After-effects of anodal transcranial direct current stimulation on the excitability of the motor cortex in rats

Abstract

Purpose: Transcranial direct current stimulation (tDCS) is increasingly seen as a useful tool for noninvasive cortical neuromodulation. A number of studies in humans have shown that when tDCS is applied to the motor cortex it can modulate cortical excitability. It is especially interesting to note that when applied with sufficient duration and intensity, tDCS can enable long-lasting neuroplastic effects. However, the mechanism by which tDCS exerts its effects on the cortex is not fully understood. We investigated the effects of anodal tDCS under urethane anesthesia on field potentials in in vivo rats.

Methods: These were measured on the skull over the right motor cortex of rats immediately after stimulating the left corpus callosum.

Results: Evoked field potentials in the motor cortex were gradually increased for more than one hour after anodal tDCS. To induce these long-lasting effects, a sufficient duration of stimulation (20 minutes or more) was found to may be required rather than high stimulation intensity.

Conclusion: We propose that anodal tDCS with a sufficient duration of stimulation may modulate transcallosal plasticity.

Keywords

Transcranial direct stimulation cerebral cortex cortical excitability neurophysiology rat

1 Introduction

Transcranial direct current stimulation (tDCS) is a promising tool for cortical neuromodulation. In many human studies, it has been successfully used for the modulation of cortical excitability (Antal, Kincses, Nitsche, & Paulus, 2003; Nitsche et al., 2007; Nitsche et al., 2003; Nitsche & Paulus, 2000). Furthermore, various clinical studies have explored its potential for treating neurologic pathologies such as Parkinson’s disease, chronic pain, stroke, depression and epilepsy (Benninger et al., 2010; Floel, 2014; Fregni et al., 2006; Hummel et al., 2005; Kuo, Paulus, & Nitsche, 2014; Nitsche, Boggio, Fregni, & Pascual-Leone, 2009; San-juan et al., 2015). However, some studies have shown negative results (Iodice, Dubbioso, Ruggiero, Santoro, & Manganelli, 2015). Several animal studies have shown that epidural or intracortical direct current stimulation (DCS) can elicit changes in motor cortex excitability that are dependent on the polarity of the stimulus (Bindman, Lippold, & Redfearn, 1962; Creutzfeldt, Fromm, & Kapp, 1962; Purpura & McMurtry, 1965). Anodal stimulation generally increases neuronal excitability, while cathodal stimulation causes the opposite effect. Interestingly, previous electrophysiological data in humans and animals also show that DCS can produce long-lasting after-effects depending on N-methyl-D-aspartate (NMDA) receptors, intracellular calcium levels, and glutamatergic synapses (Bindman et al., 1962; Bindman, Lippold, & Redfearn, 1964; Cambiaghi et al., 2010; Fritsch et al., 2010; Liebetanz, Nitsche, Tergau, & Paulus, 2002; Márquez-Ruiz et al., 2012; Nitsche et al., 2003; Nitsche & Paulus, 2000; Stagg et al., 2009).

Over the last 30 years, most animal studies of DCS and its electrophysiological effects on the cortex have used invasive methods, such as epidural in vivo approaches or direct stimulation of cortical cells in vitro, to demonstrate the effects of direct current stimulation on the cortex (Bindman et al., 1962, 1964; Creutzfeldt et al., 1962; Purpura & McMurtry, 1965). Recently, several studies (Cambiaghi et al., 2010; Liebetanz, Fregni, et al., 2006; Márquez-Ruiz et al., 2012) have attempted to develop less invasive in vivo methods of DCS. These methods have the advantage to prevent surgery-related tissue damage caused by changes in temperature, bleeding during the surgical procedure, heating, and chemical reactions from stimulation electrodes. Moreover, in vivo approaches have the advantage that these do not compromise spontaneous cortical activity as much as in vitro approaches, which is important for the plasticity effects of DCS. In accordance, in a recent in vitro study using brain slices, Fritsch et al. showed that tDCS elicits synaptic plasticity which depends on brain derived neurotrophic factor (BDNF) secretion and tropomyosin receptor kinase B (TrkB) activation. The induction of long-lasting effects through DCS required however its coupling with repetitive low-frequency synaptic activation, which suggests that DCS requires brain activity to exert its neuroplastic effects. Thus less invasive in vivo DCS procedures are relevant for establishment of an animal model which mimics stimulation in humans closely.

