Analysis of electric field stimulation of single cardiac muscle cells.

Electrical stimulation of cardiac cells by imposed extracellular electric fields results in a transmembrane potential which is highly nonuniform, with one end of the cell depolarized and the other end hyperpolarized along the field direction. To date, the implications of the close proximity of oppositely polarized membranes on excitability have not been explored. In this work we compare the biophysical basis for field stimulation of cells at rest with that for intracellular current injection, using three Luo-Rudy type membrane patches coupled together as a lumped model to represent the cell membrane. Our model shows that cell excitation is a function of the temporal and spatial distribution of ionic currents and transmembrane potential. The extracellular and intracellular forms of stimulation were compared in greater detail for monophasic and symmetric biphasic rectangular pulses, with duration ranging from 0.5 to 10 ms. Strength-duration curves derived for field stimulation show that over a wide range of pulse durations, biphasic waveforms can recruit and activate membrane patches about as effectively as can monophasic waveforms having the same total pulse duration. We find that excitation with biphasic stimulation results from a synergistic, temporal summation of inward currents through the sodium channel in membrane patches at opposite ends of the cell. Furthermore, with both waveform types, a net inward current through the inwardly rectifying potassium channel contributes to initial membrane depolarization. In contrast, models of stimulation by intracellular current injection do not account for the nonuniformity of transmembrane potential and produce substantially different (even contradictory) results for the case of stimulation from rest.     

The Role of Pulse Shape in Motor Cortex Transcranial Magnetic Stimulation Using Full-Sine Stimuli

A full-sine (biphasic) pulse waveform is most commonly used for repetitive transcranial magnetic stimulation (TMS), but little is known about how variations in duration or amplitude of distinct pulse segments influence the effectiveness of a single TMS pulse to elicit a corticomotor response. Using a novel TMS device, we systematically varied the configuration of full-sine pulses to assess the impact of configuration changes on resting motor threshold (RMT) as measure of stimulation effectiveness with single-pulse TMS of the non-dominant motor hand area (M1). In young healthy volunteers, we (i) compared monophasic, half-sine, and full-sine pulses, (ii) applied two-segment pulses consisting of two identical half-sines, and (iii) manipulated amplitude, duration, and current direction of the first or second full-sine pulse half-segments. RMT was significantly higher using half-sine or monophasic pulses compared with full-sine. Pulses combining two half-sines of identical polarity and duration were also characterized by higher RMT than full-sine stimuli resulting. For full-sine stimuli, decreasing the amplitude of the half-segment inducing posterior-anterior oriented current in M1 resulted in considerably higher RMT, whereas varying the amplitude of the half-segment inducing anterior-posterior current had a smaller effect. These findings provide direct experimental evidence that the pulse segment inducing a posterior-anterior directed current in M1 contributes most to corticospinal pathway excitation. Preferential excitation of neuronal target cells in the posterior-anterior segment or targeting of different neuronal structures by the two half-segments can explain this result. Thus, our findings help understanding the mechanisms of neural stimulation by full-sine TMS.     

Comparative psychophysical characteristics of cutaneous CO2 laser and contact heat stimulation

Psychophysical visual analog scaling can be used to reveal critical determinants of the neural processing underlying non-painful and painful heat sensations produced by radiant and contact heat stimulation. This study determined the stimulus-response (S-R) functions of cutaneous non-painful and painful heat stimuli delivered by an infra-red CO2 laser or by a contact thermode in a series of experiments in healthy volunteers. In experiment 1 ( n = 12), with the rating scale anchored at pain threshold, the S-R curve for brief (60 ms) laser pulse stimulation with a beam diameter of 10 mm was a negatively accelerating function. Transformation of laser stimulus intensity (W) into temperatures ( C) did not change the form of the S-R curve. In experiment 2 ( n=9), using the same laser stimulus parameters as in experiment 1, but without an anchored rating scale, the form of the S-R relationship did not change. In experiment 3 ( n =9), increases of the laser pulse duration up to 5 s and the beam diameter up to 18 mm produced linear S-R curves. In contrast, in experiment 4 ( n =21), the S-R curve for cutaneous contact heat stimuli applied for 5 s with an 18 mm diameter probe was best described by a positively accelerating power function with an exponent greater than 2.0. These experiments have (1) characterized the S-R functions for different parameters of infra-red laser stimulation of the skin, and (2) have shown that the form of the S-R function for innocuous and noxious heat sensation is influenced strongly by the physical conditions of heat stimulus application, including mechanical contact with the skin.     

