Sequential Stimulation Research Articles

A hemodynamic study of the effect of neuromuscular electrical stimulation on enhancing popliteal venous flow

Neuromuscular electrical stimulation has been studied to be a method of prophylaxis for deep venous thrombosis by activating the calf muscle pump. However, there is little evidence of the effects of different stimulating parameters on hemodynamics and comfort levels. The objective of this paper is to compare the effects of different stimulating parameters (current amplitude, pulse-width) on hemodynamic alterations of the popliteal vein and the comfort levels in a group of fourteen healthy subjects. Doppler ultrasound detection of peak venous velocity and blood volume were taken from baseline, twelve sequential electrical stimulations and foot dorsiflexion for each subject. A visual analogue scale was used to assess the subjects’ pain perception of neuromuscular electrical stimulation. The results showed that peak venous velocity and blood volume augmented as current amplitude and pulse-width increased while pain level also increased. A compromise was reached that parameters consisting of an amplitude of 10mA and a pulse-width of 500 μs would obtain a high-peak venous velocity and blood flow volume with a relatively comfortable perception. In addition, parameters consisting of an amplitude of 20mA and a pulse-width of 300 μs were also shown to be a promising choice. However, further studies need to be done to validate and enrich these findings.

Speech perception with interaction-compensated simultaneous stimulation and long pulse durations in cochlear implant users

Early multi-channel designs in the history of cochlear implant development were based on a vocoder-type processing of frequency channels and presented bands of compressed analog stimulus waveforms simultaneously on multiple tonotopically arranged electrodes. The realization that the direct summation of electrical fields as a result of simultaneous electrode stimulation exacerbates interactions among the stimulation channels and limits cochlear implant outcome led to the breakthrough in the development of cochlear implants, the continuous interleaved (CIS) sampling coding strategy. By interleaving stimulation pulses across electrodes, CIS activates only a single electrode at each point in time, preventing a direct summation of electrical fields and hence the primary component of channel interactions.In this paper we show that a previously presented approach of simultaneous stimulation with channel interaction compensation (CIC) may also ameliorate the deleterious effects of simultaneous channel interaction on speech perception. In an acute study conducted in eleven experienced MED-EL implant users, configurations involving simultaneous stimulation with CIC and doubled pulse phase durations have been investigated. As pairs of electrodes were activated simultaneously and pulse durations were doubled, carrier rates remained the same. Comparison conditions involved both CIS and fine structure (FS) strategies, either with strictly sequential or paired-simultaneous stimulation.Results showed no statistical difference in the perception of sentences in noise and monosyllables for sequential and paired-simultaneous stimulation with doubled phase durations. This suggests that CIC can largely compensate for the effects of simultaneous channel interaction, for both CIS and FS coding strategies.A simultaneous stimulation paradigm has a number of potential advantages over a traditional sequential interleaved design. The flexibility gained when dropping the requirement of interleaving pulses across electrodes may be instrumental in designing coding strategies for a more accurate transmission of stimulus features such as temporal fine structure or interaural time delays to the auditory nerve. Also, longer pulse phase durations may be implemented while maintaining relatively high stimulation pulse rates. Utilizing longer pulse durations may relax requirements on implant compliance and facilitate the design of more energy-efficient implant receivers for a longer battery lifetime or a reduction in implant size.This article is part of a Special Issue entitled <Lasker Award>.

Isometric exercises reduce temporal summation of pressure pain in humans.

Aerobic and isometric exercises are known to decrease pain sensitivity. The effect of different types of exercise on central mechanisms such as temporal summation of pain (TSP) is less clear. This study hypothesized that both aerobic and isometric exercises would increase pressure pain tolerance (PTT) and reduce TSP with greater effects after higher-intensity exercises. One hundred thirty-six healthy subjects (18-65 years; 68 women) participated in two randomized crossover experiments with trials on two different days. PTT and TSP were assessed before and after bicycling and a non-exercise condition (experiment 1), and after low- and high-intensity bicycling and low- and high-intensity isometric arm and leg exercises with the dominant arm/leg (experiment 2). PTT and TSP were assessed before and after each exercise condition on the non-dominant arm and leg by computer-controlled cuff algometry. TSP was assessed by visual analogue scale (VAS) scores of the pain intensity during sequential cuff-pressure stimulation at the pain tolerance intensity related to that specific time point. In experiment 1, bicycling, but not the non-exercise condition, slightly increased PTT when assessed at the leg (p < 0.05). In experiment 2, isometric arm and leg exercises increased PTT and reduced VAS scores to sequential stimulation at the arm and leg (p < 0.05). No systematic difference was found between low- and high-intensity exercises. Different manifestations of hypoalgesia between aerobic and isometric exercises were found. Isometric exercises reduced temporal summation illustrating the potential for exercise as a rehabilitation procedure also targeting the central mechanisms.

