Direct effects of transcranial electric stimulation on brain circuits in rats and humans methods
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Focused TES effect by Intersectional Short Pulse stimulation
For many experimental and clinical applications,, it would be desirable to apply TES in a spatially targeted manner and simultaneously monitor the induced electrical changes to verify online effectiveness. Because the scalp, skull, and brain conduct current in a homogenous manner, simultaneous application of TES through multiple electrode pairs cannot induce a spatially confined effect (see Supplementary Figure ). Our proposed solution to achieve spatially targeted TES effects is to apply spatio-temporally rotating Intersectional Short Pulse (ISP) stimulation. This method exploits the short integration time constant of the neuronal membrane (5-20 ms), a mechanism that can temporally integrate multiple electrical gradients with similar vector directions (Fig., Supplementary Figure ). An added advantage of fast pulse stimulation (2.5 or 10 µs...
Discussion
An experimental advantage of the ISP technique is that high frequency pulses do not saturate recording amplifiers. This feature allowed us to measure the physiological effects of scalp stimulation in human subjects. Instead of focusing on brain rhythm-entrainment effects,, in which residual artifacts are notoriously difficult to eliminate,, we examined how the amplitude of the spontaneous LFP was biased by the slowly changing forced fields. This method is similar to using tDCS at multiple current levels, where the additive/subtractive effect of the applied field can be probed on the amplitude of native network patterns. We have verified the validity of this approach previously in rodents, using both LFP and unit firing. In support of the estimated voltage gradients from the cadaver experiments and the 'minimum' fields (~1 mV/mm) in rodents to affect network ac...
Comparing the effect of TES and ISP stimulation
To compare the effects of ISP and DC stimulation in rats, the same surgery procedure was applied but the stimulation was performed in current-controlled mode (stimulus intensity 200 µA) using the high-speed analog switch-based circuits described below. The recorded signals ( n = 64 channels) were amplified (400 × gain) and stored after digitization at 20 kHz sampling rate per channel (KJE-1001, Amplipex, Szeged, Hungary). We repeated the same measurements on one awake, freely moving animal.
Measurements on human cadavers
Recordings were performed at the Department of Pathology, Faculty of Medicine, University of Szeged. Medical history of cadavers was consulted in advance and only those with no known brain disorder were selected for measurements. The corpses were kept at 4 °C after death in plastic bags until autopsy to prevent desiccation. The autopsy theater temperature was 22 °C. The routine medical autopsy procedure was done on the same day as experimental measurements. The sample size required (number of cadavers and recording sessions) was extrapolated from the results of animal studies. There was no blinding or randomization employed. No cadaver was excluded from the analysis.
Recording tACS-induced intracerebral electric fields
A needle was inserted through the skull above the prefrontal cortex and served as reference electrode. Physiologic saline solution (2-5 ml) was injected through the same hole to refill the cerebrospinal fluid lost during the drilling procedure. Recording electrodes made a watertight seal in the skull holes, thus further leakage was not significant. The chest wall was used as grounding. Subcutaneous (electrodes placed on the skull surface) alternating and direct current stimulation was performed using stimulation signals generated by either an STG 4008-16 mA (Multi Channel Systems, Reutlingen) or an NI 6343 board (National Instruments) with precision isolation amplifier circuits. The floating (cadaver) side of the isolation amplifiers were powered using two 9-V batteries for each stimulator pair. To monitor the applied current, a 100-Ω resistor was placed...
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For many experimental and clinical applications,, it would be desirable to apply TES in a spatially targeted manner and simultaneously monitor the induced electrical changes to verify online effectiveness. Because the scalp, skull, and brain conduct current in a homogenous manner, simultaneous application of TES through multiple electrode pairs cannot induce a spatially confined effect (see Supplementary Figure ). Our proposed solution to achieve spatially targeted TES effects is to apply spatio-temporally rotating Intersectional Short Pulse (ISP) stimulation. This method exploits the short integration time constant of the neuronal membrane (5-20 ms), a mechanism that can temporally integrate multiple electrical gradients with similar vector directions (Fig., Supplementary Figure ). An added advantage of fast pulse stimulation (2.5 or 10 µs pulse width with 5 or 50 µs pause, depending on the number of electrode pairs) is that the transients of high frequency pulses affect simultaneously recorded LFP or neuronal spikes (1 Hz-5 kHz; 20 kHz sampling) substantially less than conventional tACS and they do...
An experimental advantage of the ISP technique is that high frequency pulses do not saturate recording amplifiers. This feature allowed us to measure the physiological effects of scalp stimulation in human subjects. Instead of focusing on brain rhythm-entrainment effects,, in which residual artifacts are notoriously difficult to eliminate,, we examined how the amplitude of the spontaneous LFP was biased by the slowly changing forced fields. This method is similar to using tDCS at multiple current levels, where the additive/subtractive effect of the applied field can be probed on the amplitude of native network patterns. We have verified the validity of this approach previously in rodents, using both LFP and unit firing. In support of the estimated voltage gradients from the cadaver experiments and the 'minimum' fields (~1 mV/mm) in rodents to affect network activity, we found that >4.5 mA currents were required to reliably bias the amplitude of occipital alpha waves. While we designed our experiments to maximize the stimulation effects on the parietal-occipital region where alpha waves are of largest amplitude, we cannot exclude the possibili...
To compare the effects of ISP and DC stimulation in rats, the same surgery procedure was applied but the stimulation was performed in current-controlled mode (stimulus intensity 200 µA) using the high-speed analog switch-based circuits described below. The recorded signals ( n = 64 channels) were amplified (400 × gain) and stored after digitization at 20 kHz sampling rate per channel (KJE-1001, Amplipex, Szeged, Hungary). We repeated the same measurements on one awake, freely moving animal.
Recordings were performed at the Department of Pathology, Faculty of Medicine, University of Szeged. Medical history of cadavers was consulted in advance and only those with no known brain disorder were selected for measurements. The corpses were kept at 4 °C after death in plastic bags until autopsy to prevent desiccation. The autopsy theater temperature was 22 °C. The routine medical autopsy procedure was done on the same day as experimental measurements. The sample size required (number of cadavers and recording sessions) was extrapolated from the results of animal studies. There was no blinding or randomization employed. No cadaver was excluded from the analysis.
A needle was inserted through the skull above the prefrontal cortex and served as reference electrode. Physiologic saline solution (2-5 ml) was injected through the same hole to refill the cerebrospinal fluid lost during the drilling procedure. Recording electrodes made a watertight seal in the skull holes, thus further leakage was not significant. The chest wall was used as grounding. Subcutaneous (electrodes placed on the skull surface) alternating and direct current stimulation was performed using stimulation signals generated by either an STG 4008-16 mA (Multi Channel Systems, Reutlingen) or an NI 6343 board (National Instruments) with precision isolation amplifier circuits. The floating (cadaver) side of the isolation amplifiers were powered using two 9-V batteries for each stimulator pair. To monitor the applied current, a 100-Ω resistor was placed in series with the stimulating electrodes and the voltage drop across the resistor was measured by an isolation amplifier circuit. The stimulating electrodes of the two sides were paired using different parallel or diagonal arrangements. Sinusoid stimuli with varying intensities (1, 2, 3, 4, 5, and...
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