Mechanisms of cardiac cell excitation with premature monophasic and biphasic field stimuli: a model study.

The mechanisms by which extracellular electric field stimuli induce the (re)excitation of cardiac cells in various stages of refractoriness are still not well understood. We modeled the interactions between an isolated cardiac cell and imposed extracellular electric fields to determine the mechanisms by which relatively low-strength uniform monophasic and biphasic field stimuli induce premature reexcitations. An idealized ventricular cell was simulated with 11 subcellular membrane patches, each of which obeyed Luo-Rudy (phase 1) kinetics. Implementing a standard S1-S2 pulse protocol, strength-interval maps of the cellular excitatory responses were generated for rectangular monophasic and symmetric biphasic field stimuli of 2, 5, 10, and 20 ms total duration. In contrast to previously documented current injection studies, our results demonstrate that a cardiac cell exhibits a significantly nonmonotonic excitatory response to premature monophasic and, to a much lesser degree, biphasic field stimuli. Furthermore, for monophasic stimuli at low field strengths, the cell is exquisitely sensitive to the timing of the shock, demonstrating a classic all-or-none depolarizing response. However, at higher field strengths this all-or-none sensitivity reverts to a more gradual transition of excitatory responses with respect to stimulus prematurity. In contrast, biphasic stimuli produce such graded responses at all suprathreshold stimulus strengths. Similar behaviors are demonstrated at all S2 stimulus durations tested. The generation of depolarizing (sodium) currents is triggered by one or more of the sharp field gradient changes produced at the stimulus edges-i.e., make, break, and transphasic (for biphasic stimuli)-with the magnitude of these edge-induced current contributions dependent on both the prematurity and the strength of the applied field. In all cases, however, depolarizing current arises from the partial removal of sodium inactivation from at least part of the cell, because of either the natural process of repolarization or a localized acceleration of this process by the impressed field.     

Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium

A strong premature electrical stimulus (S(2)) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca(2+) concentration ([Ca(2+)](i)) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca(2+)](i) optical mapping and for vulnerable window determination. After baseline pacing (S(1)), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S(2) was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca(2+)](i) varied depending on the S(2) polarity. Relative to the level of [Ca(2+)](i) during the S(1) beat, the [Ca(2+)](i) level 40 ms after the onset of monophasic S(2) increased by 36+/-8% at virtual anodes and 20+/-5% at virtual cathodes (P<0.01), compared with 25+/-5% at both virtual cathode-anode and anode-cathode sites for biphasic S(2). The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S(2) than for either anodal or cathodal S(2) (n=7, P<0.01). Treatment with thapsigargin and ryanodine (n=6) significantly prolonged the action potential duration compared with control (255+/-22 vs. 189+/-6 ms, P<0.05) and eliminated the difference in VFT between monophasic and biphasic S(2), although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca(2+)](i) than virtual cathodes. Monophasic S(2) is associated with lower VFT than biphasic S(2), but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca(2+)](i) dynamics modulate, but are not essential, to ventricular vulnerability.     

Threshold fluctuations in an N sodium channel model of the node of Ranvier.

Computer simulations of stochastic single-channel open-close kinetics are applied to an N sodium channel model of a node of Ranvier. Up to 32,000 voltage-gated sodium channels have been simulated with modified amphibian sodium channel kinetics. Poststimulus time histograms are obtained with 1000 monophasic pulse stimuli, and measurements are made of changes in the relative spread of threshold (RS) with changes in the model parameters. RS is found to be invariant with pulse durations from 100 microseconds to 3 ms. RS is approximately of inverse proportion to square-root of N. It decreases with increasing temperature and is dependent on passive electrical properties of the membrane as well as the single-channel conductance. The simulated RS and its independence of pulse duration is consistent with experimental results from the literature. Thus, the microscopic fluctuations of single, voltage-sensitive sodium channels in the amphibian peripheral node of Ranvier are sufficient to account for the macroscopic fluctuation if threshold to electrical stimulation.     

