Electrical Stimulation of Coleopteran Muscle for Initiating Flight

Some researchers have long been interested in reconstructing natural insects into steerable robots or vehicles. However, until recently, these so-called cyborg insects, biobots, or living machines existed only in science fiction. Owing to recent advances in nano/micro manufacturing, data processing, and anatomical and physiological biology, we can now stimulate living insects to induce user-desired motor actions and behaviors. To improve the practicality and applicability of airborne cyborg insects, a reliable and controllable flight initiation protocol is required. This study demonstrates an electrical stimulation protocol that initiates flight in a beetle (Mecynorrhina torquata, Coleoptera). A reliable stimulation protocol was determined by analyzing a pair of dorsal longitudinal muscles (DLMs), flight muscles that oscillate the wings. DLM stimulation has achieved with a high success rate (> 90%), rapid response time (< 1.0 s), and small variation (< 0.33 s; indicating little habituation). Notably, the stimulation of DLMs caused no crucial damage to the free flight ability. In contrast, stimulation of optic lobes, which was earlier demonstrated as a successful flight initiation protocol, destabilized the beetle in flight. Thus, DLM stimulation is a promising secure protocol for inducing flight in cyborg insects or biobots.     

The Effect of Slow Electrical Stimuli to Achieve Learning in Cultured Networks of Rat Cortical Neurons

Learning, or more generally, plasticity may be studied using cultured networks of rat cortical neurons on multi electrode arrays. Several protocols have been proposed to affect connectivity in such networks. One of these protocols, proposed by Shahaf and Marom, aimed to train the input-output relationship of a selected connection in a network using slow electrical stimuli. Although the results were quite promising, the experiments appeared difficult to repeat and the training protocol did not serve as a basis for wider investigation yet. Here, we repeated their protocol, and compared our ‘learning curves’ to the original results. Although in some experiments the protocol did not seem to work, we found that on average, the protocol showed a significantly improved stimulus response indeed. Furthermore, the protocol always induced functional connectivity changes that were much larger than changes that occurred after a comparable period of random or no stimulation. Finally, our data shows that stimulation at a fixed electrode induces functional connectivity changes of similar magnitude as stimulation through randomly varied sites; both larger than spontaneous connectivity fluctuations. We concluded that slow electrical stimulation always induced functional connectivity changes, although uncontrolled. The magnitude of change increased when we applied the adaptive (closed-loop) training protocol. We hypothesize that networks develop an equilibrium between connectivity and activity. Induced connectivity changes depend on the combination of applied stimulus and initial connectivity. Plain stimuli may drive networks to the nearest equilibrium that accommodates this input, whereas adaptive stimulation may direct the space for exploration and force networks to a new balance, at a larger distance from the initial state.     

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.     

The Effect of the Stimulation Regime on Palaemon serratus Tail Muscle Energy Metabolism

The evolution of phosphometabolites was determined in the abdominal muscle of a crustacean Palaemon serratus during intermittent electrical stimulation at 1, 2 and 4 Hz and during natural escape behavior.The changes in AMP, IMP, phosphomonoesters, adenylate energy charge and ATP/ADP ratio were not affected by the frequency of electrical stimulation. On the contrary, changes in ATP, ADP and sum of adenylates depended on the stimulation protocol: degradation of ATP and accumulation of ADP were not significantly different after electrical stimulation at 2 and 4 Hz as compared to manual stimulation, but differed from the 1 Hz stimulation protocol values. The sum of adenylates decreased similarly after 2 and 4 Hz stimulation and manual protocols. The different exercise protocols did not produce any changes in AMP and IMP accumulation, ATP/ADP ratio and A.E.C. After manual stimulation, the phosphomonoester and phosphoarginine concentrations were similar to the variations observed in the all electrical stimulation protocols, while the Pi levels were similar to the variations observed in the 4 Hz stimulation protocol only. The NMR index decrease was significantly higher after the manual and 4 Hz stimulation protocols.     

