Abstract
Background: A recently devised test of motor cortex excitability (short latency afferent inhibition) was shown to be sensitive to the blockade of muscarinic acetylcholine receptors in healthy subjects. The authors used this test to assess cholinergic transmission in the motor cortex of patients with AD.
Methods: The authors evaluated short latency afferent inhibition in 15 patients with AD and compared the data with those of 12 age-matched healthy controls.
Results: Afferent inhibition was reduced in the patients (mean responses ± SD reduced to 85.7% ± 15.8% of the test size) compared with controls (mean responses ± SD reduced to 45.3% ± 16.2% of the test size; p < 0.001, unpaired t-test). Administration of a single oral dose of rivastigmine improved afferent inhibition in a subgroup of six patients.
Conclusions: The findings suggest that this method can be used as a noninvasive test of cholinergic pathways in AD. Future studies are required to evaluate whether short latency afferent inhibition measurements have any consistent clinical correlates.
The pathogenesis of AD appears to involve several different mechanisms, the most consistent of which is a deficit in cholinergic activity.1-4⇓⇓⇓ Indeed, the hypothesis of significant cholinergic dysfunction in AD provides a rationale for pharmacologic treatments based on drugs that may enhance cholinergic neurotransmission, such as acetylcholinesterase (AChE) inhibitors.
We recently described in normal subjects a neurophysiologic tool that is sensitive to the excitability of some cholinergic circuits in the human cerebral motor cortex.5 This test is based on coupling transcranial magnetic stimulation (TMS), a noninvasive method of stimulating the motor cortex, with peripheral nerve stimulation. Motor evoked potentials (MEP) recorded after TMS of the motor cortex can be suppressed by electrical stimulation of the median nerve if the time between stimulation of the median nerve and that of the motor cortex is 2 to 8 msec longer than the time needed by the peripheral nerve afferent input to reach the cortex. This effect, named short latency afferent inhibition of the motor cortex, is produced by interactions within the cerebral cortex.6 Because the effect is reduced or abolished by IV injection of the muscarinic antagonist scopolamine,5 we suggested that it might be a noninvasive way of testing cholinergic activity in the cerebral cortex.
The present study examined short latency afferent inhibition in patients with AD. Our hypothesis was that if afferent inhibition is a marker of cholinergic cortical activity, then it should be reduced in AD. Furthermore, it may be a way of detecting increased cholinergic transmission induced by treatment with AChE inhibitors. To determine the specificity of the test, we also examined a number of other excitatory and inhibitory cortical circuits with a range of TMS paradigms.7
Methods.
Patients.
We examined 15 patients with a diagnosis of probable AD according to the NINCDS–ADRDA criteria8 and 12 neurologically healthy age-matched controls. The main clinical and demographic characteristics of the patients with AD are reported in the table. The mean age ± SD of the patients was 69 ± 5.3 years, while that of the controls was 73.1 ± 5.4 years. The inclusion criteria were the following: 1) absence of other major medical illnesses; 2) symptom onset no longer than 5 years previously; and 3) age at onset no older than 75 years. All the AD patients selected were able to understand and carry out simple tasks required for this electrophysiologic study, such as to contract a hand muscle or to keep fully relaxed.
Table 1 Demographic characteristics and neuropsychological test results for patients with AD and cutoff scores for healthy controls matched by age and educational level in a study of cholinergic cortical circuits
None of the patients had been treated with AChE inhibitors before participating in this electrophysiologic study, which was performed according to the Declaration of Helsinki and approved by the Ethics Committee of the Medical Faculty of the Catholic University in Rome. Patients and their caregivers provided informed consent before participation.
Magnetic stimulation.
Magnetic stimulation was performed with a high-power Magstim 200 (Magstim, Whitland, Dyfed, Wales). A figure-of-eight coil with external loop diameters of 9 cm was held over the right motor cortex at the optimum scalp position to elicit motor responses in the contralateral first dorsal interosseous muscle. The induced current flowed in a posteroanterior direction.
Short latency afferent inhibition by somatosensory input from the hand.