Influence of angular acceleration-deceleration pulse shapes on regional brain strains

Recognizing the association of angular loading with brain injuries and inconsistency in previous studies in the application of the biphasic loads to animal, physical, and experimental models, the present study examined the role of the acceleration-deceleration pulse shapes on region-specific strains. An experimentally validated two-dimensional finite element model representing the adult male human head was used. The model simulated the skull and falx as a linear elastic material, cerebrospinal fluid as a hydrodynamic material, and cerebrum as a linear viscoelastic material. The angular loading matrix consisted coronal plane rotation about a center of rotation that was acceleration-only (4.5 ms duration, 7.8 krad/s/s peak), deceleration-only (20 ms, 1.4 krad/s/s peak), acceleration-deceleration, and deceleration-acceleration pulses. Both biphasic pulses had peaks separated by intervals ranging from 0 to 25 ms. Principal strains were determined at the corpus callosum, base of the postcentral sulcus, and cerebral cortex of the parietal lobe. The cerebrum was divided into 17 regions and peak values of average maximum principal strains were determined. In all simulations, the corpus callosum responded with the highest strains. Strains were the least under all simulations in the lower parietal lobes. In all regions peak strains were the same for both monophase pulses suggesting that the angular velocity may be a better metric than peak acceleration or deceleration. In contrast, for the biphasic pulse, peak strains were region- and pulse-shape specific. Peak values were lower in both biphasic pulses when there was no time separation between the pulses than the corresponding monophase pulse. Increasing separation time intervals increased strains, albeit non-uniformly. Acceleration followed by deceleration pulse produced greater strains in all regions than the other form of biphasic pulse. Thus, pulse shape appears to have an effect on regional strains in the brain.     

Current topics in clinical FES in Japan

This paper reviews recent topics of clinical application of functional electrical stimulation (FES) for the paralyzed extremities in Japan. Transcutaneous and percutaneous FES systems have been clinically used in Japan. Candidates of extremity FES arer mostly stroke and spinal cord injury patients. By using percutaneous FES system, all of the joints of the upper extremity including the shoulder have been controlled for activities of daily living in the hemiplegic patient. Simultaneous FES control of the hand and wrist and the bilateral hands have also been achieved in C5 and C6 quadriplegics, respectively. Hybrid FES systems using percutaneous and surface electrodes, where FES is used in combination with orthoses, have been applied to the paraplegics because they are highly practical for assisting their locomotive activities. Percutaneous FES have been also provided the amyotropic lateral sclerosis patients with standing up motion. A total implant FES system with 16 output channels is currently developing as a next generation FES system.     

Pulsed signal properties of free‐ranging bottlenose dolphins (Tursiops truncatus) in the central Mediterranean Sea

This study describes pulsed signals from bottlenose dolphins of the central Mediterranean Sea. Data were collected during 2011 and 2012 in 27 surveys in the Sicilian Channel, during which 163 animals were sighted. Based mainly on the pulse repetition rate, the signals were classified as Low‐frequency click (LF; single clicks without a regular pulse rate), Train click (TC; with a interclick interval of 80 ± 2 ms), Burst (with a interclick interval of 3.4 ± 0.2 ms), or Packed click (with a lower number of clicks per train and median interclick interval of 3.2 ± 0.0 ms). The measured parameters were peak sound pressure level (SPL_pk); signal duration; the 1st, 2nd, and 3rd peak of frequency; number of peaks frequency; bandwidth; centroid frequency; and the 10th, 25th, 75th, and 90th percentiles of the power spectrum distribution. Most of the parameters were significantly different among the groups, reflecting the different functions of these signals. LF clicks showed a lower peak frequency and percentiles and a longer duration and could be used to explore a wide area without a specific target focalization and with less resolution. The TC showed a higher SPL_pk, higher peak frequency, lower duration, and lower number of secondary peaks frequency, showing a better resolution to investigate a specific target.     