Calcium dependent plasticity applied to repetitive transcranial magnetic stimulation with a neural field model

The calcium dependent plasticity (CaDP) approach to the modeling of synaptic weight change is applied using a neural field approach to realistic repetitive transcranial magnetic stimulation (rTMS) protocols. A spatially-symmetric nonlinear neural field model consisting of populations of excitatory and inhibitory neurons is used. The plasticity between excitatory cell populations is then evaluated using a CaDP approach that incorporates metaplasticity. The direction and size of the plasticity (potentiation or depression) depends on both the amplitude of stimulation and duration of the protocol. The breaks in the inhibitory theta-burst stimulation protocol are crucial to ensuring that the stimulation bursts are potentiating in nature. Tuning the parameters of a spike-timing dependent plasticity (STDP) window with a Monte Carlo approach to maximize agreement between STDP predictions and the CaDP results reproduces a realistically-shaped window with two regions of depression in agreement with the existing literature. Developing understanding of how TMS interacts with cells at a network level may be important for future investigation.     

A 31P-n.m.r. study of the acute effects of beta-blockade on the bioenergetics of skeletal muscle during contraction.

1. The effects of beta-adrenoceptor antagonist administration on skeletal muscle contractile performance and bioenergetics in vivo have been investigated during unilateral sciatic nerve stimulation in the rat. 2. Two muscle stimulation protocols have been used: supramaximal stimulation at 4 Hz, or incremental supramaximal stimulation at 1, 2 and 4 Hz. Changes in high-energy phosphate concentrations were followed using 31P-n.m.r., and gastrocnemius muscle twitch characteristics were monitored continuously. 3. Under all conditions investigated, DL-propranolol administration (2.5 mg/kg body wt.) caused a significant decrease in cyclic AMP concentrations in resting and stimulated gastrocnemius muscle, prevented an increase in heart rate upon muscle stimulation, but did not affect plasma glucose, fatty acid or lactate concentrations in comparison with values obtained in control experiments. 4. Administration of DL-propranolol 5 min or 35 min before unilateral stimulation of 4 Hz had no effect on changes in muscle phosphocreatine, ATP or Pi concentrations, intracellular pH or contractile performance. 5. In contrast, animals receiving DL-propranolol 5 min before unilateral stimulation of 1, 2 and 4 Hz showed a significant deterioration in gastrocnemius muscle tension development during 2 and 4 Hz stimulation compared with control animals. Concurrent with this change in contractile performance was a higher muscle concentration of phosphocreatine, a lower concentration of Pi and no significant change in intramuscular pH compared with control experiments. 6. The changes in muscle performance and bioenergetics observed during the incremental stimulation protocol were not observed when D-propranolol was administered and could be completely circumvented by a short period of muscle stimulation of 4 Hz prior to initiation of the incremental stimulation protocol. 7. Mechanisms are discussed which may account for the failure of gastrocnemius muscle to generate the expected force during the incremental stimulation protocol in the presence of beta-blockade.     

An age-related shift in the force-frequency relationship affects quadriceps fatigability in old adults.

We examined the effect of an age-related leftward shift in the force-frequency relationship on the comparative quadriceps fatigability of nine young (27 +/- 1 yr old) and nine old men (78 +/- 1 yr old) during low-frequency electrical stimulation. Two different protocols of intermittent trains (6 pulses on, 650 ms off) of electrical stimulation at 25% maximum voluntary contraction were performed by both groups: 1) 180 trains at 14.3 Hz [constant frequency (CF) protocol], and 2) 180 trains at the frequency corresponding to 60% of each subject's force-frequency curve [normalized frequency (NF) protocol; young 14.9 +/- 0.4 vs. old 12.7 +/- 0.5 Hz; P < 0.05]. The quadriceps of the old men were weaker (approximately 31%) and relaxation was slower compared with the young men, as assessed by the maximal relaxation rate constant of the 50-Hz tetanus (young 12.1 +/- 0.2 vs. old 9.2 +/- 0.5 s(-1); P < 0.05) and a leftward shift in the force-frequency relationship. The NF protocol revealed a decreased fatigability in the quadriceps with old age (percentage of 1st contraction force remaining at 180th: old 63.4 +/- 1.5 vs. young 58.2 +/- 1.7%; P < 0.05) that was masked during the CF protocol (old 60.7 +/- 1.6 vs. young 58.6 +/- 2.3%; P > 0.05). Irrespective of the protocol, the maximal relaxation rate was reduced to approximately 73 and approximately 57% of the prefatigue value in the young and old men, respectively. The age-related leftward shift in the force-frequency relationship of the quadriceps contributed to an underestimation of the fatigue resistance with old age during the CF protocol. However, when the stimulation frequency used in the NF protocol was adjusted to account for the age-related shift in the force-frequency relationship, the quadriceps muscles of the old men were less fatigable than those of the young men. Thus we suggest that whole muscle fatigability is better examined by electrical stimulation protocols that are adjusted for inter- and intragroup differences in the force-frequency relationship.     

