peak plasma concentrations were 7– 8 mg/l, 5 min after completion of the bolus injection (Klotz et al., 1986; Roncari et al., 1986). The distribution half-life of flumazenil following single i.v. doses administered to healthy subjects was about 5 min. Peak plasma concentration rapidly declined, becoming undetectable after 4– 6 h; the bioavailability of flumazenil is also very low because of presystemic metabolism of 70 –80% (Cone and Stott, 1994). Most studies, however, were done with oral administration (Roncari et al., 1986) or only a single i.v. infusion (Neave et al., 2000; Schopf et al., 1984; Smolnik et al., 1998). The pharmacokinetics may contribute to the different results of previous studies. For our TMS study, a continuous i.v. infusion method was used because we required a flatter plasma concentration-time profile around the peak concentration, and the same or similar plasma drug concentration must be supplied for a cortical excitability study of about 30 –40 min. The intrinsic effect of flumazenil is still controversial and previous results should be interpreted cautiously. Flumazenil has been reported to have weak BZP antagonist effects (Schopf et al., 1984); no BZP effect (Breimer et al., 1991; File et al., 1982); and partial agonist properties (Neave et al., 2000). These differences are certainly due in part to the different methods and different dosages of drug administration. TMS of the cortex allows evaluation of the excitability of motor corticospinal pathways in experimental or pathologic conditions. Specifically, this technique can be used to investigate cortical motor excitability. Flumazenil’s lack of effect on all TMS measures (i.e. rMT, ICI, ICF, RC and SP) suggests that in healthy subjects, the pharmacological action of a therapeutic i.v. dose of flumazenil does not affect neuronal excitability mediated through GABAergic or glutamergic mechanisms or alternatively, there is no tonic activity at BZP receptors in the motor cortex. One possibility is that even though flumazenil competitively interacts with BZP in the motor cortex, GABAA/BZP receptors may not be directly affected by flumazenil. This is supported by the fact that BZP binding does not compete with GABA (Schofield et al., 1987), but BZPs bind to an additional site on the GABAA receptor-ion channel complex (Mohler and Okada, 1977; Schofield et al., 1987). Therefore, flumazenil may not directly modulate GABA receptors without BZPs also being present. In addition, if BZP receptors are not intrinsically very active in healthy subjects, then flumazenil can affect GABA. The second possibility is that even though reduced GABAergic activity was induced by flumazenil in the motor cortex at this therapeutic dose, it may be too small to be detected by TMS measures. Since we cannot exclude that this was a dose-related phenomenon, further studies may be needed.
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Fig. 3. Intracortical inhibition (left side) and intracortical facilitation (right side) are averaged across inhibitory interstimulus intervals (ISI 2 ms) and facilitatory intervals (15 ms), respectively (B, flumazenil; X, placebo). The x axis represents the time intervals before, during and after intravenous infusion of flumazenil. MEP size is expressed in percent as the ratio of conditioned motor evoked potential (MEP) versus control MEP amplitude (y axis).
Acknowledgements The authors thank Aaron H. Burstein, Ph.D., for his assistance in the study design and Devera G. Schoenberg, M.Sc., for skillful editing of the manuscript.
