Author response: Lifting the veil on the dynamics of neuronal activities evoked by transcranial magnetic stimulation
2017-11-11

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Transcranial magnetic stimulation (TMS) is a widely used non-invasive tool to study and modulate human brain functions. However, TMS-evoked activity of individual neurons has remained largely inaccessible due to the large TMS-induced electromagnetic fields. Here, we present a general method providing direct in vivo electrophysiological access to TMS-evoked neuronal activity 0.8–1 ms after TMS onset. We translated human single-pulse TMS to rodents and unveiled time-grained evoked activities of motor cortex layer V neurons that show high-frequency spiking within the first 6 ms depending on TMS-induced current orientation and a multiphasic spike-rhythm alternating between excitation and inhibition in the 6–300 ms epoch, all of which can be linked to various human TMS responses recorded at the level of spinal cord and muscles. The advance here facilitates a new level of insight into the TMS-brain interaction that is vital for developing this non-invasive tool to purposefully explore and effectively treat the human brain. https://doi.org/10.7554/eLife.30552.001 eLife digest Being able to tap into someone’s brain activity by holding loops of wires above their head sounds a little like the stuff of science fiction. And yet this technique, known as transcranial magnetic stimulation or TMS, is used in research and to treat many brain disorders. TMS emits a pulsed magnetic field that induces tiny electrical currents in the underlying brain tissue, activating that region of the brain. But exactly how these currents affect the individual neurons and networks within activated brain regions remains unclear. The main reason for this is that we cannot use conventional electrode-based techniques to study neuronal activity during TMS because its strong electromagnetic interferences mask the signals from the electrodes. Several groups have found ways to overcome this problem. However, their methods are technically demanding and specific to one single animal model –limitations that could present an obstacle for many laboratories. Li et al. therefore set out to develop a simple and widely accessible method to study neuronal activities under TMS. The resulting method makes it possible to measure the activity of individual neurons roughly 1/1,000th of a second after applying TMS. To show that the technique works, Li et al. induced small movements in the forelimbs of rats by applying TMS to the brain region that controls the forelimbs, while measuring the activity of neurons at the same time. This revealed, for the first time, how the neurons responsible for the forelimb movements responded to TMS. The observed TMS-triggered neuronal activity continued long after the TMS pulse had ended. The activity also varied depending on the direction of TMS-induced currents in the brain. This new method opens up the possibility to conveniently study – in rodents or other animals – how TMS procedures that are used in patients affect neuronal activity. Li et al. hope this will make it easier to develop, study and refine these procedures, and lead to advances in TMS therapies. https://doi.org/10.7554/eLife.30552.002 Introduction In 1985, when Barker et al. (1985) applied a pulsed magnetic field to a subject’s head to selectively evoke responses in a hand muscle, the scientific community was fascinated by this pain-free, electrodeless, and non-invasive tool for brain stimulation. Since then, transcranial magnetic stimulation (TMS) has attracted enormous interest in neurology, psychiatry, and applied neuroscience research for its unique capability of non-invasively activating neuronal populations and inducing plasticity. Despite its impressive array of applications and fast-growing popularity (Cohen et al., 1998; Walsh and Cowey, 2000; Reis et al., 2008; Funke and Benali, 2011; Lefaucheur et al., 2014), TMS is poorly understood physiologically as we know very little about how TMS interacts with the brain at the level of neurons and circuitries. Although various sources of indirect evidence obtained from human studies support the use of TMS in multiple contexts (Di Lazzaro et al., 2008; Chung et al., 2015; Suppa et al., 2016), our very limited insight into the neurophysiology of TMS remains a bottleneck that hampers the utilization and the development of TMS applications, blocking the exciting potential of this non-invasive brain stimulation tool. One critical reason behind this inadequacy is the absence of a research platform on which TMS-evoked neuronal activities can be investigated in vivo in real time. Extracellular electrophysiology (EEP) with microelectrodes is the gold standard for studying brain activities at the level of neurons (Scanziani and Häusser, 2009). However, a single TMS pulse, characterized by an alternating tesla-level