The purpose of the current in vivo animal study was to investigate whether tDCS over the motor cortex can induce long-term after-effects as reported in previous studies, which were primarily conducted in humans. Animal studies applying tDCS are valuable as a means of uncovering the mechanisms by which tDCS exerts its effects and assess its potential as a treatment for neurologic disorders. Most previous animal studies applied epidural or intracortical methods to investigate the effects of DCS on the cortex. We developed a simple and less invasive experimental model with minimal damage in the motor cortex to record. DCS-induced alterations of cortical field potentials were monitored in anesthetized rats using a metallic electrode on the skull over the motor cortex. Evoked field potentials in the motor cortex were obtained by stimulating the contralateral corpus callosum before and after tDCS. In addition, a paired-pulse test was performed to determine whether presynaptic processes that are involved in long-term excitability alterations (Nitsche & Paulus, 2000) are affected by tDCS. To minimize invasiveness, we used the same electrode for recording evoked field potentials and for tDCS.

2 Material and methods

2.1 Subjects

Experiments were performed on male Sprague Dawley rats (n = 23; 300∼450 g; 9∼14 weeks; Samtako, Osan, Korea). The animals were maintained on a 12-hour light and 12-hour dark cycle with a continuously controlled temperature of 22°C and humidity 50%. All surgical procedures were approved by the Institutional Animal Care and Use Committee of Wonkwang University.

2.2 Surgery, stimulation, and recording

All rats were anesthetized with a 20% urethane solution (1.3 g/kg, I.P.). Anesthetized animals were then placed in a stereotactic frame and their body temperature was kept constant at 37.5°C by using a homeothermic heating device (TR-100, Fine Science Tools Inc., Foster City, CA). A craniotomy and durotomy were conducted to place an electrode in the corpus callosum. The electrode was placed by passing it through the cortex. During experiments, artificial cerebrospinal fluid (ACSF; 135 mM NaCl, 5.4 mM KCL, 1 mM MgCl₂, 1.8 mM CaCl₂, and 5 mM HEPES) was applied to the exposed cortex to prevent dehydration.

A Teflon-coated stainless-steel twisted-wire electrode (125 μm exposed tips; 0.5 mm tip separation) was placed in the left corpus callosum (3.0 mm anterior to bregma, 2.0 mm lateral to midline, 3.0 mm below the dural surface). The electrode position was adjusted in depth to maximize evoked field potentials at the surface of the skull (Fig. 1. (A, B)). Reference and ground electrodes were attached to the exposed scalp tissue and to a stereotaxic screw.

To evoke field potentials in the right motor cortex, biphasic, square-wave constant current stimulation pulses with a duration of 0.1 ms and an intensity of 300 μA were delivered to the left corpus callosum at 30 seconds intervals using an A365 stimulus isolator (World Precision Instruments, Sarasota, USA).”