Spatiotemporal interactions in the visual cortex following paired electrical stimulation of the retina.

Retinal prostheses use spatiotemporal patterns of electrical stimulation across multiple electrodes to provide visual percepts to blind patients. It is generally assumed that percepts produced by individual electrodes are independent of one another, which may not be the case. In this study, we aimed to quantify interactions between pairs of electrical stimuli delivered to the retina. Normally sighted cats were implanted with a suprachoroidal electrode array. The retina was stimulated with a paired-pulse paradigm that consisted of a conditioning stimulus followed by a test stimulus, while recording multiunit activity in the visual cortex. Conditioning current, and spatial and temporal separation between the conditioning and test stimuli were varied. Cortical interactions were quantified by changes in multiunit activity elicited by stimulation with the paired-pulse paradigm, compared to stimulation of the test stimulus alone (control). Interactions varied as a function of conditioning current and temporal separation between the two stimulating pulses. Cortical activity increased compared to the control condition at an interstimulus delay of 1.025 ms and was significantly suppressed for delays between 20 and 90 ms, returning to near control levels for longer delays. The level of interactions increased when the conditioning current was increased. Interactions were found to be similar for electrode separations up to 3 mm. Interactions between sequential stimulation of pairs of electrodes in a suprachoroidal retinal prosthesis occur for delays up to 100 ms and electrode separations of several millimeters. Knowledge of these spatiotemporal interactions is essential for developing effective patterns of stimulation for retinal prostheses.

Operational challenges of retinal prostheses

Two computational models for research on retinal implants are presented. In the first model, the electric field produced by a multi-electrode array in a uniform retina is calculated. It is shown how cross talk of activated electrodes and the resulting bunching of field lines in monopole and dipole activation prevent high resolution imaging with retinal implants. Furthermore, it is demonstrated how sequential stimulation and multipolar stimulation may overcome this limitation. In the second model a target volume, i.e., a probe cylinder approximating a bipolar cell, in the retina is chosen, and the passive Heaviside cable equation is solved inside this target volume to calculate the depolarization of the cell membrane. The depolarization as a function of time indicates that shorter signals stimulate better as long as the current does not change sign during stimulation of the retina, i.e., mono-phasic stimulation. Both computational models are equally applicable to epiretinal, subretinal, and suprachoroidal vision implants.

Peripheral chemoreceptors tune inspiratory drive via tonic expiratory neuron hubs in the medullary ventral respiratory column network.

Models of brain stem ventral respiratory column (VRC) circuits typically emphasize populations of neurons, each active during a particular phase of the respiratory cycle. We have proposed that "tonic" pericolumnar expiratory (t-E) neurons tune breathing during baroreceptor-evoked reductions and central chemoreceptor-evoked enhancements of inspiratory (I) drive. The aims of this study were to further characterize the coordinated activity of t-E neurons and test the hypothesis that peripheral chemoreceptors also modulate drive via inhibition of t-E neurons and disinhibition of their inspiratory neuron targets. Spike trains of 828 VRC neurons were acquired by multielectrode arrays along with phrenic nerve signals from 22 decerebrate, vagotomized, neuromuscularly blocked, artificially ventilated adult cats. Forty-eight of 191 t-E neurons fired synchronously with another t-E neuron as indicated by cross-correlogram central peaks; 32 of the 39 synchronous pairs were elements of groups with mutual pairwise correlations. Gravitational clustering identified fluctuations in t-E neuron synchrony. A network model supported the prediction that inhibitory populations with spike synchrony reduce target neuron firing probabilities, resulting in offset or central correlogram troughs. In five animals, stimulation of carotid chemoreceptors evoked changes in the firing rates of 179 of 240 neurons. Thirty-two neuron pairs had correlogram troughs consistent with convergent and divergent t-E inhibition of I cells and disinhibitory enhancement of drive. Four of 10 t-E neurons that responded to sequential stimulation of peripheral and central chemoreceptors triggered 25 cross-correlograms with offset features. The results support the hypothesis that multiple afferent systems dynamically tune inspiratory drive in part via coordinated t-E neurons.