Short latency afferent inhibition of the motor cortex was studied using a technique that we recently described.6 Conditioning stimuli were single pulses of electrical stimulation applied through bipolar electrodes to the median nerve at the wrist. The intensity of the conditioning stimulus was set at just over the motor threshold for evoking a visible twitch of the thenar muscles. The intensity of the test cortical magnetic shock was adjusted to evoke muscle responses in relaxed first dorsal interosseous muscles with amplitudes of ≈1 mV peak to peak.
The conditioning stimulus to the peripheral nerve preceded the magnetic test stimulus. Interstimulus intervals (ISI) were determined relative to the latency of the N20 component of the somatosensory evoked potential evoked by stimulation of the right median nerve. The active electrode for recording the N20 potential was attached 3 cm posterior to C3 (10–20 system), and the reference was 3 cm posterior to C4. Five hundred responses were averaged to identify the latency of the N20 peak. ISI from the latency of the N20 component plus 2 msec to the latency of the N20 component plus 8 msec were investigated. Five stimuli were delivered at each ISI. The subject was given audiovisual feedback at high gain to assist in maintaining complete relaxation. Trials were rejected from the average when there was no complete relaxation. The amplitude of the conditioned MEP was expressed as the percentage of the amplitude of the test MEP. The percentage inhibition of the conditioned responses at the seven different ISI was averaged to obtain a grand mean to reduce the variation in the data.
Threshold and amplitude of MEP, central motor conduction time (CMCT), silent period duration, intracortical inhibition, and short latency intracortical facilitation.
In addition to short latency afferent inhibition, we evaluated the following TMS parameters: 1) threshold and amplitude of MEP, which reflect the intrinsic and extrinsically modulated excitability properties of corticospinal neurons; 2) CMCT, which reflects the integrity of the corticospinal tract; 3) intracortical facilitation to paired TMS, which is thought to depend upon the activity of intracortical glutamatergic excitatory circuits; and 4) intracortical inhibition to paired TMS and the cortical silent period, which are both believed to reflect the excitability of inhibitory GABAergic cortical circuits.7
The resting motor threshold (RMT) was defined as the minimum stimulus intensity that produced a liminal MEP (≈50 μV in 50% of trials) at rest. The active motor threshold (AMT) was defined as the minimum stimulus intensity that produced a liminal MEP (≈200 μV in 50% of trials) during isometric contraction of the tested muscle at ≈20% maximum. We evaluated the excitability of the motor cortex at increasing stimulus intensity by measuring the average response to five stimuli at 100% AMT, 150% AMT, and 200% AMT during a 50% maximum contraction. CMCT was calculated by subtracting the peripheral conduction time from the spinal cord to muscles from the latency of responses evoked by cortical stimulation. The silent period was elicited while subjects held a tonic voluntary contraction of ≈50% of MVC. Five stimuli at 150% AMT and 200% AMT were given. Intracortical inhibition was studied using a paired pulse magnetic stimulation paradigm.9 Two magnetic stimuli were given through the same stimulating coil, using a Bistim module, over the motor cortex, and the effect of the first (conditioning) stimulus on the second (test) stimulus was investigated. The conditioning stimulus was set at an intensity of 5% (of the stimulator output) below the active threshold. The second (test) shock intensity was adjusted to evoke an MEP in relaxed first dorsal interosseous muscle with an amplitude of ≈1 mV peak to peak. The timing of the conditioning shock was altered in relation to the test shock. ISI between 1 and 3 msec were investigated. Five stimuli were delivered at each ISI. Subjects were given audiovisual feedback at high gain to assist in maintaining complete relaxation. The amplitude of the conditioned MEP was expressed as the percentage of the amplitude of the test MEP. Inhibition of the conditioned responses at the three different ISI studied was averaged to give grand mean values.
In 12 of the patients, we also analyzed the facilitatory interaction that occurs between pairs of threshold magnetic stimuli given over the motor cortex at short ISI.10-13⇓⇓⇓ This is not the same as the facilitation that follows the period of intracortical inhibition described above at ISI of 10 to 15 msec. Short latency intracortical facilitation occurs at much shorter intervals and is seen when both stimuli have an intensity that is above the active threshold. It may represent interaction between the mechanisms that produce repetitive I wave firing of pyramidal neurons.10-13⇓⇓⇓ Two magnetic stimuli were given through the same stimulating coil, using a Bistim module, over the motor cortex, and the effect of the first stimulus on the second stimulus was investigated. The intensity of the first stimulus was set to be 4% (of the stimulator output) above AMT. The second stimulus had an intensity of 2% (of the stimulator output) above AMT. The timing of the conditioning shock was altered in relation to the test shock. ISI of 1, 1.2, and 1.4 msec were investigated. Five stimuli were delivered at each ISI. The responses to five single and five paired stimuli were averaged. When both magnetic shocks were applied, the MEP evoked by the first stimulus alone were subtracted off line from the responses recorded using paired stimulation to evaluate the additional activity evoked by the second stimulus.
