TWO PHASES OF INTRACORTICAL INHIBITION EXPLORED BY TRANSCRANIAL MAGNETIC THRESHOLD TRACKING

3.1 Introduction

The paired-pulse test, in which a subthreshold TMS pulse is used to condition the EMG response to a larger test stimulus, was described by Kujirai et al. (1993) as a method to study inhibitory mechanisms in the human motor cortex. Further information about the intracortical mechanisms involved has come from more invasive studies. Indirect activation o f the corticospinal tract with a single test stimulus evokes multiple descending volleys in the spinal cord, termed I-waves (Nakamura et al. 1997; Di Lazzaro et al. 1998a,b). When a conditioning stimulus preceded the test stimulus by 1-5 ms, the later I- waves evoked by the test stimulus were inhibited. Suppression o f the L-wave by a conditioning stimulus has also been inferred, without exposure o f the spinal cord, from recordings o f single motor units (Hanajima et al. 1998).

The mechanisms of I-wave generation and inhibition by TMS are still far from clear (Ziemann and Rothwell 2000). Kujirai et al. (1993) proposed that the intracortical inhibition is mediated by GABAergic intemeurones. Hanajima et al. (1998) showed that I-wave suppression lasts up to 20 ms, a period that corresponds with the activation of

GABAareceptors. Further support for this theory has come from studies in which several

GABA-potentiating compounds were found to enhance intracortical inhibition (Ziemann et al. 1996a,b). However, not all GABAergic compounds had this effect (Ziemann et al. 1996a), and tiagabine, a GAB A uptake blocker, has been found to reduce intracortical inhibition (Werhahn et al. 1999). This apparently contradictory result might be explained by enhanced activation of GAB As receptors, acting pre-synaptically to inhibit the

GABAamediated inhibition (Werhahn et al. 1999; Sanger et al. 2001).

The paired-pulse test has been extensively used in patient studies to test for alterations in intracortical inhibition in neurological disease. The results were initially promising, but since a similar reduction in intracortical inhibition has now been found in a wide range o f neurological conditions (Ridding et al. 1995a; Ikoma et al. 1996; Abbruzzese et al. 1997; Ziemann et al. 1997; Greenberg et al. 1998; 2000), the current test has little diagnostic value and its implications for disease pathophysiology are as yet unclear. Factors that may have helped obscure disease-specific alterations in intracortical inhibition include: (i) the use o f a measure of inhibition (response reduction) that is restricted in range (since reduction is limited to 100 %), (ii) the averaging together o f inhibition levels over a range of interstimulus intervals (ISIs), and (iii) the dependence o f paired-pulse inhibition on other factors than the integrity of particular synaptic pathways in the cortex (e.g. the resting level of synaptic drive on PTNs).

Recently, Awiszus et al. (1999) addressed the first of these issues by applying a threshold tracking procedure (Bostock et al. 1998) to the paired-pulse test. Inhibition was measured by the increase in test stimulus required to maintain a constant response, rather than by the reduction in response to a constant stimulus. The present study has additionally addressed the other two issues to further develop the paired-pulse test, in the hope of improving its clinical usefulness. In normal subjects, the dependence of cortical excitability on the timing and amplitude of a subthreshold conditioning stimulus, and on voluntary muscle activity, has revealed two distinct components of intracortical inhibition, which may differ in their susceptibility to disease.

3.2 Methods (1)

S. 2.1 Subjects

16 healthy volunteers (8 males and 8 females, 23-55 years old) were the subjects. Informed written consent was obtained from all subjects. A local ethics committee approved the experimental procedures, which were performed according to the Declaration o f Helsinki.

3.2.2 EMG recordings

Surface EMGs were amplified (ImV/V), filtered (2 kHz with a time constant of 100 ms) and sampled at 10 kHz. Recordings were taken from the right abductor pollicis brevis (AbPB) using 5 mm Ag-AgCl surface electrodes (Dantec, UK). Sweeps of data 300 ms in length (100 ms pre-stimulus) were saved via a custom made interface and computer program (QTRAC, H Bostock) onto a computer. Subjects were provided with audio feedback of the EMG signal, to ensure total EMG silence in the experiments performed at rest. For active muscle conditions, subjects were instructed to perform a low level of voluntary activity by simple thumb abduction. The activity was quantified by full-wave rectification and leaky integration of the EMG signal (time constant 1 s). A target level was provided, by a simple dial scale (calibrated to maximum voluntary contraction), and the subject was requested to maintain a -1 0 % maximum contraction.