A multiple switching diarylethene with a phenyl-linked rhodamine B unit and its application as chemosensor for Cu2+

A new photochromic diarylethene with a phenyl-linked rhodamine B unit was synthesized and its photochemcial properties were systematically studied. The diarylethene could be used as a multiple fluorescence switch by sequential stimulations with protons and light. As a result, an INHIBIT logic gate was constructed by using the stimulation of trifluoroacetic acid and ultraviolet as inputs and the fluorescence intensity at 599 nm as output. Additionally, the diarylethene could act as a naked-eye chemosensor for the recognition of Cu2+ in aqueous acetonitrile. When triggered by Cu2+, the fluorescence intensity of the diarylethene at 594 nm was dramatically enhanced by 133-fold with a significant fluorescence change from dark to bright yellow.

Erratum to: Sequential (hFSH + recFSH) vs homogenous (hFSH or recFSH alone) stimulation: clinical and biochemical (cumulus cell gene expression) aspects

Erratum to: Sequential (hFSH + recFSH) vs homogenous (hFSH or recFSH alone) stimulation: clinical and biochemical (cumulus cell gene expression) aspects

Multimodal cortical sensory pathways revealed by sequential transcranial electrical stimulation in mice

We investigated polysynaptic cortical pathways linking primary to multimodal sensory association areas in mice using transcranial flavoprotein imaging combined with sequential application of transcranial electrical stimulation (TES). Stimulation of primary visual cortex (V1) elicited activity in lateral and medial areas of secondary visual cortices (V2), which were reciprocally connected. Stimulation of V2 areas elicited activity in area 2. Similarly, corticocortical pathways from primary somatosensory cortex (S1) through the corresponding secondary somatosensory areas (S2) to area 2 were observed. Auditory pathways from primary auditory area (A1) through peripheral region (area 22) to area 2 and from anterior auditory field to area 2 were also found. Stimulation in area 2 elicited activity in part of parietal association cortex (PtA), which was reciprocally connected with area 2, and in some areas near the midline including retrosplenial cortex (RSA). A cortical pathway from RSA through anterior cingulate cortex (aCC) to frontal areas was also visualized. These results indicate that area 2, surrounded by visual, somatosensory and auditory cortices, may receive inputs from all three primary sensory areas, and may send outputs through the parietal association cortex to frontal areas, suggesting that area 2 may have an important role in multimodal sensory integration in mice.

P827: Beyond P300: an auditory ERP paradigm with sequential stimulation

P827: Beyond P300: an auditory ERP paradigm with sequential stimulation

Combining physical training with transcranial direct current stimulation to improve gait in Parkinson’s disease: a pilot randomized controlled study

Objective: To improve gait and balance in patients with Parkinson’s disease by combining anodal transcranial direct current stimulation with physical training. Design: In a double-blind design, one group (physical training; n = 8) underwent gait and balance training during transcranial direct current stimulation (tDCS; real/sham). Real stimulation consisted of 15 minutes of 2 mA transcranial direct current stimulation over primary motor and premotor cortex. For sham, the current was switched off after 30 seconds. Patients received the opposite stimulation (sham/real) with physical training one week later; the second group (No physical training; n = 8) received stimulation (real/sham) but no training, and also repeated a sequential transcranial direct current stimulation session one week later (sham/real). Setting: Hospital Srio Libanes, Buenos Aires, Argentina. Subjects: Sixteen community-dwelling patients with Parkinson’s disease. Interventions: Transcranial direct current stimulation with and without concomitant physical training. Main measures: Gait velocity (primary gait outcome), stride length, timed 6-minute walk test, Timed Up and Go Test (secondary outcomes), and performance on the pull test (primary balance outcome). Results: Transcranial direct current stimulation with physical training increased gait velocity (mean = 29.5%, SD = 13; p < 0.01) and improved balance (pull test: mean = 50.9%, SD = 37; p = 0.01) compared with transcranial direct current stimulation alone. There was no isolated benefit of transcranial direct current stimulation alone. Although physical training improved gait velocity (mean = 15.5%, SD = 12.3; p = 0.03), these effects were comparatively less than with combined tDCS + physical therapy (p < 0.025). Greater stimulation-related improvements were seen in patients with more advanced disease. Conclusions: Anodal transcranial direct current stimulation during physical training improves gait and balance in patients with Parkinson’s disease. Power calculations revealed that 14 patients per treatment arm (α = 0.05; power = 0.8) are required for a definitive trial.