After removing the scalp and underlying tissues, a circle electrode (diameter 3 mm) was positioned on the skull over the right motor cortex (AP 3.0 mm, L 2.0 mm). The space between this electrode and the skull was filled with a highly conductive electrolyte gel (Signa gel, Parker Laboratories, NJ, USA). To block that electrolyte gel spread over skull, a transparent hollow cylinder (3.4 mm diameter) was filled by the electrolytic gel and then located on the place for tDCS. After that, an electrode for tDCS was inserted into its hollow with filled the electrolytic gel. A rubber pad (4 cm²) was placed onto the chest as a counter electrode. Anodal tDCS current was applied at an intensity of 250 μA for 20 minutes (n = 8) or 500 μA for 10 minutes (n = 5) using a direct current stimulator (Cybermedic Corp., Iksan, Korea). These intensities for tDCS were based on previously reported limits (52400 C/m²) for safe stimulation (Liebetanz et al., 2009). To test the effects of coupling of tDCS with repetitive low-frequency synaptic activation, the left corpus callosum of five rats was stimulated at 0.1 Hz during tDCS (n = 5) (Fritsch et al., 2010). The electrode for recording evoked field potentials was also used for tDCS. The current intensity was ramped up at the start of stimulation and ramped down at stimulation termination for 10 seconds to prevent stimulation make and break effects that might be produced by switching it abruptly on and off (Bindman et al., 1964; Liebetanz, Klinker, et al., 2006). For sham stimulation (n = 5), the current intensity of tDCS was zero. For the rest, we performed the same experimental protocols. Just one session was performed with each animal. Signals from recording electrodes were pre-amplified and filtered between 0.1 Hz-300 Hz (CyberAmp 320, Axon instruments, Foster City, CA) and then digitized at 10,000 samples per second (1401 plus, CED, Cambridge, UK). Signals were collected on a computer with a Cambridge Electronic Design (CED) interface and Signal, a data acquisition and analysis package (CED, Cambridge, UK). For baseline measurements, evoked field potentials were measured for 15 minutes before beginning of tDCS. Application of tDCS was continued for 20 minutes, and then recording of potentials was resumed for 60 minutes (Fig. 1. (C)).

The stimulation of the corpus callosum generated waveforms composed of population action potential spikes and excitatory postsynaptic potentials (EPSP) that have been described previously (Teskey, Monfils, VandenBerg, & Kleim, 2002; Wawryko, Ward, Whishaw, & Ivanco, 2004). We collected EPSP amplitudes of evoked field potentials for analysis, but did not attempt to analyze spike population data, because spike populations often disappeared due to waveform distortion (Fig. 2).

2.3 Paired-pulse test

For input and output tests, three responses were evoked and recorded by stimulating the left corpus callosum of the animals for each intensity with the interval of 0.1 mA in a series of increasing intensities (0.1∼1 mA). Moreover, for paired-pulse tests, two pulses were delivered to the left corpus callosum at various intervals (20, 50, 100, and 200 ms) before tDCS and 1 hour after it. For any given intensity, a paired pulse test was repeated three times with 60-s intervals. The paired pulse ratio (PPR) was calculated as the peak EPSP amplitude of the response waveform produced by the second stimulation pulse, divided by the peak EPSP amplitude of the response waveform produced by the first stimulation pulse(Fig. 3).

2.4 Histology

One hour after tDCS, to identify the effect of tDCS on the histopathologic changes in the cortex, the brain was fixed, with a cardiac perfusion fixation under deep urethane anesthesia and then was removed from the skull after end of the recording. After that, the brain was cryoprotected with 30% sucrose solution until the brain sinks to the bottom and then sectioned at 40 μm with a freezing microtome. The brain slices were stained by Hematoxylin and eosin (HE) staining protocol and was visualized under a light microscope.

3 Data analysis

The Friedman test (non-parametric analysis of repeated-measures ANOVAs) was used to evaluate changes of amplitude in responses field potential. Significant results were more analyzed with Wilcoxon signed rank test as post hoc test. The post hoc comparisons were performed after correcting by the Bonferroni method. SPSS statistical software (Version 12.0; SPSS Inc., Chicago, IL, USA) was used for all statistical analysis.