Conditioning of beta(1)-adrenoceptor effect via beta(2)-subtype on L-type Ca(2+) current in canine ventricular myocytes

We investigated the roles of beta(1)- and beta(2)-receptors (beta-AR) in adrenergic enhancement of L-type Ca(2+) current (I(CaL)) in canine ventricular myocytes. Isoproterenol and l-norepinephrine produced a monophasic and a biphasic concentration-I(CaL) relationship (CR), respectively. alpha(1)-AR inhibition with prazosin and beta(2)-AR stimulation with zinterol or l-epinephrine shifted the CR of l-norepinephrine leftward. Zinterol (50 nM) and l-epinephrine (10 nM), but not prazosin, altered the biphasic CR of l-norepinephrine to a monophasic CR. Zinterol and l-epinephrine applied after l-norepinephrine had no effect on I(CaL). beta(2)-AR inhibition with ICI-118551 reduced the E(max) of isoproterenol and l-norepinephrine by 60% and abolished the augmentation of l-norepinephrine by zinterol and l-epinephrine. Carbachol (100 nM) modestly reduced the I(CaL) response to beta(1)-AR stimulation but abolished the enhancement via beta(2)-AR. Zinterol augmented the enhancement of I(CaL) by forskolin, IBMX, and theophylline, but not in the presence of CGP-20712A. We conclude that selective beta(2)-AR stimulation does not increase I(CaL) but enhances adenylyl cyclase activity when stimulated via beta(1)-AR and with forskolin. beta(2)-AR activity preconditions adenylyl cyclase for beta(1)-AR stimulation.     

Paced monophasic and biphasic waveforms alter transmembrane potentials and metabolism of human fibroblasts

Resting transmembrane potential (TMP) of primary human fibroblast cells was altered in predictable directions by subjecting cell cultures to specific monophasic and biphasic waveforms. Cells electrically stimulated with an anodal pulse resulted in hyperpolarization while a cathodal waveform depolarized the TMP to below that of non-paced control cells. The biphasic waveform, consisting of an anodal pulse followed immediately by an inverse symmetric cathodal pulse, also lessened the TMP similar to that of the cathodal pulse. The effect of short-term pacing on the TMP can last up to 4 h before the potentials equilibrate back to baseline. While subjecting the cells to this electrical field stimulation did not appear to damage the integrity of the cells, the three paced electrical stimulation waves inhibited expansion of the cultures when compared to non-paced control cells. With longer pacing treatments, elongation of the cells and electrotaxis towards the anodal polarity were observed. Pacing the fibroblasts also resulted in modest, yet very statistically significant (and likely underestimated) changes to cellular adenosine-5'-triphosphate (ATP) levels, and cells undergoing anodal and biphasic (anodal/cathodal) stimulation also exhibited altered mitochondrial morphology. These observations indicate an active role of electrical currents, especially with anodal content, in affecting cellular metabolism and function, and help explain accumulating evidence of cellular alterations and clinical outcomes in pacing of the heart and other tissues in general.     

Alternating stimulation of synergistic muscles during functional electrical stimulation cycling improves endurance in persons with spinal cord injury

Therapeutic effects of functional electrical stimulation (FES) cycling for persons with spinal cord injury (SCI) are limited by high rates of muscular fatigue. FES-cycling performance limits and surface mechanomyography (MMG) of 12 persons with SCI were compared under two different stimulation protocols of the quadriceps muscles. One strategy used the standard “co-activation” protocol from the manufacturer of the FES cycle which involved intermittent simultaneous activation of the entire quadriceps muscle group for 400 ms. The other strategy was an “alternation” stimulation protocol which involved alternately stimulating the rectus femoris (RF) muscle for 100 ms and the vastus medialis (VM) and vastus lateralis (VL) muscles for 100 ms, with two sets with a 400 ms burst. Thus, during the alternation protocol, each of the muscle groups rested for two 100 ms “off” periods in each 400 ms burst. There was no difference in average cycling cadence (28 RPM) between the two protocols. The alternation stimulation protocol produced longer ride times and longer virtual distances traveled and used lower stimulation intensity levels with no differences in average MMG amplitudes compared to the co-activation protocol. These results demonstrate that FES-cycling performance can be enhanced by a synergistic muscle alternation stimulation strategy.     

