Surface EMG was recorded from the right first dorsal interosseus muscle. The raw signal was amplified, filtered (time constant of 10 ms, high cut-off filter of 1 kHz), and recorded onto a PC using a 12-bit AD interface (sampling rate of 5 kHz) for off-line analysis. TMS was performed using a figure-of-eight magnetic coil (external loop diameter, 7 cm) connected to two Magstim 200 stimulators via a Bistim module (The Magstim Company, Dyfed, UK). This device allows two magnetic stimulators to discharge through the same coil but attenuates the maximum power output by about 30% (peak magnetic field, 1.5 T). In the text, stimulus intensities are expressed as a percentage of the maximum output when connected via the Bistim unit.
We first determined the optimal coil position over the left motor cortex, with the handle held posteriorly, and laterally at an angle of 45 deg to the sagittal plane, using suprathreshold stimulus intensities. This position was marked directly on the scalp with a soft-tip pen, to ensure accurate repositioning of the coil. At the optimal site, resting motor threshold (RMT) was defined as the stimulator intensity needed to produce a response of more than 50 μV in at least five of ten consecutive trials using an intensity resolution of 1% of the maximal stimulator output.
Several measures of cortical excitability were obtained. Paired-pulse inhibition (PPI3) and facilitation (PPF) were tested at short intervals (see below) and at rest, using the method described by Kujirai and colleagues (1993). In brief, the effect of a conditioning stimulus delivered prior to a second (test) stimulus was investigated. The conditioning stimulus was set at an intensity of 70% of RMT, and, thus, at an intensity known to produce no changes of excitability in the spinal cord (Kujirai et al. 1993; Di Lazzaro et al. 1998). The intensity of the test stimulus was adjusted to evoke unconditioned muscle responses of 1-2 mV peak-to-peak amplitude. The timing of the conditioning shock was varied in relation to the test shock. Single test stimuli and paired stimuli with interstimulus intervals (ISIs) of 3 or 10 ms were delivered 9 s apart in random order generated by the computer. Twenty trials were recorded for each ISI and the control condition (single stimuli). The conditioned response CR3 (or CR10) was defined as the mean amplitude of the conditioned responses at 3 ms (or 10 ms) ISI, expressed as a percentage of the mean amplitude of the unconditioned test responses. The amplitudes of the motor-evoked potentials (MEPs) were measured peak to peak. Resting amplitudes (RAs) were defined as the mean amplitudes of the unconditioned MEPs.
Paired-pulse inhibition was also tested in another protocol using two shocks of equal intensities delivered at an ISI of 160 ms through the same coil (Valls-Soléet al. 1992). The stimulus intensity was 130% of the RMT and twenty trials were sampled at the optimal scalp site while subjects were at rest. CR160 was defined as the mean amplitude of the conditioned (second) responses expressed as a percentage of the mean amplitude of the responses to the first pulse.
Absence of involuntary EMG activity was monitored in all double-shock protocols by audiovisual feedback, and trials with background EMG activity were rejected.
The cortical stimulation-induced silent period (CSSP) was elicited while subjects held a tonic voluntary contraction of approximately 30% of maximum voluntary contraction. Ten trials were conducted at the optimal scalp site using a stimulus intensity of 130% of RMT. The duration of the CSSP was measured in individual trials from stimulus onset to the end of the CSSP, which was defined as the point where the first burst of continuous EMG activity was seen following the period of EMG silence. Measurements of the duration of the CSSP were made, for the most part, in a blinded fashion by a single investigator, and independently confirmed by a second investigator. Active amplitudes (AA) were defined as the mean amplitudes of the MEPs recorded in the same trials and as measured peak to peak.
The silent period induced by peripheral nerve stimulation (PSSP) was studied with the first dorsal interosseus muscle contracted at about 30% of maximal force. Ten electrical stimuli were delivered to the ulnar nerve through a Digitimer D180 electrical stimulator (maximum stimulator output, 750 V, 1 A; Digitimer Ltd, Welwyn Garden City, Hertfordshire, UK) using a stimulus width of 100 μs and a stimulus intensity of 375 V. At this intensity a maximal compound muscle action potential was elicited in all subjects studied. Similar to CSSP, PSSP duration was assessed from the time of stimulation until the first occurrence of continuous EMG activity.
In a first series of experiments, the effect of different doses of TGB were investigated in three subjects (all males). Subjects took 5, 10 or 15 mg or an equivalent of 63, 125 or 188 al of 25 μg (kg body weight)−1 of TGB. The order of doses was balanced across subjects. At least 48 h elapsed between any two sessions. Following the baseline (BSL) testing of all variables (RMTBSL, RABSL, AABSL, PSSPBSL, CSSPBSL, CR3BSL, CR10BSL, CR160BSL) subjects were studied again at 90-120 min after oral administration of TGB (RMTTGB, RATGB, AATGB, PSSPTGB, CSSPTGB, CR3TGB, CR10TGB, CR160TGB). The return to baseline levels was ascertained by a separate measurement each time before taking another dose, and on some occasions, on the following day.
In a second series, eight subjects (6 males and 2 females), received an equivalent of approximately 200 μg TGB (kg body weight)−1 (15 mg, 6 males, or 10 mg, 2 females). In this series, RMT, RA, AA, CSSP, CR3 and CR10 were measured. The results of the experiments testing the effect of 15 mg TGB were similar in the three subjects who had already taken 15 mg in the first series. Therefore, in those cases, the mean of the two experiments was used for further analysis.