Large Animal Model for Development of Functional Restoration Paradigms Using Epidural and Intraspinal Stimulation

Restoration of movement following spinal cord injury (SCI) has been achieved using electrical stimulation of peripheral nerves and skeletal muscles. However, practical limitations such as the rapid onset of muscle fatigue hinder clinical application of these technologies. Recently, direct stimulation of alpha motor neurons has shown promise for evoking graded, controlled, and sustained muscle contractions in rodent and feline animal models while overcoming some of these limitations. However, small animal models are not optimal for the development of clinical spinal stimulation techniques for functional restoration of movement. Furthermore, variance in surgical procedure, targeting, and electrode implantation techniques can compromise therapeutic outcomes and impede comparison of results across studies. Herein, we present a protocol and large animal model that allow standardized development, testing, and optimization of novel clinical strategies for restoring motor function following spinal cord injury. We tested this protocol using both epidural and intraspinal stimulation in a porcine model of spinal cord injury, but the protocol is suitable for the development of other novel therapeutic strategies. This protocol will help characterize spinal circuits vital for selective activation of motor neuron pools. In turn, this will expedite the development and validation of high-precision therapeutic targeting strategies and stimulation technologies for optimal restoration of motor function in humans.     

Combined (thalamotomy and stimulation) stereotactic surgery of the VIM thalamic nucleus for bilateral Parkinson disease

Stereotactic thalamotomy of the thalamic nucleus ventralis intermedius (VIM) is routinely used for movement disorders. During this procedure, it has been observed that high-frequency (100 Hz) stimulation of VIM was able to stop the extrapyramidal tremor. In patients with bilateral tremor of extrapyramidal origin, who were resistant to drug therapy, the therapeutic protocol associated (1) a radiofrequency VIM thalamotomy for the most disabled side, and (2) a continuous VIM stimulation for the other side using stereotactically implanted electrodes, connected to subcutaneous stimulators. VIM thalamotomy relieved the tremor in all operated cases. Side effects were mild and regressive. VIM stimulation strongly decreased the tremor but failed to suppress it as completely as thalamotomy did. This was due in part to the fact that programmable stimulator frequency rate is limited to 130 Hz, while it appeared that the optimal stimulation frequency was 200 Hz. This therapeutic protocol appears to be of interest for patients with bilateral extrapyramidal movement disorders.     

Delay-Dependent Response in Weakly Electric Fish under Closed-Loop Pulse Stimulation

In this paper, we apply a real time activity-dependent protocol to study how freely swimming weakly electric fish produce and process the timing of their own electric signals. Specifically, we address this study in the elephant fish, Gnathonemus petersii, an animal that uses weak discharges to locate obstacles or food while navigating, as well as for electro-communication with conspecifics. To investigate how the inter pulse intervals vary in response to external stimuli, we compare the response to a simple closed-loop stimulation protocol and the signals generated without electrical stimulation. The activity-dependent stimulation protocol explores different stimulus delivery delays relative to the fish’s own electric discharges. We show that there is a critical time delay in this closed-loop interaction, as the largest changes in inter pulse intervals occur when the stimulation delay is below 100 ms. We also discuss the implications of these findings in the context of information processing in weakly electric fish.     