Functional electrical stimulation of intrinsic laryngeal muscles under varying loads in exercising horses

Bilateral vocal fold paralysis (BVCP) is a life threatening condition and appears to be a good candidate for therapy using functional electrical stimulation (FES). Developing a working FES system has been technically difficult due to the inaccessible location and small size of the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle. A naturally-occurring disease in horses shares many functional and etiological features with BVCP. In this study, the feasibility of FES for equine vocal fold paralysis was explored by testing arytenoid abduction evoked by electrical stimulation of the PCA muscle. Rheobase and chronaxie were determined for innervated PCA muscle. We then tested the hypothesis that direct muscle stimulation can maintain airway patency during strenuous exercise in horses with induced transient conduction block of the laryngeal motor nerve. Six adult horses were instrumented with a single bipolar intra-muscular electrode in the left PCA muscle. Rheobase and chronaxie were within the normal range for innervated muscle at 0.55±0.38 v and 0.38±0.19 ms respectively. Intramuscular stimulation of the PCA muscle significantly improved arytenoid abduction at all levels of exercise intensity and there was no significant difference between the level of abduction achieved with stimulation and control values under moderate loads. The equine larynx may provide a useful model for the study of bilateral fold paralysis.     

Stimulation characteristics that determine arteriolar dilation in skeletal muscle

To determine the skeletal muscle stimulation parameters that are most important in establishing vasodilation in the microvasculature, I tested whether arteriolar diameter during 2 min of repetitive, short-duration, tetanic skeletal muscle contractions increased with changes in stimulus frequency, stimulation train duration, and contraction frequency. To test this, the diameter of transverse arterioles approximately perpendicular to small bundles of cremaster muscle fibers in situ of anesthetized Golden Syrian hamsters was used as a bioassay system. Arteriolar diameter was measured before and during different stimulation patterns that consisted of a contraction frequency [6, 12, or 24 contractions per minute (cpm)], a stimulation train duration (250, 500, or 750 ms) and a stimulus frequency (4, 8, 10, 15, 20, 30, 40, 60, and 80 Hz). The magnitude of the dilation significantly increased with stimulus frequency but not in a simple linear manner. The average rate of increase was 0.32 +/- 0.02 microm/Hz from 4 to 20 Hz and 0.09 +/- 0.02 microm/Hz from 30 to 80 Hz. The magnitude of the dilation increased significantly with the contraction frequency where the dilation at 6 cpm was significantly smaller than the dilation at 24 cpm across all stimulus frequencies. Changing the train duration from 250 to 750 ms did not significantly affect the magnitude of the dilation. These observations suggest that stimulation parameters are important in determining the magnitude of the microvascular dilation and that the magnitude of the dilation was dependent on both the contraction frequency and stimulus frequency but was independent of train duration.     

Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection

Cryopreservation, directed differentiation, and genetic manipulation of human embryonic stem cells (hESCs) all require the transport of exogenous small molecules, proteins, or DNA into the cell. The absence of standard small and macromolecule loading techniques in hESCs as well as the inadequacies of current DNA transfection techniques have led us to develop electroporation as an efficient loading and transfection methodology. The electroporation parameters of pulse voltage, duration, and number have been explored and evaluated in terms of cell viability, molecular loading, and transfection efficiency on a per cell basis. Small molecule loading was assessed using propidium iodide (PI) and the disaccharide trehalose. Additionally, protein loading was investigated using a glutathione-S-transferase green fluorescent protein (GST-GFP) conjugate, and DNA transfection optimization was performed by constitutive expression of GFP from a plasmid. The optimum pulse voltage must balance cell viability, which decreases as voltage increases, and loading efficiency, which increases at higher voltages. Short pulse times of 0.05 ms facilitated PI and trehalose loading, whereas 0.5 ms or more was required for GST-GFP loading and DNA transfection. Multiple pulses increased per cell loading of all molecules, though there was a dramatic loss of viability with GST-GFP loading and DNA transfection, likely resulting from the longer pulse duration required to load these molecules.     

Observer-based tracking control of abnormal oscillations in demyelination symptom

Fast axonal conduction of action potentials in mammals relies on myelin insulation. Demyelination can cause slowed, blocked, desynchronized, or paradoxically excessive spiking that underlies the symptoms observed in demyelination diseases. Feedback control via functional electrical stimulation (FES) seems to be a promising treatment modality in such diseases. However, there are challenges to implementing such method for neurons: high nonlinearity, biological tissue constrains and unobservable ion channel states. To address this problem, we propose an estimating and tracking control strategy for systems based on Kalman filter, in order to enhance the action potential propagation reliability of demyelinated neuron via FES. Unscented Kalman filter (UKF) is employed to estimate the unobservable states and parameters in the demyelination neuron model from membrane potential dynamics. Our method could promote the design of new closed-loop electrical stimulation systems for patients suffering from different nerve system dysfunctions.     

