• Users Online: 224
  • Print this page
  • Email this page


 
 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 39  |  Issue : 2  |  Page : 68-73

Weak transcranial direct current effect on i waves: A single motor unit recording study of healthy controls


1 Department of Neurology, Division of Clinical Neurophysiology, Gazi University Faculty of Medicine, Ankara, Turkey
2 Department of Neurology, Division of Algology, Gazi University Faculty of Medicine, Ankara, Turkey

Date of Submission19-Nov-2021
Date of Decision25-Dec-2021
Date of Acceptance05-Feb-2022
Date of Web Publication28-Apr-2022

Correspondence Address:
Asli Akyol Gurses
Department of Neurology, Division of Clinical Neurophysiology, Faculty of Medicine, Gazi University, Besevler 06500, Ankara
Turkey
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/nsn.nsn_221_21

Rights and Permissions
  Abstract 


Background: A single transcranial magnetic pulse over the motor cortex is known to generate repetitive descending activity along the corticospinal tract. With respect to the origin; the earliest volley is named direct wave and the subsequent activity forms indirect (I) waves. I waves are assumed to originate from corticocortical afferents; they can be modulated by several methods and are practical parameters for evaluating motor cortex excitability. Weak transcranial direct current stimulation (tDCS), which has been widely used in human studies since the early 2000s, is a noninvasive and painless modulatory method for studying cortical excitability. We aimed to investigate the modulatory effects of anodal and cathodal tDCS on I waves of healthy controls, as a component of the motor evoked potential response generator. Materials and Methods: Twelve healthy volunteers were enrolled in the study. One mA tDCS was applied for 10 min and; single motor unit (SMU) recording technique was used for the identification of I waves. Two conditions were analyzed for each SMU in both anodal and cathodal current polarities; before tDCS and after tDCS. Separate peristimulus time histograms were constituted for each condition. Total peak duration, early peak latency, early peak duration, and early peak discharge rate were calculated. Results: Total peak duration, early peak latency, and early peak duration did not differ between pre- and post-tDCS conditions in either polarity. However, I1 peak discharge rate was found to be significantly decreased after cathodal tDCS (P: 0.017) and increased after anodal tDCS (P: 0.003). Conclusion: Our results confirm polarity-specific effects of tDCS of the primary motor cortex on I waves. According to our knowledge, this is the first study evaluating modulatory effects of tDCS on I waves using SMU recording technique.

Keywords: I waves, single motor unit recording, transcranial direct current stimulation


How to cite this article:
Gurses AA, Boran HE, Vuralli D, Cengiz B. Weak transcranial direct current effect on i waves: A single motor unit recording study of healthy controls. Neurol Sci Neurophysiol 2022;39:68-73

How to cite this URL:
Gurses AA, Boran HE, Vuralli D, Cengiz B. Weak transcranial direct current effect on i waves: A single motor unit recording study of healthy controls. Neurol Sci Neurophysiol [serial online] 2022 [cited 2022 Dec 2];39:68-73. Available from: http://www.nsnjournal.org/text.asp?2022/39/2/68/344307




  Introduction Top


The discovery of 600 Hz descending volleys recorded from ipsilateral bulbar and contralateral corticospinal tracts of mammalians after a single electrical stimulus in 1954, provided a critical milestone for understanding corticospinal electrophysiology.[1] In the following years, these components of motor evoked potential (MEP) response generator were demonstrated in epidural recordings of vertebral colon surgery patients with the aid of transcranial magnetic stimulation (TMS) and transcranial electrical stimulation.[2]

Approximately 50 years later, Nitche and Paulus reintroduced direct current application as a modulator of motor cortex excitability and identified polarity-specific effects in humans. The researchers observed increase in TMS-induced MEP amplitudes both during and after anodal transcranial direct current stimulation (tDCS) depending on the stimulation duration and, the reverse effects following cathodal tDCS. While shifts in neuronal resting membrane potential were responsible for the effects during tDCS, more complex mechanisms accounted for the aftereffects including changes in spontaneous neuronal firing rate and synaptic modulation similar to long term potentiation and long-term depression phenomena.[3]

Expectedly; tDCS induced cortical excitability investigations have gained acceleration within the last few decades and the effects on motor cortex excitability have been extensively evaluated based on surface recorded MEP amplitudes, as a conventional method. Besides few studies that examine subjects with invasive spinal cord procedures, there has been a little effort to target I waves, as a primary response element.