magnetic field with a center frequency of approximately 4 kHz (Ilmoniemi et al., 1999; Wagner et al., 2007), generates an array of strong interferences disturbing the acquisition of EEP signal. As evidenced by the pioneering works on TMS-EEP (Moliadze et al., 2003; Pasley et al., 2009), artifacts from TMS resulted in a significant amount of data loss that precluded the investigation of TMS-evoked neuronal activities for up to 100 ms after each TMS pulse. Several groups used innovative imaging methods to circumvent this problem (Allen et al., 2007; Kozyrev et al., 2014; Murphy et al., 2016); however, their investigations were restricted to the upper cortical layers and they were unable to reach the single-neuron and millisecond precision needed to decipher the intricate interactions between TMS and neurons. Recently, two groups reported TMS-EEP methods for non-human primate research. One of the methods utilized custom-built TMS coil and offline correction to minimize the TMS-induced data loss to a median time of 2.5 ms (Tischler et al., 2011), while the other used a combination of custom-built coil, amplifier modifications, field sensing, active compensation, and offline correction to minimize the data loss to 1 ms (Mueller et al., 2014). Despite their success in artifact reduction, these methods face a major limitation that the technical expertise required for their implementations, especially custom-building TMS coils and field sensing, is not widely accessible to the neuroscience community. More importantly, these methods were developed solely for non-human primate research, which is used for investigating the neuronal underpinnings of high-level cognitive functions and therefore is not best suited for investigations concerning basic neurophysiology on the level of cells and detailed microcircuits. With the aim of establishing a widely applicable in vivo experimental platform to study the dynamics of TMS-evoked neuronal activities and to further develop the scientific and clinical applications of this powerful technique, we engineered a novel method for TMS-EEP that is suitable for, but not limited to, laboratory rodents, which are widely accessible and offer a rich repertoire of experimental techniques including transgenic and optogenetic tools. The method is compatible with existing standard TMS coils and allows the recording of neuronal activities 0.8–1 ms after the onset of various types of TMS stimuli by attenuating artifacts resulting from magnetic induction, electric field coupling, and vibrations. Furthermore, the method allows for the instantaneous determination of TMS-driven inadvertent charge injection into the neural tissue, a problem that has been overlooked by almost all prior TMS-EEP studies. In the following sections, we will present this methodological advance and demonstrate its potential by unveiling neuronal activities in the layer V of the primary motor cortex (M1) that accompany cortically evoked unilateral muscle activations by monophasic single-pulse TMS (mspTMS). Additionally, we will also demonstrate that mspTMS modulates different neuronal circuits depending on the orientation of the stimulating current or the time-window of investigation. Results Attenuation of the induction artifact The induction artifact (Figure 1, period indicated in blue) is created as coil-generated rapid magnetic flux change induces voltages within loops formed along an EEP recording assembly. Owing to the large rate of flux change, the induction artifact, if transmitted to the high-gain and filter stages of an EEP amplifier, can easily lead to signal saturation and data loss (Figure 1 inset). To address this, we developed a gated multi-stage TMS-EEP amplifier (Figure 2). It consists of a differential preamplifier (Pamp) stage of gain four and a filter-amplifier (Famp) stage of gain 500, separated by an ultra-low capacitance/charge injection solid-state analog switch (SSSW) controlled by optically coupled digital signal synchronized to TMS. The components of the amplifier were chosen to provide the optimal balance between voltage and current noise with the source impedance of EEP microelectrodes. The Pamp stage must be able to maintain its high impedance character when being perturbed by TMS. Failing to do so will result in excessive induction current in the input wires that leads to electrical stimulation of the brain. In our design, the electronic components and supply voltage of the Pamp stage were chosen so it can tolerate ±7.9 V input during TMS. Due to its high-gain and filters, the Famp stage must be protected from TMS by SSSW that grounds the input to Famp for a user-defined time interval around TMS onset (e.g. from 0.2 ms pre- to 0.8 ms post-TMS). The SSSW was strategically placed behind the input capacitor of a high-pass filter of the Famp so that the input capacitor is preconditioned to any DC bias in the microelectrodes before the end of the grounding period. To