Sequential (hFSH + recFSH) vs homogenous (hFSH or recFSH alone) stimulation: clinical and biochemical (cumulus cell gene expression) aspects

FSH is a key hormone in the regulation of follicular development. Together with the EGF network, these molecules mediate oocyte maturation and competence in preparation for the action of LH. FSH isoforms regulate distinct biological pathways and have specific effects on granulosa cell function and maturation of the ovarian follicle. Their dynamic interactions occur during the follicular cycle; short-living forms are predominant in the pre-ovulatory phase, whereas long-acting molecules characterize the luteal-follicular transition. Recombinant FSH (rFSH) molecules have a reduced number of isoforms and are less acidic, with a shorter half-life. We have investigated sequential stimulation, comparing hFSH + rFSH, vs. rFSH alone and hFSH alone for the entire stimulation phase. Sequential stimulation leads to an E2 per MII oocyte ratio that is much lower than is seen during treatment with the two drugs individually. Although there is a positive tendency in favor of the sequential treatment, there was no significant difference in pregnancy rates, even taking frozen embryos into consideration. The cumulus cell transcriptome varies considerably between the treatments, although with no clear significance. When comparing pregnant vs. non-pregnant patients, in general a decrease in mRNA expression can be observed in the pregnant patients, especially in expression of folic acid receptor 1 and ovostatin 2. This indicates that material has been transferred from CC to the oocyte. However, a common observation in the literature is that variations in the transcriptome of the cumulus cells are highly dependent upon the patient genotype; the potential for applying this strategy as a basis for selecting embryos is, at the very least, questionable.

Improvement of language functions in a chronic non-fluent post-stroke aphasic patient following bilateral sequential theta burst magnetic stimulation

In chronic non-fluent aphasia patients, inhibition of the intact right hemisphere (RH), by transcranial magnetic stimulation (TMS) or similar methods, can induce improvement in language functions. The supposed mechanism behind this improvement is a release of preserved left hemisphere (LH) language networks from RH transcallosal inhibition. Direct stimulation of the damaged LH can sometimes bring similar results too. Therefore, we developed a novel treatment approach that combined direct LH (Broca’s area (BA)) stimulation, by intermittent theta burst stimulation (TBS), with homologue RH area’s inhibition, by continuous TBS. We present the results of application of 15 daily sessions of the described treatment approach in a right-handed patient with chronic post-stroke non-fluent aphasia. The intervention appeared to improve several language functions, but most notably propositional speech, semantic fluency, short-term verbal memory, and verbal learning. Bilateral TBS modulation of activation of the language-related areas of both hemispheres seems to be a feasible and promising way to induce recovery in chronic aphasic patients. Due to potentially cumulative physiological effects of bilateral stimulation, the improvements may be even greater than following unilateral interventions.

Stimulation Selectivity of the “Thin-Film Longitudinal Intrafascicular Electrode” (tfLIFE) and the “Transverse Intrafascicular Multi-Channel Electrode” (TIME) in the Large Nerve Animal Model

Neural prostheses are limited by the availability of peripheral neural electrodes to record the user's intention or provide sensory feedback through functional electrical stimulation. Our objective was to compare the ability of the novel “transverse intrafascicular multi-channel electrode” (TIME) and an earlier generation “thin-film longitudinal intrafascicular electrode” (tfLIFE) to selectively stimulate nerve fascicles and activate forelimb muscles in pigs. TIME was designed to access a larger subpopulation of fascicles than tfLIFE and should therefore be able to selectively activate a larger number of muscles. Electrodes were implanted in the median nerve, and sequential electric stimulation was applied to individual contacts. The compound muscle action potentials of seven muscles were recorded to quantify muscle recruitment. As expected, TIME was able to recruit more muscles with higher selectivity than tfLIFE (significant difference when comparing the performance of an entire electrode); a similar activation current was used (no significant difference). Histological analysis revealed that electrodes were located between fascicles, which influenced the selectivity and activation current level. In conclusion, TIME is a viable neural interface for selective activation of multiple fascicles in human-sized nerves that may assist to pave the way for future neuroprosthesis applications.

HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS–JNK–autophagy pathway

The Bcl-2 inhibitor ABT-737 has shown promising antitumor efficacy in vivo and in vitro. However, some reports have demonstrated that HCC cells are resistant to ABT-737, and the corresponding molecular mechanisms of this resistance are not well known. In this study, we found that HCC cells with high levels of Bcl-2 were markedly resistant to ABT-737 compared to HCC cells with low levels of Bcl-2. In HCC cells with high levels of Bcl-2 (such as HepG2 cells), ABT-737 induced protective autophagy via the sequential triggering of reactive oxygen species (ROS) accumulation, short-term activation of JNK, enhanced phosphorylation of Bcl-2, and dissociation of Beclin 1 from the Bcl-2/Beclin 1 complex. Moreover, autophagy suppressed the overactivation of the ROS–JNK pathway and protected against apoptosis. In HCC cells with low levels of Bcl-2 (i.e., Huh7 cells), ABT-737 induced apoptosis via the sequential stimulation of ROS, sustained activation of JNK, enhanced translocation of Bax from the cytosol to the mitochondria, and release of cytochrome c. In sum, this study indicated that the activation of the ROS–JNK–autophagy pathway may be an important mechanism by which HCC cells with high levels of Bcl-2 are resistant to ABT-737.

Reducing muscle fatigue during transcutaneous neuromuscular electrical stimulation by spatially and sequentially distributing electrical stimulation sources

PurposeA critical limitation with transcutaneous neuromuscular electrical stimulation is the rapid onset of muscle fatigue. We have previously demonstrated that spatially distributed sequential stimulation (SDSS) shows a drastically greater fatigue-reducing ability compared to a single active electrode stimulation (SES). The purposes of this study were to investigate (1) the fatigue-reducing ability of SDSS in more detail focusing on the muscle contractile properties and (2) the mechanism of this effect using array-arranged electromyogram (EMG).MethodsSDSS was delivered through four active electrodes applied to the plantarflexors, sending a stimulation pulse to each electrode one after another with 90° phase shift between successive electrodes. In the first experiment, the amount of exerted ankle torque and the muscle contractile properties were investigated during a 3 min fatiguing stimulation. In the second experiment, muscle twitch potentials with SDSS and SES stimulation electrode setups were compared using the array-arranged EMG.ResultsThe results demonstrated negligible torque decay during SDSS in contrast to considerable torque decay during SES. Moreover, small changes in the muscle contractile properties during the fatiguing stimulation using SDSS were observed, while slowing of muscle contraction and relaxation was observed during SES. Further, the amplitude of the M-waves at each muscle portion was dependent on the location of the stimulation electrodes during SDSS.ConclusionWe conclude that SDSS is more effective in reducing muscle fatigue compared to SES, and the reason is that different sets of muscle fibers are activated alternatively by different electrodes.

Mental, Autonomic, and Neurophysiological Impairments in Patients with Obesity and Their Correction Using Transcranial Electrical Stimulation

A total of 85 patients aged 45–68 years with grade I and II obesity for more than five years with concomitant arterial hypertension and atherosclerosis were observed. Patients were divided into two randomized groups. The study group underwent transcranial electrical stimulation (TES) on the background of standard treatment with antiaggregant vascular agents and low-calorie diet. The control group underwent a placebo TES procedure (with the electrodes disconnected). TES therapy was performed using the fronto-mastoid method with sequential stimulation of the right and left hemispheres at a frequency of 10 Hz; patients received a total of 10–12 daily procedures. Patients’ status was evaluated using a series of psychometric scales; autonomic nervous system status was determined by rhythmocardiography; brain bioelectrical activity (EEG) was also studied; the activity of the lipid peroxidation system was assessed. TES produced improvements in most indicators which were significantly greater than average improvements in the control group. Changes in clinical and laboratory parameters were accompanied by a mean 9.8 % decrease in body mass index.