Repeated BOLD-fMRI Imaging of Deep Brain Stimulation Responses in Rats

Functional magnetic resonance imaging (fMRI) provides a picture of the global spatial activation pattern of the brain. Interest is growing regarding the application of fMRI to rodent models to investigate adult brain plasticity. To date, most rodent studies used an electrical forepaw stimulation model to acquire fMRI data, with α-chloralose as the anesthetic. However, α-chloralose is harmful to animals, and not suitable for longitudinal studies. Moreover, peripheral stimulation models enable only a limited number of brain regions to be studied. Processing between peripheral regions and the brain is multisynaptic, and renders interpretation difficult and uncertain. In the present study, we combined the medetomidine-based fMRI protocol (a noninvasive rodent fMRI protocol) with chronic implantation of an MRI-compatible stimulation electrode in the ventroposterior (VP) thalamus to repetitively sample thalamocortical responses in the rat brain. Using this model, we scanned the forebrain responses evoked by the VP stimulation repeatedly of individual rats over 1 week. Cortical BOLD responses were compared between the 2 profiles obtained at day1 and day8. We discovered reproducible frequency- and amplitude-dependent BOLD responses in the ipsilateral somatosensory cortex (S1). The S1 BOLD responses during the 2 sessions were conserved in maximal response amplitude, area size (size ratio from 0.88 to 0.91), and location (overlap ratio from 0.61 to 0.67). The present study provides a long-term chronic brain stimulation protocol for studying the plasticity of specific neural circuits in the rodent brain by BOLD-fMRI.     

Ultrasonic neuromodulation by brain stimulation with transcranial ultrasound

Brain stimulation methods are indispensable to the study of brain function. They have also proven effective for treating some neurological disorders. Historically used for medical imaging, ultrasound (US) has recently been shown to be capable of noninvasively stimulating brain activity. Here we provide a general protocol for the stimulation of intact mouse brain circuits using transcranial US, and, using a traditional mouse model of epilepsy, we describe how to use transcranial US to disrupt electrographic seizure activity. The advantages of US for brain stimulation are that it does not necessitate surgery or genetic alteration, but it confers spatial resolutions superior to other noninvasive methods such as transcranial magnetic stimulation. With a basic working knowledge of electrophysiology, and after an initial setup, ultrasonic neuromodulation (UNMOD) can be implemented in less than 1 h. Using the general protocol that we describe, UNMOD can be readily adapted to support a broad range of studies on brain circuit function and dysfunction.     

Sucrose acceptance threshold: a way to measure sugar perception in ants

Variation in the perception of sweet taste is a well-known phenomenon in the animal kingdom. Well-established protocols for measuring sucrose responsiveness in non-social insects and honeybees have made it possible to understand many aspects of their biology and behaviour. Ants are also advanced social insects that present a plethora of life histories with diverse strategies and behaviours; however, a universal paradigm possible to measure this response in different ant species has not yet been developed. Here, we present a protocol for measuring the sucrose acceptance threshold (SAT) under controlled conditions in harnessed ants with different feeding habits. By testing the response to antennal and palp sucrose stimulation and using the occurrence of licking as the response, we developed a non-ambiguous evaluation that allowed easy detection of threshold changes. The results showed that the response to both antennal and palp stimulation varied widely among species. Some species licked in response to antennal stimulation while others did so in response to palp stimulation. Using the appropriate kind of stimulation, we tested the SAT protocol in ants of different genera and ants of the same species with different levels of sugar reserve. The differences detected in both cases imply that the protocol is appropriate for measuring and detecting variations in sugar perception in ants.     