4 Results

Continuously monitoring evoked field potentials for 15 minutes during baseline measures prior to tDCS and for 1 hour after its completion enabled us to observe progressive changes in EPSP amplitudes after tDCS alone and tDCS delivered in combination with LFS (low frequency stimulation). Gradual increases of EPSP amplitude were found after tDCS at 250 μA for 20 minutes as well as tDCS (250 μA for 20 minutes) in conjunction with LFS. The For up to one hour after tDCS, EPSP amplitudes increased by 45.9±14% in comparison with the average baseline EPSP amplitude (p = 0.008). In case of simultaneous LFS during tDCS, the EPSP amplitude after tDCS was up to 20.9±7.5% larger than the baseline value (p = 0.09). These results show that LFS may attenuate the induction of long-term tDCS effects. By contrast, sham tDCS produced no significant changes in EPSP amplitude (in relation to baseline (Fig. 2). The results also show that the observed tDCS-induced elevation of EPSP amplitudes was affected by the duration of tDCS. With a current intensity of 250 μA for 20 minutes, tDCS progressively increased EPSP amplitude for more than one hour, whereas tDCS with a current intensity of 500 μA for 10 minutes only slightly increased EPSP amplitudes (7.1±12.1%; p = 0.255). Moreover, in difference to the 250 μA for 20 minutes condition, 500 μA tDCS for 10 minutes, increased field potentials for a relevantly shorter duration (Fig. 2). To evaluate possible changes in PPR after tDCS (250 μA for 20 minutes), we compared PPRs before with PPRs measured 1 hour after tDCS (Fig. 3). We could not find any significant differences between PPR before and after tDCS. PPR applied with a 20-ms inter-pulse interval showed a tendency towards facilitation, while PPR at other paired pulse intervals (50 ms, 100 ms and 200 ms) did not show any noticeable tendency. PPRs at paired-pulse intensities of 300 μA and 400 μA decreased after tDCS, whereas PPR at other paired pulse intensities showed a tendency towards being facilitated tendency after it. From histological analysis (HE staining), there were no morphological changes in the brain tissue after anodal tDCS at 250 μA for 20 minutes (Fig. 4).

5 Discussion

The results of this study show that anodal tDCS over the in vivo rat motor cortex induces long-lasting after-effects on cortical excitability. The response of the motor cortex to stimulation of the corpus callosum was gradually increased after anodal tDCS, regardless of whether or not low frequency stimulation of the corpus callosum caused repeated externally driven synaptic activation during tDCS. A sufficient duration of stimulation may play an important role to induce this long-term effect.

To demonstrate long-lasting effects of tDCS on the motor cortex, the majority of previous studies in humans (Pellicciari, Brignani, & Miniussi, 2013) and a recent animal study (Cambiaghi et al., 2010) investigated alterations of motor-evoked potentials (MEPs) after tDCS. They showed that anodal tDCS increases MEP amplitude, while cathodal tDCS decreases it.

To monitor the excitability of motor cortex, stimuli of afferents from the somatosensory cortex (Hasan et al., 2013; Atusushi Iriki, Pavlides, Keller, & Asanuma, 1989) or thalamus (Atusushi Iriki et al., 1989; ATSUSHI Iriki, Pavlides, Keller, & Asanuma, 1991) were mainly used. In the current experiments, we stimulated the transcallosal pathway with one bipolar pulse every 30 seconds to evoke field potentials in the contralateral motor cortex. This method is generally a useful tool for investigating places in the brain that we cannot access through peripheral stimulation (Hoffmeyer, Enager, Thomsen, & Lauritzen, 2007; Racine, Wilson, Teskey, & Milgram, 1994).

Therefore, in the present study, we directly estimated tDCS-induced excitability changes at the cortical level by measuring field potentials on the skull over the motor cortex evoked by stimulating, the corpus callosum of the contralateral hemisphere.