The amplitude of the MEP to both stimuli was expressed as the percentage of the amplitude of the MEP to the second stimulus on its own. Facilitation of the conditioned responses at the three different ISI studied was averaged to give grand mean values.
Effects of AChE inhibition.
To test if short latency afferent inhibition was sensitive to changes in AChE activity, we examined the motor threshold and short latency afferent inhibition in the last six consecutive patients before and after the administration of a single dose (3 mg) of rivastigmine, an AChE inhibitor commonly used for treatment of AD. Measurements were made before and 2.4 hours after the administration, when AChE inhibition in the CSF is maximal.14
One of these six patients died about 3 months later of pneumonia, and the remaining five patients started receiving long-term treatment with rivastigmine (6 to 9 mg/d) immediately after the electrophysiologic tests. During follow-up 1 year later, scores of the Mini-Mental State Examination and neuropsychological examination were slightly improved for four patients and worse for one patient.
The effects of a single oral dose of rivastigmine were also evaluated in three controls (mean age ± SD, 30.6 ± 2.3 years).
Neuropsychological examination.
All patients enrolled in the study underwent the Mini-Mental State Examination and an extensive neuropsychological test battery including tests of episodic verbal memory (Rey Auditory Verbal Learning Test),15 immediate visual memory, constructional praxis, verbal fluency, abstract reasoning (Raven Progressive Matrices 1947),16 and a test of executive function sensitive to frontal lobe damage (Temporal Rule Induction).17
In Rey Auditory Verbal Learning Test, the examiner reads a list of 15 words aloud. After each presentation, the subject is asked to recall as many words as possible. The immediate recall score is the sum of all words correctly recalled immediately after the five administrations. After a 15-minute delay, the subject is asked to recall as many words of the list as possible, without any further presentation. The delayed recall score is the number of correctly recalled words after such delay. After a further 15-minute delay, the subject is requested to recognize the 15 words (forced delayed recognition) belonging to the list presented in the Rey Auditory Verbal Learning Test, among 45 items (15 target words and 30 distractor words). Two scores are taken into account: 1) the number of correctly recognized target words (hits); and 2) the number of incorrectly recognized distractor words (false alarms).
Statistical analysis.
Motor cortex excitability parameters were analyzed separately. The amplitude of motor responses at different stimulus intensities was analyzed using repeated measures multivariate analysis of variance. The threshold for EMG responses, duration of the cortical silent period, intracortical inhibition, short latency intracortical facilitation, and short latency inhibition produced by somatosensory input from the hand were analyzed using an unpaired t-test.
The effects of rivastigmine on the threshold of MEP and short latency inhibition produced by somatosensory input from the hand were assessed by a two-tailed paired Student’s t-test.
Linear regression analysis (forward stepwise method) was performed between neurophysiologic parameters and disease duration.
Results.
Electrophysiologic results are summarized in figure 1. An example of afferent inhibition in one patient and in one control is shown in figure 2.
Figure 1. Resting motor threshold (RMT), active motor threshold (AMT), motor evoked potential (MEP) amplitude, cortical silent period, short latency afferent inhibition, intracortical inhibition, and short latency intracortical facilitation in AD patients and controls. Histograms show mean values for threshold, afferent inhibition, and short latency intracortical facilitation; error bars are SE. The amplitude of MEP and duration of the silent period at increasing stimulus intensities are shown for patients (squares) and controls (diamonds); points represent means ± SE. RMT (*p < 0.05, unpaired t-test) and short latency afferent inhibition (**p < 0.001, unpaired t-test) were significantly reduced in AD patients.