New protocol for commencing the GnRH antagonist in assisted conception treatment cycles: elimination of the premature LH surge with similar pregnancy rates

GnRH antagonists have been used with increasing frequency in assisted reproduction treatments over the past few years and have been associated with quicker and more profound LH suppression and shorter treatment cycles than conventional GnRH agonists. Usually, these are commenced on day 6 of FSH stimulation without allowing for patient variation in response to treatment. The study was aimed at individualising this protocol to the patients' ovarian response. The control group included 215 treatment cycles where the GnRH antagonist was commenced on day 6 of FSH stimulation. A new individualised protocol was formulated, applied to practice and 172 treatment cycles following that were analysed. The study group had no premature LH surges (LH > 10 iu x mL(-1)) compared to the control group who had a rate of 4.1%. There was also a higher fertilisation and clinical pregnancy rate in the study group (P = 0.06). It is concluded that the new individualised GnRH antagonist protocol eliminates premature LH surges in assisted conception treatment cycles and may improve clinical pregnancy rates compared to the conventional protocol of "day 6 commencement".     

Electrical stimulation of human forearm extensor muscles as an indicator of handgrip fatigue and recovery

A set-up for percutaneous electrical stimulation of the forearm extensor muscles and measurement of wrist extension force is described. The frequency-force relationship and pulse duration-force relationship are described together with an experimental protocol showing that brief electrical test stimulations do not produce fatigue. In another set of experiments carried out a few weeks later, the subjects performed handgrip contractions: protocol A at 25% of maximal voluntary contraction (MVC) continuously until exhaustion, protocol B at 25% MVC intermittent (contraction + relaxation = 10 + 2 s) until exhaustion, and protocol C at 25% MVC intermittent until half the time to exhaustion. In all experiments, brief electrical stimulations were used to test the degree of fatigue during and up to 24 h after the experiments. There were marked changes in the force during stimulation at 20 and 100 Hz and these changes did not correlate with the increase in intramuscular temperature. Low frequency fatigue persisted for at least 24 h after protocol A and 1 h after protocols B and C. The significance of this is discussed and it is suggested that low frequency fatigue could be used as a sensitive indicator of muscle dysfunction after low and medium intensity exercise.     

Nonparallel secretion of enzymes by the rabbit pancreas

Since nonparallel secretion of enzymes by the exocrine pancreas has been demonstrated with several experimental models, we were interested in verifying a recent claim that enzyme secretion remained strictly proportional (parallel) upon stimulation of the in vivo rabbit pancreas. Pancreatic juice was collected by extraduodenal cannulation of the pancreatic duct, in two different protocols. In the first protocol the administration of pentobarbital induces a mild anesthesia. Under this condition, amylase and chymotrypsin secretion remained parallel after cholecystokinin stimulation. In a second protocol, a deeper and constant anesthesia was attained with Fluothane resulting in a lower basal protein output than in the first protocol. Pancreatic secretion was collected under intravenous secretin perfusion (4.5 clinical units X kg-1 X h-1). After stabilization and basal collection periods, pancreatic secretion was stimulated with an i.v. bolus injection of either cholecystokinin (2 Ivy dog units/kg), caerulein (0.1 micrograms/kg), or carbachol (6 micrograms/kg). Upon stimulation of the pancreas, protein output increased an average of 30-fold and there was a concomitant 20-25% decrease in the ratio of the specific activities of amylase to chymotrypsin which resulted from a greater increase in the specific activity of chymotrypsin in pancreatic juice after stimulation of secretion. Thus, under appropriate conditions, nonparallel secretion of enzymes by the exocrine pancreas can be demonstrated in yet another experimental model. Furthermore, the proportion of amylase and chymotrypsin activities in pancreatic juice are once more shown to be dependent, up to a threshold, upon the rate of protein output by this exocrine gland.     

Estrogen and gender do not affect fatigue resistance of extensor digitorum longus muscle in rats.