5.1 Comparison with the results of other studies

Our results are similar to those of other previously published studies that report a gradual increase in neuronal excitability after anodal tDCS. In a recent human study, the corticospinal excitability of young adults was largest immediately after anodal tDCS, while corticospinal excitability in older adults was delayed (Fujiyama et al., 2014). These results from older adults are similar to our experimental data. In an in vitro mouse brain slice study, electrophysiological data obtained were also similar to our results (Fritsch et al., 2010). However, in difference to our results, low-frequency synaptic activation during DCS was required to induce a long-lasting effect in vitro. In this study, low-frequency synaptic activation during tDCS was not vitally required to elicit long-term effects after tDCS. To explain the differences between this in vitro study and our in vivo study, we note that brain slices do not experience variations in blood supply, or network effects from distant neurons. In this respect, previous research showed that tDCS could cause widespread variations in blood flow (Stagg et al., 2013) and alterations in the functional network (Notturno, Marzetti, Pizzella, Uncini, & Zappasodi, 2014). Especially, spontaneous activities of neuron reduce in brain slice. Its enhancement facilitates synaptic plasticity. We cannot exclude these potential factors when attempting to explain our results in which we induced long-term effects after tDCS.

According to our experimental results, tDCS for 20 minutes or more was required to induce long-term effects. This fits well to previous animal data (Bindman et al., 1964). A sufficient duration of stimulation was found to be more critical for producing this effect than intensity of stimulation. For two experiments with equal charge densities, tDCS applied at an intensity of 250 μA for 20 minutes was more effective than tDCS applied at an intensity of 500 μA for 10 minutes for eliciting long-term effects (Fig. 2). In this study, tDCS was applied at a charge density of 42480 C/m², while the mean current density applied in humans is 171∼480 C/m² (Brunoni, Fregni, & Pagano, 2011; Liebetanz et al., 2009). Although it may need more intensity because of lower spontaneous neuronal activity under anesthesia, current densities of tDCS in our experiment greatly exceed those being used for humans. Thus, the duration of stimulation may be a more significant factor to be considered regardless of the current intensities used because both current intensities of 250 μA and 500 μA used in this experiment were veryhigh.

A paired pulse test was used to evaluate whether presynaptic mechanisms were affected by tDCS (Zucker & Regehr, 2002). Presynaptic LTP is associated with an increase in presynaptic neurotransmitter release probability (Bliss, Errington, Lynch, & Williams, 1990). According to previous studies, a decrease in PPR is associated with an increase in the probability of transmitter release, which means that LTP expression involves a presynaptic process (Kabakov, Muller, Pascual-Leone, Jensen, & Rotenberg, 2012; Schulz, Cook, & Johnston, 1994). However, we could not find significant changes in PPR after tDCS. Thus we conclude that presynaptic processes may not play a critical role in long-lasting after-effects of tDCS, as induced by the protocols in our study. This result is in accordance with other studies applying different LTP-inducing stimulation protocols (Manabe, Wyllie, Perkel, & Nicoll, 1993; McNaughton, 1982).