References Brasil-Neto JP, Cohen LG, Panizza M, Nilsson J, Roth BJ, Hallett M. Optimal focal transcranial magnetic activation of the human motor cortex: effects of coil orientation, shape of the induced current pulse, and stimulus intensity. J Clin Neurophysiol 1992;9:132 –6. Breimer LT, Hennis PJ, Burm AG, Danhof M, Bovill JG, Spierdijk J, Vletter AA. Pharmacokinetics and EEG effects of flumazenil in volunteers. Clin Pharmacokinet 1991;20:491–6. Bu¨tefisch CM, Davis BC, Wise SP, Sawaki L, Kopylev L, Classen J, Cohen LG. Mechanisms of use-dependent plasticity in the human motor cortex. Proc Natl Acad Sci USA 2000;97:3661–5. Cone AM, Stott SA. Flumazenil. Br J Hosp Med 1994;51:346 –8. Di Lazzaro V, Oliviero A, Meglio M, Cioni B, Tamburrini G, Tonali P, Rothwell JC. Direct demonstration of the effect of lorazepam on the excitability of the human motor cortex. Clin Neurophysiol 2000;111: 794–9. File SE, Lister RG, Nutt DJ. Intrinsic actions of benzodiazepine antagonists. Neurosci Lett 1982;32:165–8. Gomez-Fernandez L. Cortical plasticity and restoration of neurologic functions: an update on this topic. Rev Neurol 2000;31:749 –56. Hallett M. Motor cortex plasticity. Electroenceph clin Neurophysiol Suppl 1999;50:85 –91. Hallett M, Chen R, Ziemann U, Cohen LG. Reorganization in motor cortex in amputees and in normal volunteers after ischemic limb deafferentation. Electroenceph clin Neurophysiol Suppl 1999;51: 183–7. Hoffman EJ, Warren EW. Flumazenil: a benzodiazepine antagonist. Clin Pharm 1993;12:641–56.
Klotz U, Ziegler G, Rosenkranz B, Mikus G. Does the benzodiazepine antagonist Ro 15-1788 antagonize the action of ethanol? Br J Clin Pharmacol 1986;22:513 –20. Liepert J, Graef S, Uhde I, Leidner O, Weiller C. Training-induced changes of motor cortex representations in stroke patients. Acta Neurol Scand 2000;101:321–6. Mohler H, Okada T. Properties of 3H-diazepam binding to benzodiazepine receptors in rat cerebral cortex. Life Sci 1977;20:2101–10. Neave N, Reid C, Scholey AB, Thompson JM, Moss M, Ayre G, Wesnes K, Girdler NM. Dose-dependent effects of flumazenil on cognition, mood, and cardio- respiratory physiology in healthy volunteers. Br Dent J 2000;189:668–74. Nudo RJ, Wise BM, Sifuentes F, Milliken GW. Neural substrates for the effects of rehabilitative training on motor recovery after ischemic infarct. Science 1996;272:1791– 4. Palmieri MG, Iani C, Scalise A, Desiato MT, Loberti M, Telera S, Caramia MD. The effect of benzodiazepines and flumazenil on motor cortical excitability in the human brain. Brain Res 1999;815:192 –9. Rex N, Brogden KLG. Flumazenil: a preliminary review of its benzodiazepine antagonist properties, intrinsic and therapeutic use. Drugs 1988;35:448 –67. Roncari G, Ziegler WH, Guentert TW. Pharmacokinetics of the new benzodiazepine antagonist Ro 15-1788 in man following intravenous and oral administration. Br J Clin Pharmacol 1986;22:421 –8. Schofield PR, Darlison MG, Fujita N, Burt DR, Stephenson FA, Rodriguez H, Rhee LM, Ramachandran J, Reale V, Glencorse TA, Seeburg PH, Barnard EA. Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 1987;328:221–7. Schopf J, Laurian S, Le PK, Gaillard JM. Intrinsic activity of the benzodiazepine antagonist Ro 15-1788 in man: an electrophysiological investigation. Pharmacopsychiatry 1984;17:79–83. Smolnik R, Pietrowsky R, Fehm HL, Born J. Enhanced selective attention after low-dose administration of the benzodiazepine antagonist flumazenil. J Clin Psychopharmacol 1998;18:241–7. Taub E, Morris DM. Constraint-induced movement therapy to enhance recovery after stroke. Curr Atheroscler Rep 2001;3:279 –86. Ziemann U, Lonnecker S, Steinhoff BJ, Paulus W. The effect of lorazepam on the motor cortical excitability in man. Exp Brain Res 1996;109:127–35.