Figure 2. Raw data traces showing short latency afferent inhibition by somatosensory input from the hand in one control and one AD patient. The top traces show the average (of five trials each) of motor evoked potentials (MEP) recorded in the first dorsal interosseous muscle by cortical stimulation alone and cortical stimulation conditioned by a median nerve stimulus with an interstimulus interval corresponding to the N20 latency plus 2 and 3 msec. In the control, the median nerve conditioning stimulus suppressed the MEP evoked by transcranial stimulation, while in the AD patient, the MEP evoked by the test stimulus was not inhibited by median nerve stimulation.
Short latency afferent inhibition produced by somatosensory input from the hand.
MEP in controls were inhibited when the median nerve stimulus was given before TMS of the cerebral cortex at an interval corresponding to the N20 latency plus 2 msec to the N20 latency plus 8 msec. The amount of inhibition over this period (see figure 1) was significantly smaller in AD patients (mean responses ± SD reduced to 85.7% ± 15.8% of the test size) than in controls (mean responses ± SD reduced to 45.3% ± 16.2% of the test size), with a highly significant difference between the two groups (p < 0.001, unpaired t-test).
Threshold and amplitude of EMG responses, CMCT, silent period duration, intracortical inhibition, and short latency intracortical facilitation.
The mean RMT ± SD was significantly lower in AD patients (50.2% ± 6.4%; p < 0.05) than in controls (57.9% ± 11.7%). There was no significant difference between controls and AD patients in terms of the threshold or amplitude of motor responses evoked during voluntary activity, although responses evoked at higher stimulus intensities tended to be larger in AD patients (see figure 1). There was also a tendency for AD patients to have less pronounced intracortical inhibition and more pronounced short latency intracortical facilitation than controls, but these differences between the two groups were not significant (p < 0.05; see figure 1). CMCT and silent period duration were similar in both groups (see figure 1).
Effects of AChE inhibition.
In six AD patients, the electrophysiologic study was carried out before and after a single oral dose of rivastigmine. Administration of the single dose of rivastigmine did not produce any detectable clinical effect in the patients.
RMT and AMT were not significantly modified by the administration of rivastigmine (mean RMT ± SD before administration, 50.5% ± 7%; mean RMT ± SD after administration, 49.7% ± 8.3%; mean AMT ± SD before administration, 41.8% ± 7%; mean AMT ± SD after administration, 40.5% ± 8.1%). In contrast, short latency afferent inhibition from the median nerve was significantly increased by the administration of rivastigmine (p < 0.05, paired t-test): the mean amplitudes of the conditioned response ± SD were 83.2% ± 15% (range, 95%–53.8%) of the control size before the administration and 59.4% ± 25.4% (range, 84.2%–19%) after administration of rivastigmine (figure 3). One of the patients had only a minor change after rivastigmine administration, with an increment of inhibition of only 8% (the amplitude of the conditioned response was 92.3% of the control size before the administration and 84.2% after administration of rivastigmine). This latter patient was the one who had worse results on neuropsychological examination after 1 year of treatment.
Figure 3. Mean short latency afferent inhibition in AD (six patients): effects of rivastigmine on afferent inhibition. Histograms show mean values; error bars are SD. Rivastigmine significantly increased afferent inhibition (*p < 0.05, paired t-test).
We also tested the effect of a single oral dose of rivastigmine in three controls. In contrast to the patients, their baseline afferent inhibition was relatively strong (mean ± SD, 47.6% ± 20.7% of the control size) but was little changed after rivastigmine administration (mean ± SD, 42.2% ± 11%; p = 0.6, paired t-test).
Correlation of electrophysiologic parameters and disease duration.
No electrophysiologic measure correlated with disease duration.
Discussion.
This study showed that short latency afferent inhibition of the motor cortex, a putative marker of cholinergic cortical activity, is significantly reduced in AD patients compared with controls. A single dose of the AChE inhibitor rivastigmine significantly increased afferent inhibition in the six patients in whom it was tested. These findings, together with the results of a previous study of normal subjects that showed that short latency afferent inhibition of the motor cortex can be reduced or abolished by IV injection of the muscarinic antagonist scopolamine,5 support the view that afferent inhibition may be a useful marker of cholinergic activity in some motor circuits of the cerebral cortex.