minimize ground bounce and to reduce ground loop, the external digital signal that controls SSSW is connected to the amplifier circuit through an optocoupler (OC). In addition, to protect the amplifier circuit from TMS-induced fields, the circuit board of the amplifier is mounted inside a 1.5-mm-thick grounded aluminum enclosure. The Pamp, as well as DC-DC converters, is housed inside a metal shield for additional protection. Furthermore, to minimize artifacts from vibrations due to the loud click sound of TMS coils, solder contacts, instead of spring-loaded connectors, were used whenever possible, as well as polyphenylene sulfide film capacitors, since they do not generate piezoelectric voltages from vibration. The default frequency response of the amplifier was set from 300 Hz (−3 dB) to 5 kHz (−6 dB), but the lower bound of the passband can be adjusted to 4 Hz as needed for different applications. A simplified circuit diagram of the amplifier, including the model number of its critical components, is shown in Figure 2. Figure 1 Download asset Open asset Simultaneous TMS-EEP recording requires artifact attenuation in multiple dimensions. The full waveform of a TMS artifact recorded differentially under low-gain (x4) using a high impedance amplifier. The artifact consists of a series of sharp deflections (induction and fast E-field coupling artifacts) and a long tail (polarization decay artifact resulted from E-field coupling). The slow polarization decay artifact renders the signal out of range (indicated by the green area) until ca. 30 ms post-TMS in a standard EEP system. Vibration artifacts (see Figure 4B) are not visible here due to low amplification. The inset shows that under high-gain (x2000) needed for EEP, TMS artifacts lead to long signal saturation in a standard EEP system (bandpass 300–5000 Hz) while producing negligible interference in our method. Lightning symbol, TMS onset (at 0 ms). E-field, electric field. https://doi.org/10.7554/eLife.30552.003 Figure 2 Download asset Open asset Simplified circuit diagram of the TMS-EEP amplifier. Model numbers of the most critical electronic components are noted in red, the values of certain elementary components are noted in grey (units: ohm for resistor; farad for capacitor), and the amplification factor for each stage is indicated in parenthesis. Pamp, pre-amplifier stage; Famp, filter-amplifier stage; OC, optocoupler; SSSW, solid-state analog switch; LGM, low-gain monitoring channel. https://doi.org/10.7554/eLife.30552.004 Attenuation of electric field coupling artifacts The coil-emitted electric field gives rise to another type of artifacts. When a TMS pulse is triggered, a large current driven by a kV-level voltage pulse flows through the coil. Inadvertently, this process emits an electric field that injects a displacement current into the EEP recording assembly through capacitive coupling (Figure 3A). In a short time-interval (<0.4 ms; Figure 1, period indicated in orange), as the coil current rapidly fluctuates, the displacement current generates a fast-changing artifact in EEP signal. In a long time-interval (tens of ms; Figure 1, period indicated in yellow), a decay-like artifact is observed as the displacement current polarizes the electrochemical double-layer of microelectrode tips and thereby generates a decaying waveform while the double-layer returns to its equilibrium potential. Depending on stimulation intensity, electrode impedance, and filter settings, the decay may persist with relatively high signal values for tens of milliseconds before re-entering the input range of a standard EEP amplifier (green area in Figure 1), contributing to an extended period of data loss. To address this problem, we developed an electrical shield for the TMS coil that substantially attenuates the coil-emitted electric field (Figure 3B). One important consideration in shield construction is that the amount of eddy current in the shield should remain low; otherwise, strong vibration or even magnetic attenuation may occur. Therefore, we applied a layer of weakly conducting material in shield construction and the resulted shield possesses an electrical resistance of 10 kΩ (see Materials and methods) that does not result in vibration and magnetic attenuation. Figure 3 Download asset Open asset Electric field coupling in TMS-EEP and its attenuation. (A) A schematic illustrating how electric field coupling interferes with the EEP recording circuit. Here, the loop between one microelectrode and the ground is used as an example. The microelectrode is modeled as a parallel resistor-capacitor for simplification. Note how displacement current (ID), generated by electric field coupling, propagates in both directions once it enters the circuit, while the magnetically induced current (Iind) only propagates in a circular manner. The branch of displacement current (ID1) that flows toward the input end of the amplifier opposes the