I waves which refer to high-frequency repetitive discharge of corticospinal fibers,[4] are practical parameters for studying corticospinal excitability in healthy controls and patients. In contrast to D waves which appear first after cortical stimulation and originate from direct excitation of initial segments of pyramidal axons, I waves are generated transsinaptically through excitatory corticocortical afferents. Premotor-supplementary motor areas and somatosensory cortex are suggested to be the most probable sources and,[5] they can easily be stimulated by TMS of the motor cortex. The occurrence of marked suppression in I waves after GABAergic medications supports their powerful GABAergic control.[6] They can be demonstrated through direct or indirect methods and modulation can be performed via pharmacological interventions, different paired-pulse TMS paradigms, or direct currents.

Here, we evaluated the effects of weak tDCS of the primary motor cortex on I waves through an indirect method. We used single motor unit (SMU) recording technique and according to our knowledge; this is the first study that evaluated modulatory effects of tDCS on I wave behavior with this method.


  Materials and Methods Top


Participants

The study was conducted in the Motor Control Laboratory of Gazi University School of Medicine and consisted of two separate experiments with at least 24 h intervals. Twelve volunteers, aged between 29 and 45 were enrolled in the study. Previous diagnosis of a central nervous system (CNS) disorder (e.g., epilepsy); medication which have a potential effect on CNS; the presence of intracranial aneurysm clip, cochlear implant, and pacemaker; history of syncope, hearing loss due to any acoustic trauma and pregnancy were the exclusion criteria. All of the volunteers were informed about the details and probable adverse effects of the experimental procedures and all gave informed consent. Ten of them were subjected to both anodal and cathodal tDCS in two different sessions with a minimum 24 h interval. Two of the participants did not attend the second session and, subject 11 received only anodal tDCS while subject 12 received only cathodal tDCS in a single session. After obtaining baseline parameters of firing probability by TMS; subjects were preconditioned with either anodal or cathodal tDCS in each session. The participants were blinded to the polarity in all sessions. Experimental procedures were approved by the ethics committee.

Experimental procedures

Transcranial magnetic stimulation

90 mm circular magnetic coil (Magstim) was used for monophasic stimulation. Cz point was used to stimulate the left motor cortex in a counterclockwise manner. Resting motor threshold for the right first dorsal interosseoz (FDI) muscle was identified as the minimum stimulator output level which provided ≥50 microvolt MEP response in at least three of five stimuli. Active motor threshold (AMT) was identified as the minimum stimulator output level which provided ≥100 microvolt MEP response in at least three of five stimuli, while the participant slightly contracted the right FDI muscle (which enabled a SMU discrimination). We first applied posteroanterior (PA) directed 150% AMT stimulus intensity for the identification of D waves (150% AMTpa).[7],[8],[9] Later, PA directed 100–150 magnetic stimuli were applied with an intensity of 110% AMT, as the motor unit continued to fire tonically (110% AMTpa). The latency of the earliest peak evoked by 110% AMTpa stimulation was 1.15–1.7 ms later than that of 150% AMTpa, in 8 of the participants. In the remaining 4 subjects, both stimulation intensities evoked responses with similar latencies. Analog signal was transferred to the computer via analog-digital transducer. Signal 5.05 program was used for the analysis.

Weak transcranial direct current stimulation

Electrical stimulation of the motor cortex was performed through a battery-driven constant-current stimulator. 4 cm × 3 cm electrodes were placed in a pair of saline-soaked sponges (5–7 cm). The active electrode was placed over the left FDI motor cortex area (approximately 4 cm lateral to the Cz) and the reference electrode was placed over the contralateral supraorbital region. tDCS was given with an intensity of 1 mA for 10 min duration. Most of the participants defined a slight itching under the electrode. 100–150 magnetic stimuli (110% AMTpa) were applied during tDCS period and immediately afterward (post tDCS). After an interval of minimum 24 h, the same steps were repeated with reverse current polarity in 10 of the participants, who were both subjected to anodal and cathodal tDCS in two different sessions. Two of the participants were subjected to either cathodal or anodal tDCS in a single session (subject 11 only anodal and subject 12 only cathodal). Participants were blinded to the polarity in all sessions.