In some experiments, single motor units were recorded from the right first dorsal interroseous (IDI) muscle with a concentric needle electrode (Medelec). Signals were

amplified (100 fiV/V), filtered at 1 or 3 kHz with a time constant o f 3 ms and sampled at

5000 Hz. Data was sampled in 100 ms sweeps via a 1401 interface system and computer running Sig-Av software (CED, Cambridge UK). Subjects were instructed to activate a single motor unit at a steady, low rate with the aid of audio-visual feedback. Action

potential waveform was monitored with an oscilloscope to ensure that the same motor unit was recorded during each experimental session.

3.2.3 Stimulation

TMS was applied over the hand area o f the left motor cortex through a figure-of-eight- shaped coil (outer diameter o f each loop, 9cm) using two high-power Magstim 200 magnetic stimulators (Magstim Company Ltd, Dyfed UK). The coil was oriented to evoke a current within the cortex flowing in an anterior-posterior direction. Both stimulators were connected to the same coil through a Y connector or bistim module (Magstim Company Ltd, Dyfed UK). For the surface EMG experiments, subjects were laid on a bed and the coil was held in a fixed position with a clamp. In the single motor unit recordings the coil was held on the head by an experimenter and care was taken to retain a constant position.

Experiments were conducted in a conditioning-test design and the ISI was varied. For the purposes o f threshold tracking (see below), resting motor threshold (RMT) was defined as the stimulus intensity needed to produce a 0.2 mV (peak-to-peak) MEP response. Active motor threshold (AMT) was defined as the intensity needed to produce the same response during low level voluntary activity ( - 1 0 % maximum). The 0.2 mV ‘threshold’ response level was about twice as high as that commonly used (0.05 mV rectified EMG), and was preferred for two reasons: it enabled more efficient threshold tracking (see below), and it enabled AMT to be tracked in the presence of low level spontaneous activity without signal averaging.

3.2.4 Surface EMG threshold tracking

In order to measure intracortical inhibition the paired-pulse paradigm was used. This involved three conditions: a single conditioning stimulus intensity, a single test stimulus intensity and a paired-pulse consisting o f the conditioning stimulus preceding the test stimulus (Figure 3.1 A). Conventionally in the paired-pulse test, the conditioning stimulus is set at an intensity below threshold and the test stimulus is suprathreshold and set to evoke an MEP o f approximately 1-2 mV. The ISI between the stimuli is usually between 1-5 ms and 3 conditions are usually mixed randomly during each experimental block. When the test stimulus is preceded by a conditioning stimulus, the MEP evoked is smaller than the response elicited by the test stimulus alone (Figure 3.IB). In the conventional paired-pulse test, quantification of how much the MEP elicited by the paired-pulse has been reduced, in comparison to the test stimulus response alone, indicates the inhibitory effect o f the conditioning stimulus (Figure 3. ID).

For this study the paired-pulse test was modified. A fixed target MEP response (MEP amplitude) was set at a threshold level o f 0.2 mV (peak-to-peak in unrectified EMG). Inhibition produced by the conditioning stimulus was measured as the increase in test stimulus required to achieve the target response (Figure 3.1C,E). This technique is analogous to the measurement o f impulse-dependent excitability changes in peripheral nerve by threshold tracking (Bostock et al. 1998) and in order for this test to be optimal, conditions could not be randomised. Motor thresholds were measured by automatic adjustment of the test stimulus intensity by computer to maintain the MEP at the target level of 0.2 mV. MEPs were measured online, peak-to-peak and from raw unrectified EMG data, on a trial to trial basis. To achieve this the computer program set up a sampling window in the EMG data of approximately 15 ms in length, starting 20-30 ms after the stimulus. This was an appropriate latency to record MEP responses in each subject. Each paired-pulse combination comprised a fixed conditioning stimulus delivered by one magnetic stimulator followed by a varying test stimulus from the other magnetic

B

Test stimulus alone

A

Conditioning stimulus precedes test stimulus

C

Test stimulus increased

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