The effects of estrogen on skeletal muscle fatigue are controversial. To determine the effects of estrogen and gender on rat extensor digitorum longus (EDL) muscle, we either injected 40 microg beta-estradiol 3/benzoate.kg BW(-1) to female rats or sham injected male or female rats for 14 days. Subsequently a 90 min fatigue protocol consisting of electrical stimulation at 10 Hz delivered in 500 ms trains was administered. Force was recorded for a 5 s period at the start of the protocol (0 min) and at 5 min intervals until completion following 90 min of stimulation. After 90 min, EDL force generation at 10 Hz stimulation declined in all groups to between 50-60 % of initial values. However, no significant difference in fatigue rate or final 10 Hz stimulated force was seen between females administered estrogen, sham injected females or males. Hence, estrogen administration and gender did not significantly affect EDL muscle fatigue in this model.     

Electrical stimulation as an adjunct to spinal fusion: a meta-analysis of controlled clinical trials

This study was a meta-analysis to examine whether electrical stimulation has a specific effect on spinal fusion. Little evidence exists on the efficacy of electrical stimulation for improving fusion rate of spinal fusion surgery. Using MEDLINE (1966-2000) and EMBASE (1985-1999), a search for articles was carried out using the Medical Subject Headings: (1) electric stimulation or electromagnetic fields, (2) spinal fusion, (3) controlled or clinical trial, and (4) human. Data were extracted from all the hit articles and additionally collected from appropriate journal lists. A total of five randomized controlled trials (RCT) on bones assessing healing of spinal fusion were identified and scored on methodological quality. All the identified studies reported positive findings, but the quality score of each trial showed wide flaws. Because of relatively homogenous subjects who had spine fusion and radiographic assessment from these studies, pooling of the data was able to be performed. Excluding one trial with the lowest score, the combined results of four trials, whose major endpoints were the success rate of the fusion, revealed a statistically significant effect of electrical stimulation with various techniques, but the selected trials still showed wide variation in view of stimulation modalities and treatment protocol. The pooled result of the studies in this review revealed the efficacy of electrical stimulation based on proved methodological quality. As problems on therapeutic modality and protocol remain, there is a further need for improvement in design to constitute acceptable proof and to establish treatment programs that better demonstrate electrical stimulation effects on spinal fusion.     

Mathematical models for fatigue minimization during functional electrical stimulation

We previously reported the development of a force- and fatigue-model system that predicted accurately forces during repetitive fatiguing activation of human skeletal muscles using brief duration (six-pulse) stimulation trains. The model system was tested in the present study using force responses produced by longer duration stimulation trains, containing up to 50 pulses. Our results showed that our model successfully predicted the peak forces produced when the muscle was repetitively activated with stimulation trains of frequencies ranging from 20 to 40 Hz, train durations ranging from 0.5 to 1 s, and varied pulse patterns. The predicted peak forces throughout each protocol matched the experimental peak forces with r² values above 0.9 and predicted successfully the forces at the end of each protocol with <15% error for all protocols tested. The success of our model system further supports its potential use for the design of optimal stimulation patterns for individual users during functional electrical stimulation.     

A Protocol for the Use of Remotely-Supervised Transcranial Direct Current Stimulation (tDCS) in Multiple Sclerosis (MS)

Transcranial direct current stimulation (tDCS) is a noninvasive brain stimulation technique that uses low amplitude direct currents to alter cortical excitability. With well-established safety and tolerability, tDCS has been found to have the potential to ameliorate symptoms such as depression and pain in a range of conditions as well as to enhance outcomes of cognitive and physical training. However, effects are cumulative, requiring treatments that can span weeks or months and frequent, repeated visits to the clinic. The cost in terms of time and travel is often prohibitive for many participants, and ultimately limits real-world access. Following guidelines for remote tDCS application, we propose a protocol that would allow remote (in-home) participation that uses specially-designed devices for supervised use with materials modified for patient use, and real-time monitoring through a telemedicine video conferencing platform. We have developed structured training procedures and clear, detailed instructional materials to allow for self- or proxy-administration while supervised remotely in real-time. The protocol is designed to have a series of checkpoints, addressing attendance and tolerability of the session, to be met in order to continue to the next step. The feasibility of this protocol was then piloted for clinical use in an open label study of remotely-supervised tDCS in multiple sclerosis (MS). This protocol can be widely used for clinical study of tDCS.