Single motor unit studies and peristimulus time histograms

SMUs were recorded from the right FDI with a concentric needle electrode. Signals were amplified through filters set at 20 and 500 Hz. Participants mildly contracted the right FDI muscle with a firing rate of 10 Hz; audiovisual feedback with an oscilloscope monitor was provided to follow the same motor unit during each session and eliminate the others. Two different peristimulus time histograms (PSTH) were constituted for every SMU in each experiment; reflecting baseline (pre-tDCS) and post-tDCS properties of firing probability.

All of the participants completed the study; no significant side effects except burning and itching sense under the electrode and mild headache (in 2 participants) were observed.

Statistical analysis

Statistical analyses were performed using SPSS.20 (SPSS, Inc., Chicago, Illinois) package software. Numerical data were reported as mean ± standard deviation. Shapiro–Wilk test was used to test the data for normality. Total peak duration, early peak latency, early peak duration, early peak discharge rate (early peak discharge/total peak discharge), late peak latency, late peak duration, and late peak discharge rate (late peak discharge/total peak discharge) were calculated for pre-tDCSand post-tDCSconditions. Difference between pre and post-tDCS measurements was assessed using paired t-test or Wilcoxon signed-rank test according to the normality of data. P < 0.05 was considered statistically significant.


  Results Top


Anodal stimulation

Under the stimulating condition, there was a single peak in most of the PSTHs (7 out of 11 subjects). SMU analyses of subjects 3, 6, 8, 9 demonstrated a second peak and only subject 6 demonstrated an additional third peak. In 11 SMU examined; parameters concerning pretDCS condition were found as follows: total peak duration 2.91 ± 1.72 ms, early peak latency 24.23 ± 1.85 ms, and early peak duration 1.77 ± 0.69 ms. Post-tDCS measures were 2.77 ± 0.66 ms for total peak duration, 24.66 ± 1.81 ms for early peak latency, and 2.05 ± 0.78 ms for early peak duration. The differences between the two conditions were not statistically significant regarding the mentioned parameters (P > 0,05).

However, the early peak discharge rate was 65.75 ± 24.53% before tDCS and 76.77 ± 18.43% after tDCS, which revealed a significant increase after stimulation when compared to baseline (P = 0.003) [Figure 1].{Figure 1}

Late peaks could be elicited in only 4 of the participants (subject 3, 6, 8, 9). Late peak latency was 26.42 ± 0.76 ms vs 26.42 ± 1.18 ms, late peak duration was 1.08 ± 0.38 ms vs 0.92 ± 0.14 ms, and late peak discharge was 29.18 ± 23.2 9% vs 30.44 ± 1.14% for the pre- and post tDCS conditions, respectively. Since the data regarding late peak parameters were small, it was not subjected to further analysis.

Cathodal stimulation

Similar to the anodal stimulation, 7 out of the 11 PSTHs demonstrated a single peak. Subject 3, 4, 6, 9 demonstrated a second peak and subject 3 had an additional third peak. In the 11 SMU studied, baseline measurements were calculated as follows: total peak duration 3.14 ± 1.26 ms, early peak latency 25.08 ± 2.52 ms, and early peak duration 2.25 ± 0.86 ms. Corresponding values in post-tDCS conditions were2.77 ± 0.91 ms for total peak duration, 25.28 ± 2.02 ms for early peak latency, and 1.48 ± 1.24 ms for early peak duration. Concerning the mentioned parameters, paired t-test revealed no significant difference between the two conditions (P > 0,05).

On the other hand, the early peak discharge rate was 75.15 ± 29.41% before tDCS and 45.49 ± 37.81% after tDCS; which demonstrated a significant decrease after stimulation when compared to the baseline condition (P: 0.017) [Figure 2].{Figure 2}

Similar to anodal stimulation, late peaks could be elicited in only 4 of the participants (subject 3, 4, 6, 9). Pre- and post-tDCS values of late peak latency, late peak duration, and late peak discharge rate were 26.69 ± 3.09 ms versus 27.68 ± 1.43 ms; 1.87 ± 0.59 ms versus 1.03 ± 0.44 ms and 30.9 ± 26.12% versus 42.4 ± 26.88%, respectively. Since the data regarding late peak parameters were obtained from a small group of subjects, the data were not further analyzed.