Information processing in a cricket ganglion: The response of giant fibres to sound pulses

The response of giant fibres in the ventral nerve cord to stimulation of cercal afferents with pulses of sound was studied in the domestic cricket, Acheta domesticus. Pulses at 450 Hz gave the highest frequency response in several classes of units, and were therefore used as stimuli in subsequent experiments. In intact animals the response of the giant fibres to bilateral cercal stimulation showed a characteristic high frequency ‘on’ response followed by steady firing of some units for the duration of the sound pulse. The end of each pulse was followed by a short period of inhibition of the tonic units.Cercal amputation and other experiments showed that input from cercal afferents excites both large and small ipsilateral giants, and excites small and inhibits large contralateral giants. Descending input from higher neural centres in intact animals tends to reduce the responses to the stimuli. It is suggested that a function of the contralateral excitatory and inhibitory effects is to sharpen the ‘on’ response of the giant fibres to sound stimuli in intact animals.     

An electronic device for accelerating bone formation in tissues surrounding a dental implant

A dental implant is a unique structure which can be used with a noninvasive method because it is inserted into the bone in part and extended extracorporally. This study presents an electronic device that is temporarily connected with the dental implant, and reports its effect on accelerating bone formation in the surrounding tissues in a canine mandibular model. A small sized and low power consumption biphasic electrical current (BEC) stimulator ASIC was developed and the surrounding tissue was exposed to continuous BEC stimulation for 7 days with the parameters of 20 µA/cm², 125 µs duration, and 100 pulses/s. After 2 (n = 5) and 5 weeks (n = 5), animals were sacrificed and the specimens were histomorphometrically evaluated. The newly formed bone area (BA) was 1.30 times (3 weeks, P < 0.05) and 1.35 times (5 weeks, P < 0.05) higher in the experimental group compared to the control group, respectively. Bone‐implant contact (BIC) in 3‐week specimens was 1.62 times (P < 0.05) greater in the experimental group, while there was no statistically significant difference in 5‐week specimens. Based on these results showing accelerated bone formation on and around the dental implant, it could be suggested that the latent time for osseointegration in dental implants can be reduced, and the success rate of implants in poor quality bone can be increased by using our device with BEC. Bioelectromagnetics 30:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

Selection of stimulus parameters for enhancing slow wave sleep events with a neural-field theory thalamocortical model

Slow-wave sleep cortical brain activity, conformed by slow-oscillations and sleep spindles, plays a key role in memory consolidation. The increase of the power of the slow-wave events, obtained by auditory sensory stimulation, positively correlates with memory consolidation performance. However, little is known about the experimental protocol maximizing this effect, which could be induced by the power of slow-oscillation, the number of sleep spindles, or the timing of both events’ co-occurrence. Using a mean-field model of thalamocortical activity, we studied the effect of several stimulation protocols, varying the pulse shape, duration, amplitude, and frequency, as well as a target-phase using a closed-loop approach. We evaluated the effect of these parameters on slow-oscillations (SO) and sleep-spindles (SP), considering: (i) the power at the frequency bands of interest, (ii) the number of SO and SP, (iii) co-occurrences between SO and SP, and (iv) synchronization of SP with the up-peak of the SO. The first three targets are maximized using a decreasing ramp pulse with a pulse duration of 50 ms. Also, we observed a reduction in the number of SO when increasing the stimulus energy by rising its amplitude. To assess the target-phase parameter, we applied closed-loop stimulation at 0°, 45°, and 90° of the phase of the narrow-band filtered ongoing activity, at 0.85 Hz as central frequency. The 0° stimulation produces better results in the power and number of SO and SP than the rhythmic or random stimulation. On the other hand, stimulating at 45° or 90° change the timing distribution of spindles centers but with fewer co-occurrences than rhythmic and 0° phase. Finally, we propose the application of closed-loop stimulation at the rising zero-cross point using pulses with a decreasing ramp shape and 50 ms of duration for future experimental work.