P 180. Bilateral sequential motor cortex stimulation improves skilled task learning

Objective Increase of excitability and activation of the primary motor cortex (M1) can be achieved either by direct M1 stimulation with an excitatory TMS protocol or by stimulation of contralateral M1 with an inhibitory TMS protocol. The aim of the study was to check whether bilateral sequential stimulation of the primary motor cortex (M1) with both types of protocols, excitatory one over target M1 and inhibitory one over contralateral M1, would be able to improve performance on a skilled task more than unilateral stimulations could do. Methods 40 right-handed healthy subjects (mean age 25.7 ± 2.4 y, 16 women) participated in the study. They were divided into 4 matched experimental groups (10 subject each) according to the intervention they had: (1) facilitatory, intermittent TBS (iTBS) protocol over target M1-the iTBSt group; (2) inhibitory, continuous TBS (cTBS) protocol over contralateral M1-the cTBSc group; (3) contralateral cTBS followed by iTBS over target M1-the cTBSc/iTBSt group, and 4) placebo, sham iTBS protocol-Placebo group. During iTBS protocol, short bursts of 50 Hz stimulation (3 pulses of 80% aMT) were applied at 5 Hz in 2 s trains every 10 s, for a total of 600 pulses. The cTBS protocol was the same except that bursts were applied continuously (600 pulses in total). The sham iTBS protocol was the same as the iTBS protocol, but a placebo coil was used. Learning was evaluated by comparing scores on Purdue peg-board (PPB) test (number of pegs positioned by hand contralateral to target M1), before (B) and immediately after (T0) experimental interventions. Results In all groups, the PPB test scores increased at T0 in comparison to B showing clear learning effect ( Fig. 1 ). However, ANOVA showed significant Group effect (F(3,36) = 4.53, p = 0.008) for relative increase in number of pegs positioned on PBB test at T0. Post-hoc t-tests showed as significantly different from placebo only increase following bilateral sequential cTBSc/iTBSt protocol. Conclusion Neither direct stimulation of the target M1 nor indirect stimulation through release from contralateral M1 inhibition (by inhibiting contralateral M1) were able to increase motor skill learning above the level seen with placebo. Only bilateral sequential stimulation consisting of inhibition of contralateral M1 followed by stimulation of target M1 was able to significantly improve learning of a skilled motor task in healthy subjects. The findings may have implications for use of TMS neuromodulatory methods in neurorehabilitation.

Quantification of effectiveness of bilateral and unilateral neuromodulation in the rat bladder rhythmic contraction model

BackgroundUsing the isovolumetric bladder rhythmic contraction (BRC) model in anesthetized rats, we have quantified the responsiveness to unilateral and bilateral stimulation of the L6 spinal nerve (SN) and characterized the relationship between stimulus intensity and inhibition of the bladder micturition reflex.MethodsA wire electrode was placed under either one or both of the L6 SN roots. A cannula was placed into the bladder via the urethra and the urethra was ligated. Saline infusion induced BRC.ResultsAt motor threshold (Tmot) intensity, SN stimulation of both roots (10 Hz) for 10 min reduced bladder contraction frequency from 0.63 ± 0.04 to 0.17 ± 0.09 contractions per min (26 ± 14% of baseline control; n = 10, p < 0.05). However, the same intensity of unilateral stimulation (n = 15) or sequential stimulation of both SNs (e.g. 5 min per side alternatively for a total of 10 min or 20 min) was less efficacious. The greater sensitivity to bilateral stimulation is not dependent upon precise bilateral timing of the stimulation pulses. Bilateral stimulation also produced both acute and prolonged- inhibition on bladder contractions in a stimulation intensity dependent fashion.ConclusionsUsing the bladder rhythmic contraction model, bilateral stimulation was more effective than unilateral stimulation of the SN. Clinical testing should be conducted to further compare efficacies of unilateral and bilateral stimulation. Bilateral stimulation may allow the use of lower stimulation intensities to achieve higher efficacy for neurostimulation therapies on urinary tract control.

Schooling Fish Under Attack Are Not All Equal: Some Lead, Others Follow

Animal groups such as fish schools, bird flocks and insect swarms appear to move so synchronously that they have long been considered egalitarian, leaderless units. In schooling fish, video observations of their spatial-temporal organization have, however, shown that anti-predator manoeuvres are not perfectly synchronous and that individuals have spatial preferences within the school. Nonetheless, when facing life-or-death situations, it is not known whether schooling fish react to a threat following a random or a hierarchically-based order. Using high-speed video analysis, here we show that schooling fish (Golden grey mullet, Liza aurata) evade a threat in a non-random order, therefore individuals that are first or last to react tend to do so repeatedly over sequential stimulations. Furthermore, startle order is strongly correlated with individual positional preferences. Because school members are known to follow individuals that initiate a manoeuvre, early responders are likely to exert the strongest influence on the escape strategy of the whole school. Our results present new evidence of the intrinsic heterogeneity among school members and provide new rules governing the collective motion of gregarious animals under predator attack.