Our findings indicated a positive effect of anodal stimulation on the firing probability of a SMU within the early subpeak, while cathodal stimulation caused the opposite effect [Figure 3].{Figure 3}


  Discussion Top


The results of our study confirm that tDCS of the primary motor cortex has polarity-specific effects on I waves which generate corticospinal descending activity. Since I waves are known to arise from intracortical circuits; the location of this modulation effect seems to be also intracortical. The analysis of PSTH graphics indicates that early peak discharge, which refers to I1 wave, decrease after cathodal tDCS, whilst anodal tDCS produce the opposite effect.

The effects during tDCS are assumed to depend on subthreshold shifts in membrane polarization mainly, as they can be abolished by calcium and sodium channel blockers.[10] In addition to the duration and intensity of the stimulation; the orientation of stimulated neurons relative to the electrical field has also influence on these effects.[11],[12] On the other hand, aftereffects that last beyond the stimulation period is not limited to an electrical phenomenon and require further mechanisms including protein synthesis.[13],[14] Anodal stimulation has been demonstrated to enhance early gene expression and intracellular calcium levels which act on the synaptic plasticity of glutamatergic neurons, and blockade of the NMDA receptors are known to result in the diminution of tDCS-induced aftereffects.[10],[15] Besides this, local GABA transmission was shown to be reduced by tDCS irrespective of polarity and considering the close relationship between these two neurotransmitters, it could be another contributor to the changes in glutamatergic plasticity.[16],[17]

After 2000, when Nitsche and Paulus reintroduced direct current application as a modulator of motor cortex excitability and demonstrated a significant alteration of surface recorded MEP amplitudes with respect to polarity; many subsequent studies using different stimulation parameters and outcome measures, have shown similar results. Anodal tDCS resulted in facilitation whereas cathodal tDCS resulted in inhibition.[3] However, this effect had not been investigated on the basis of I waves until 2005, when Nitsche et al. demonstrated raise of I1 peak amplitude following tDCS by using SICF protocol, in which a test stimulus is preceded by a subtreshold conditioning magnetic pulse.[18] Few years later, Lang et al. performed direct epidural recordings and reported increase in mean I wave amplitude following anodal tDCS and decrease in later I wave amplitudes after cathodal tDCS.[19] Here we studied the effects of tDCS on I waves with a different method; we used SMU recording technique and revealed early I1 wave suppression after cathodal tDCS, while anodal tDCS evoked facilitatory effect. In general, I1 wave is postulated to be resistant to any conditioning stimulus, such as a subtreshold magnetic stimulus through the same coil,[20],[21],[22] a conditioning magnetic stimulus applied to the motor cortex of the opposite hemisphere[23] or short-latency afferent inhibition produced by the stimulation of the median nerve.[24] It is assumed to originate from different cortical neuronal chains then the later ones. Nevertheless, the aforementioned studies investigated this modulatory effect by using different TMS paradigms and tDCS has not been the preference until 2005.[18] Thus, there is not enough evidence to conclude about early I wave behavior in response to tDCS conditioning.

Besides these, I wave generation and modulation is a complex process and still needs further investigation. Cellular morphology including the number and length of the dendritic tree within the layer V pyramidal neurons, as well as the location of synaptic arrivals from layers 2 and 3 are all known to affect consequent I wave response.[25] In contrast to the common assumption of I1 wave resistivity to any modulatory effect, Nitsche et al. demonstrated increase in the first I wave amplitude in response to both anodal and cathodal tDCS using the SICF protocol.[18] Lang et al. observed a trend toward suppression of I1 wave amplitudes after cathodal stimulation.[19] Similarly, Rusu et al. reported increase in I wave amplitude (without discrimination of either early or later components) at >5 ms ISI intervals, as a response to paired-pulse TMS paradigm, in which a supratreshold stimulus is preceded by a subtreshod stimulus.[25]

However, we can not completely exclude the effect of stimulus direction in our results; since PA stimulation recruits I1 waves preferentially whereas AP stimulation recruits the later ones.[26] Since we mainly stimulated I1 waves, we might observe the modulatory effects of tDCS on I1 waves.

