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,*
aDepartment of Rehabilitation Medicine, Inha University College of Medicine, Inchon, South Korea
bHuman 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
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 pharmaco-
logically, it might be a new strategy to enhance physical
therapyinstrokepatients.However,sinceclinically there isno
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 paired-
pulse paradigm in TMS (Di Lazzaro et al., 2000) while
flumazenil reverses the effect of diazepam-induced intra-
cortical 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
Clinical Neurophysiology 115 (2004) 325–329
www.elsevier.com/locate/clinph
* Corresponding author. Tel.:þ1-301-496-9526; fax:þ1-301-480-2286.
E-mail address: hallettm@ninds.nih.gov (M. Hallett).

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 investi-
gation. 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 approxi-
mately perpendicularly toward the position of the central
sulcus. This is thought to be the best position for acti-
vating 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 stimu-
lations in the relaxed FDI muscle were performed, and
F responses were recorded and averaged.
H.Y. Jung et al. / Clinical Neurophysiology 115 (2004) 325–329326