5.2 Mechanisms of action – a hypothesis

Some previous studies have shown that tDCS modulates membrane potentials, which results in alteration of the probability of action potential generation in case of spontaneous brain activity, and the timing of neuronal depolarization (Bikson et al., 2004). In several clinical experiments, it has been shown that tDCS-induced plasticity depends on NMDA receptors (Liebetanz et al., 2002; Nitsche et al., 2003; Nitsche et al., 2004) and is calcium-dependent. Thus most probably, combination of depolarization with spontaneous activity enhancement increases probability of NMDA receptor opening, which increases calcium influx and then results in LTP in case of anodal tDCS. Depolarization-induced opening of voltage-gated calcium channel might also contribute. Although the induction of LTP-like effects depends on changes in NMDA receptor-dependent glutamatergic interneurons (Aroniadou & Keller, 1995; Castro-Alamancos, Donoghue, & Connors, 1995; Hess & Donoghue, 1996) and GABAergic interneurons (Stagg & Nitsche, 2011; Trepel & Racine, 2000). As mentioned above, Fritsch et al. (2010) in their in vitro mouse brain slice study, were only able to induce long-term effects when tDCS was accompanied by LFS. However, in our in vivo study, we showed long-term effects after tDCS regardless of LFS. We believe that the difference between these sets of results is due to the absence of neuronal spontaneous activities and blood flow or to the lack of connections with broader neuronal networks in the slice preparation. They may have a connection to the mechanisms of the long-term effects. One possible hypothesis is that astrocytes, a type of glial cells known to play a pivotal role in cerebral perfusion, may be crucial (Metea & Newman, 2006). A recent theoretical analysis demonstrated that tDCS may be sufficient to depolarize astrocytes (Ruohonen & Karhu, 2012). Changes in neuronal plasticity produced by tDCS could also cause changes in astrocytic activity. Furthermore, cortical layer 1 consists predominantly of glial cells that extend to layers 2– 4. They could easily be influenced by tDCS because of their proximity to the stimulation electrode. Additionally, because the same electrode was used for recording and stimulating, electrophysiological changes caused by astrocytic activity in superficial cortex may be prevalent when we measure evoked field potentials because the distance between the stimulated astrocytes in superficial cortex and the recording electrode is very small. Accordingly, we cannot ignore a role for astrocytes in explaining our results. Recently, several studies have proposed that astrocytes play a role in synaptic plasticity (Takata et al., 2011). An increase in intracellular calcium concentration in astrocytes is known to effect the induction of LTP, and D-serine from astrocytes is necessary for LTP in nearby synapses (Henneberger, Papouin, Oliet, & Rusakov, 2010). However, whether or not astrocytes play a direct role in synaptic plasticity remains controversial.

5.3 Limitations

Evoked potential recordings were performed in animals under urethane anesthesia to continuously measure field potentials for several hours. Although urethane may cause a slight decrease in neuronal activity by the leaked potassium current, it has comparatively fewer influences on synaptic transmission than other anesthetics, like ketamine and pentobarbital (Sceniak & MacIver, 2006; Thimm & Funke, 2015). Nevertheless, this pharmacological agent may impact on the results of tDCS on cortical excitability, and hamper direct comparability to studies in awake humans.

We used the same metallic electrode for both recording evoked field potentials and for administering tDCS. A single electrode is often used for both recording and stimulation in closed-loop systems or in vitro studies using multi-electrode arrays (Rolston, Gross, & Potter, 2010). Combining recording and stimulation with one electrode makes recording during stimulation unfeasible, and may cause stimulation artifacts, which prevents recording also for some time after the end of intervention. The voltages measured when recording neuronal signals are roughly 10 μV, whereas the stimulation potentials are measured in volts. The scale difference between recording and stimulation is thus roughly 100,000 fold. When recording follows stimulation, the magnitude of the signal can result in saturation of the recording channel because the recording scale is much smaller than the stimulation scale. Thus, within the present protocol we were not able to record neuronal signals for several seconds after tDCS. Field potential recordings were started after one minute after termination of tDCS. At this time stimulation artifacts were no longer present. Moreover, the impedance increased instantaneously by tDCS and then returned to pre-stimulation impedance within 10 minutes after tDCS. Thus, it was no problem to investigate the tendency of cortical excitability for more than onehour.

6 Conclusions

In this study, we demonstrated long-lasting LTP-like effects after anodal tDCS in vivo, using a less invasive experimental model. This model may contribute to improving our understanding of the mechanisms of tDCS action on the motor cortex. It also sets the grounds for investigations with various transcranial electrical stimulation methods such as tDCS, transcranial alternating current (tACS), and transcranial random noise stimulation (tRNS) (Paulus, 2011) through via in vivo animal models of various human neurologic disorders. Therefore, this animal experimental model may help us to verify various clinical implications oftDCS.

Disclosures

MAN is member of the advisory board of Neuroelectrics.

Footnotes

Acknowledgments

This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2014R1A2A2A01002501).

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