There may be some possible limitations in this study. First, we did not have a sham group. Nevertheless, we believe that a potential bias is unlikely because our participants were blinded to current polarity and our results were consistent with the previous reports. Second, the circular coil we used activates a larger area than the figure of eight coil and results in a nonfocal stimulation.

Unlike the previous studies, we used SMU recording technique in the current study to demonstrate the modulatory effects of tDCS on I waves. One could expect that direct recordings from epidural space enable more precise assessment of descending volleys. However, there is limited opportunity to find sufficient time and eligible patients for direct recordings, who are generally candidates for invasive spinal cord procedures. In addition, most of these patients are already using medications which act at the synaptic level[19] and have potential influences on neurotransmitter systems. This could cause confusion while interpreting the electrophysiological findings. SMU recording technique can be applied to healthy controls and it could be an advantage when evaluating normal physiology.


  Conclusion Top


In the current study, in which we evaluated the modulatory effect of weak tDCS on I waves, our results demonstrated polarity-specific effects of tDCS of the primary motor cortex on I waves. Cathodal tDCS showed inhibitory effect within the discharge rate of I1 subpeak, whereas anodal tDCS produced the facilitatory effect. According to our knowledge, this is the first study evaluating modulatory effects of tDCS on I waves by using SMU recording technique.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Patton HD, Amassian VE. Single and multiple-unit analysis of cortical stage of pyramidal tract activation. J Neurophysiol 1954;17:345-63.  Back to cited text no. 1
    
2.
Boyd SG, Rothwell JC, Cowan JM, Webb PJ, Morley T, Asselman P, et al. A method of monitoring function in corticospinal pathways during scoliosis surgery with a note on motor conduction velocities. J Neurol Neurosurg Psychiatry 1986;49:251-7.  Back to cited text no. 2
    
3.
Nitsche MA, Paulus W. Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J Physiol 2000;527:633-9.  Back to cited text no. 3
    
4.
Ziemann U, Rothwell JC. I-waves in motor cortex. J Clin Neurophysiol 2000;17:397-405.  Back to cited text no. 4
    
5.
Patton HD, Amassian VE. The pyramidal tract: Its excitation and functions. In: Field J, editor. The Handbook of Physiology. Sec. I., Vol. II. Washington, DC: American Physiology Society; 1960. p. 837-61.  Back to cited text no. 5
    
6.
Hicks R, Burke D, Stephen J, Woodforth I, Crawford M. Corticospinal volleys evoked by electrical stimulation of human motor cortex after withdrawal of volatile anaesthetics. J Physiol 1992;456:393-404.  Back to cited text no. 6
    
7.
Burke D, Hicks R, Gandevia SC, Stephen J, Woodforth I, Crawford M. Direct comparison of corticospinal volleys in human subjects to transcranial magnetic and electrical stimulation. J Physiol 1993;470:383-93.  Back to cited text no. 7
    
8.
Kaneko K, Kawai S, Fuchigami Y, Shiraishi G, Ito T. Effect of stimulus intensity and voluntary contraction on corticospinal potentials following transcranial magnetic stimulation. J Neurol Sci 1996;139:131-6.  Back to cited text no. 8
    
9.
Di Lazzaro V, Restuccia D, Oliviero A, Profice P, Ferrara L, Insola A, et al. Effects of voluntary contraction on descending volleys evoked by transcranial stimulation in conscious humans. J Physiol 1998;508 (Pt 2):625-33.  Back to cited text no. 9
    
10.
Nitsche MA, Fricke K, Henschke U, Schlitterlau A, Liebetanz D, Lang N, et al. Pharmacological modulation of cortical excitability shifts induced by transcranial DC stimulation. J Physiol 2003;553:293-301.  Back to cited text no. 10
    
11.
Purpura DP, Mcmurtry JG. Intracellular activities and evoked potential changes during polarization of motor cortex. J Neurophysiol 1965;28:166-85.  Back to cited text no. 11
    