Iind, counteracting the magnetically induced voltage change across the amplifier input resistance (Rin). The other branch (ID2) flows toward the electrode and can cause polarization at the microelectrode tip. Abbreviations: B, magnetic field; CEL, electrode capacitance; Cin, amplifier input capacitance; Icoil, TMS coil current; REL, electrode resistance. (B) The electrical shield constructed for the Magstim D25 coil. The shield fits tightly with the coil and is grounded through the EEP recording system. (C) Induction waveforms from a pickup probe positioned right below the coil center, along the X-, Y- and Z- axis, with or without the shield, under mspTMS at maximum intensity. Along each axis, the waveforms obtained under shielded and no shield condition overlap, confirming that the shield does not attenuate the magnetic output of the TMS coil. (D) Input voltage to a high impedance buffer (AD825, Vs =±15V), measured with a 1.5 MΩ (1 kHz) microelectrode, and an Ag/AgCl ground electrode in normal saline under mspTMS at maximum intensity with or without the shield. Signal in the ‘Removed’ condition was obtained by taking the difference between the waveforms in ‘No shield’ and ‘Shielded’ condition. The shield restored the correct induction waveform and abolished the voltage offset that leads to the decay. https://doi.org/10.7554/eLife.30552.005 To verify the performance of our shield, we first used a magnetic pickup probe to confirm that at 10 kΩ, the shield does not attenuate the magnetic output of our TMS coil. As Figure 3C illustrates, induction voltage waveforms, with and without the shield, overlap perfectly, confirming the absence of any noticeable magnetic attenuation. Subsequently, using a high-impedance buffer, we measured the voltage between an EEP microelectrode and a ground electrode, both electrode tips immersed in a saline bath, under mspTMS at 100% maximum stimulator output (MSO) delivered with or without the shield. We expected that the shield would remove, to a large extent, voltage signal that is due to electric field coupling between the TMS coil and the EEP recording assembly. Figure 3D illustrates the results from these measurements. Without the shield, the captured waveform was drastically different from the induction waveforms resulted by mspTMS (as seen in Figure 3C), and it ended with a strong voltage bias (polarization). With the shield in place, the captured waveform appeared consistent with the induction waveforms and the voltage bias was no longer visible. These findings confirm that by interrupting electric field coupling, the shield is effective in preventing polarization and the decay artifact that follows. Attenuation of vibration artifacts Upon elimination of artifacts resulted from induction and electric field coupling, vibration artifacts, which are normally masked by the other artifacts, become visible (Figure 4B). Vibration can be generated by magnetic force, as well as by sound pressure perturbation. For the magnetically mediated vibration, an avoidance of ferromagnetic materials and large conductive surfaces in the close vicinity of the coil is adequate. However, the elimination of vibration artifacts driven by sound pressure is not straightforward. When a TMS pulse is triggered, a loud click sound is produced by coil wires due to the attractive forces between them. This sound is problematic as it generates micro-vibration in the amplifier input cables. The extremely weak signal (μV-level) these cables carry can be easily perturbed by micro-vibrations through the triboelectric effect (Fowler, 1976). Since the generation of such click sound is inevitable, we attenuated the vibration artifacts by using a special type of low-noise miniature coaxial cable with a semiconducting layer added between its dielectric and braided shield (Figure 4A). The addition of this semiconducting layer provides a path that drains triboelectric charges, rendering the cables insensitive to vibration (Figure 4C). Figure 4 Download asset Open asset Low-noise miniature coaxial cable attenuates vibration artifacts. (A) A schematic illustration of the 36-gauge low-noise miniature coaxial cable. A semiconducting layer of graphite is added between the braided shield and the dielectric of the cable to drain triboelectric charges, rendering the cable insensitive to vibration. (B) An example of vibration artifacts recorded under the standard EEP conditions (x2000 using our TMS-EEP amplifier; 1.5 MΩ microelectrode pair; Ag/AgCl ground electrode; normal miniature coaxial in a saline after induction and electric field coupling artifacts were The vibration artifacts can in multiple types of depending on the of the recording assembly that are perturbed and the of these (C) The of low-noise miniature coaxial cables attenuated the artifacts. Signal recorded under conditions to in (B) the cables. Despite the impressive performance of the low-noise miniature coaxial cable in attenuating