Short latency afferent inhibition of the motor cortex is thought to depend on the integrity of corticocortical inhibitory circuits.6 The reduction or suppression of inhibition induced by muscarinic blockade in healthy subjects is likely to occur via cholinergic modulation of inhibitory circuits rather than direct cholinergic involvement in such inhibitory processes. If so, then given the widespread cholinergic innervation of the cerebral cortex, tests of short latency afferent inhibition of the motor cortex might reflect the activity of inhibitory processes in large areas of the cerebral cortex.
No correlation was found in our AD patients between the level of afferent inhibition and disease duration. However, this would be better addressed in a longitudinal study of individual patients since the rate of disease progression may differ between them.
The fact that short latency afferent inhibition of the motor cortex could be increased only 2.4 hours after inhibition of AChE by rivastigmine is consistent with the hypothesis that postsynaptic muscarinic acetylcholine (ACh) receptors are not greatly affected in AD and that the cholinergic deficit is mainly presynaptic.
A clinically significant response to AChE inhibitors is observed in only 30% to 60% of AD patients, depending on the compound.18 To date, there is no laboratory test to identify patients who are likely to respond to these drugs. Our preliminary data for the six patients in whom we evaluated the effects of a single dose of rivastigmine suggest that the assessment of short latency afferent inhibition in AD patients, before and after the administration of cholinergic drugs, might be a useful tool for evaluating the pharmacologic effects of cholinergic drugs or even predicting the clinical response to treatment. However, study of a larger number of patients is needed to substantiate this hypothesis.
In controls, we observed only a slight increase in afferent inhibition after rivastigmine administration. One possible explanation for this finding is that the afferent inhibition in healthy subjects is prone to “floor” effects because the baseline levels of inhibition are strong.
In healthy subjects, IV injection of scopolamine not only reduced short latency afferent inhibition of the motor cortex but also decreased RMT.5 This finding is consistent with the reduced RMT seen in our AD patients and in other studies.19,20⇓ In one of these studies,20 the investigators also found that there was a small reduction in AMT and larger MEP, after stimulation at a constant intensity, in AD patients compared with controls. These latter effects were also detected in our AD patients, although the findings were not significant. Since spinal excitability, as tested with H reflexes and F waves, is considered to be normal in AD patients,19 all such effects are likely to be due to an increased excitability of motor cortical circuits in AD. It has been reported20 that intracortical inhibition is not impaired in AD patients. However, this was not confirmed by a recent study21 that described reduced intracortical inhibition in AD patients. In our study, we found a tendency for AD patients to have less pronounced intracortical inhibition than controls, but this difference between the two groups was not significant.
There are several possible explanations for these smaller effects on noncholinergic systems in AD patients. For example, experimental studies on slices of rat auditory cortex showed that the endogenous release of ACh can reduce the amplitude of glutamatergic excitatory postsynaptic potentials evoked in layer III, after electrical stimulation in layer VI.22 Perhaps reduced ACh neurotransmission in the cerebral cortex of AD patients might lead to the converse, increased excitability of some motor cortical circuits. This in turn would lower motor thresholds and increase the size of MEP. An alternative explanation is that in AD patients reduced short latency afferent inhibition might produce an imbalance in the activity of excitatory and inhibitory circuits of the cerebral cortex, resulting in increased excitability of pyramidal cells. The slight and not significant reduction in intracortical inhibition is not clear since it has been demonstrated that the GABA system is relatively spared in AD.23 However, cortical interneurons containing GABA have nicotinic as well as muscarinic receptors and receive rich cholinergic afferents.24 Binding studies and receptor autoradiography showed a decrease of up to 70% in nicotine-binding sites in AD.25 This finding, together with the evidence that intracortical inhibition is not affected by the muscarinic antagonist scopolamine in normal subjects,5 leads us to speculate that the changes in intracortical inhibition observed in some AD patients may be due to altered nicotinic receptor function. Impaired nicotinic activity may deprive GABAergic inhibitory neurons of a source of excitatory inputs and reduce corticocortical inhibition.
Acknowledgments
Supported by the Ministero della Sanità (Programma di ricerca finalizzata–Malattia di Alzheimer, 2000–Regione Lazio Assessorato per le Politiche della Sanità).
- Received September 12, 2001.
- Accepted April 6, 2002.
References
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