12.
Scholfield CN. Properties of K-currents in unmyelinated presynaptic axons of brain revealed revealed by extracellular polarisation. Brain Res 1990;507:121-8.  Back to cited text no. 12
    
13.
Gartside IB. Mechanisms of sustained increases of firing rate of neurons in the rat cerebral cortex after polarization: Role of protein synthesis. Nature 1968;220:383-4.  Back to cited text no. 13
    
14.
Gartside IB. Mechanisms of sustained increases of firing rate of neurons in the rat cerebral cortex after polarization: Reverberating circuits or modification of synaptic conductance? Nature 1968;220:382-3.  Back to cited text no. 14
    
15.
Liebetanz D, Nitsche MA, Tergau F, Paulus W. Pharmacological approach to the mechanisms of transcranial DC-stimulation-induced after-effects of human motor cortex excitability. Brain 2002;125:2238-47.  Back to cited text no. 15
    
16.
Stagg CJ, Best JG, Stephenson MC, O'Shea J, Wylezinska M, Kincses ZT, et al. Polarity-sensitive modulation of cortical neurotransmitters by transcranial stimulation. J Neurosci 2009;29:5202-6.  Back to cited text no. 16
    
17.
Lefaucheur JP, Antal A, Ayache SS, Benninger DH, Brunelin J, Cogiamanian F, et al. Evidence-based guidelines on the therapeutic use of transcranial direct current stimulation (tDCS). Clin Neurophysiol 2017;128:56-92.  Back to cited text no. 17
    
18.
Nitsche MA, Seeber A, Frommann K, Klein CC, Rochford C, Nitsche MS, et al. Modulating parameters of excitability during and after transcranial direct current stimulation of the human motor cortex. J Physiol 2005;568:291-303.  Back to cited text no. 18
    
19.
Lang N, Nitsche MA, Dileone M, Mazzone P, De Andrés-Arés J, Diaz-Jara L, et al. Transcranial direct current stimulation effects on I-wave activity in humans. J Neurophysiol 2011;105:2802-10.  Back to cited text no. 19
    
20.
Di Lazzaro V, Restuccia D, Oliviero A, Profice P, Ferrara L, Insola A, et al. Magnetic transcranial stimulation at intensities below active motor threshold activates intracortical inhibitory circuits. Exp Brain Res 1998;119:265-8.  Back to cited text no. 20
    
21.
Hanajima R, Ugawa Y, Terao Y, Sakai K, Furubayashi T, Machii K, et al. Paired-pulse magnetic stimulation of the human motor cortex: Differences among I waves. J Physiol 1998;509:607-18.  Back to cited text no. 21
    
22.
Nakamura H, Kitagawa H, Kawaguchi Y, Tsuji H. Intracortical facilitation and inhibition after transcranial magnetic stimulation in conscious humans. J Physiol 1997;498 (Pt 3):817-23.  Back to cited text no. 22
    
23.
Di Lazzaro V, Oliviero A, Profice P, Insola A, Mazzone P, Tonali P, et al. Direct demonstration of interhemispheric inhibition of the human motor cortex produced by transcranial magnetic stimulation. Exp Brain Res 1999;124:520-4.  Back to cited text no. 23
    
24.
Tokimura H, Di Lazzaro V, Tokimura Y, Oliviero A, Profice P, Insola A, et al. Short latency inhibition of human hand motor cortex by somatosensory input from the hand. J Physiol 2000;523 Pt 2:503-13.  Back to cited text no. 24
    
25.
Rusu CV, Murakami M, Ziemann U, Triesch J. A model of TMS-induced I-waves in motor cortex. Brain Stimul 2014;7:401-14.  Back to cited text no. 25
    
26.
Di Lazzaro V, Oliviero A, Saturno E, Pilato F, Insola A, Mazzone P, et al. The effect on corticospinal volleys of reversing the direction of current induced in the motor cortex by transcranial magnetic stimulation. Exp Brain Res 2001;138:268-73.  Back to cited text no. 26
    




 

Top
 
 
  Search
 
Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References

 Article Access Statistics
    Viewed994    
    Printed77    
    Emailed0    
    PDF Downloaded135    
    Comments [Add]    

Recommend this journal


[TAG2]
[TAG3]
[TAG4]