vibration artifacts, its in an EEP recording assembly should be limited as the with the amplifier input and electrode impedance, as a voltage that attenuates EEP signal. In our we the of our cables at to the signal attenuation at 1 and determination of inadvertent charge injection TMS-driven inadvertent charge injection is another major which has been overlooked by most prior using EEP under TMS (Moliadze et al., Pasley et al., 2009). into the brain and to the multiple loops of electric circuit are formed (Figure When being to alternating electric and magnetic voltages can be developed along these loops that current injection into the brain through microelectrodes. the amount and the of the current are to the reported in charge from to current waveform approximately to that of and and and such current will neuronal around the microelectrode tips and therefore the of TMS Therefore, it is that the development of voltages along these loops be Since a large of the electric field had been by the coil shield, were for magnetic These a of microelectrodes as well as cable (Figure 1), both minimize the area of induction loops to TMS. More importantly, we a low-gain monitoring as seen in Figure in our amplifier that to conveniently the amount of inadvertent current without any additional under each experimental The from which is measured by the LGM, to current is possible since input and its voltage are and the amount of current through the input is to the amount of current in the circuit. A detailed of this is in Materials and methods and Figure 2. Figure 5 with 2 all Download asset Open asset TMS inadvertent charge injection in multiple loops formed by an EEP recording assembly. (A) A schematic illustration of the induction loop formed between the recording and the microelectrode under TMS. In the that TMS-induced voltage is high a amount of electrical current can within the loop and this may neuronal around the microelectrode (B) A schematic illustration of the induction loop formed between a microelectrode and the ground electrode under TMS. In vivo method under various types of TMS stimuli In we and our method. Figure an of our recording and the the performance of the method in vivo under a single monophasic (Figure and (Figure TMS pulse, as well as a of Hz (Figure which is the of stimulation et al., The stimuli delivered here can be as the as the used magnetic up to four that are one of the TMS (see Materials and the EEP signal between 0.8 and 1 ms after the onset of each TMS pulse and was from artifacts. Furthermore, the amount of inadvertent charge injection under each condition was below a factor of or Figure the or reported in and and confirming the of our measurements. Figure 6 with 1 all Download asset Open asset TMS-EEP recording and rapid signal under the TMS (A) A schematic illustration of our recording direction of coil current; inset direction of induced current in the forelimb area to forelimb in A of in vivo under the (see Materials and methods) and The short to during which the amplifier is protected from the induction artifact is indicated in Lightning symbol, TMS onset (at 0 (see Materials and mspTMS in the layer V of forelimb a multiphasic of neuronal activities With the developed we to address the are the neuronal of TMS cortically evoked muscle In another of rats by we recorded in vivo neuronal activities in the layer V (Figure of the forelimb area to the forelimb area of primate et al., With the coil center positioned the and the induced current from the to the of the brain Figure mspTMS evoked unilateral of the right Simultaneous of the and right muscle motor in right to the Figure and the of Figure The onset of the was around to that found in our single-pulse (Figure and in the single-pulse et al., et al., confirming the cortical of TMS-evoked muscle Figure with 4 all Download asset Open asset mspTMS evoked multiphasic response alternating between excitation and (A) and 1 of activity evoked by mspTMS orientation intensity onset at 0 recorded in layer V of the from one (B) of evoked to in obtained by in the muscle and to the of in the layer V of evoked by mspTMS of intensity. The were by a for example of evoked in the from one by the 2.5 or of the of (see Materials and methods for the neuronal in layer V of the mspTMS evoked a of neuronal activities alternating between excitation and inhibition that until approximately 300 ms Figure illustrates the and its time of rate from one Figure show the evoked by Materials and methods) with stimulation intensity and motor across all were on the 2.5 and of the of during ms Materials and methods for to rate We the evoked significant and into excitation period of that around inhibition in after the and excitation period of following the To the of stimulation intensity on the of each we constructed intensity the for the excitation and the excitation while the for the inhibition It is important to here that the response in the