Tetanic Responses Research Articles

Assessing Contractility of 3D iPSC‐derived Muscle Models for Safety and Discovery Using a Novel, High‐throughput, and Label‐free Instrumentation Platform

Stem cell models hold great promise for improving the predictive power of preclinical in vitro assays for new therapies, drug discovery, basic scientific research, and disease modeling. Complex 3D platforms, such as Engineered Muscle Tissues (EMTs) fabricated from primary or iPSC‐derived cells, can directly measure tissue contractility, which is challenging in conventional 2D platforms where cells are rigidly attached to a surface. However, traditional methods to fabricate EMTs demand extensive bioengineering expertise, and measuring contractility often involves laborious, serial, and low‐throughput optical measurements.Here, we report on the design, fabrication, and validation of a novel 3D EMT platform that uses 1) facile and scalable bioengineering approaches to generate tissues from a variety of cell sources, and 2) a label‐free parallel measurement technique. Our tissue casting approach has a success rate of >96% (n > 100) and produces consistently‐sized constructs with a standard deviation of +/‐ 9% across 6 experiments. Tissue casting, media changes, and drug dosing are also highly amenable to automation.The substrate features an embedded magnet; as tissues contract, the magnet’s displacement is quantitatively detected in a highly‐parallel manner using specialized sensors. We detected 24 contractions simultaneously with a measurement rate of 100Hz, which is suitable for measuring various aspects of contractility such as upstroke velocity, decay time, and fatigability. We demonstrated that the signal voltage changes are linear with respect to EMT contraction.We will present data showing acute effects of drugs measured minutes after EMT dosing and chronic effects of structural cardiotoxicants like doxorubicin, sunitinib, and BMS‐986094 on EMTs. All chronically‐dosed tissues showed statistically significant dose‐dependent reduction in twitch frequency over a multi‐day time course (p <0.05). We will also show that our platform can be used to generate physiologically‐relevant skeletal muscle constructs and achieve tetanic responses upon stimulation.In addition to modeling healthy tissues, our platform can also be used to study disease models. We are currently developing patient‐specific Duchenne Muscular Dystrophy models for the development of personalized gene therapies. EMTs can be made from cells sourced from patients and used to test whether a new therapy will improve or recover functional contraction.We have designed a novel system that can leverage the complexity of 3D cellular models in a scalable format and can be tailored for specific applications, including personalized medicine or disease modeling. The Mantarray platform will provide a stand‐alone tool capable of screening significant numbers of compounds for the rapid safety evaluation of drug candidates thereby accelerating drug discovery and development.

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.63 AAV.CAPN3 gene therapy for Limb-girdle muscular dystrophy type 2A (LGMD2A): Phenotypic improvements in the calpain3 (CAPN3)-null mouse

LGMD2A is caused by mutations in CAPN3 gene. CAPN3 loss of function is shown to play a role in disease pathogenicity, which might be improved by CAPN3 gene therapy. rAAVrh74.tMCK.CAPN3 vector was systemically delivered to young (YC, 6-10 week-old) and old cohorts (OC, 21-23 week-old) of CAPN3-KO mice. Each cohort included two treatment groups receiving low dose (LD; 3E12 vg, n=12) and high dose (HD; 6E12 vg, n=12) of the vector and age-matched untreated (UT) control groups (UT-YC, n=13;UT-OC, n=15). Outcome measures were performed at 20 weeks post-injection. AAV.CAPN3 improved treadmill performance in both cohorts, at both LD and HD subgroups (p<0.0001). There was no significant age-related effect on treadmill activity and the average performance of the treatment groups, HD vs. LD did not differ significantly. In the OC however, females (n=5) from the HD group run longer distance than males from same group (n=7; p<0.01). In the LD and HD treatment groups from OC, in vivo muscle contractility assay showed an improvement in both Twitch (p=0.001) and Tetanic Max responses (p=0.047) at the endpoint. The average of percent change between baseline and endpoint tetanic responses increased in the treated-YC while there was a decline in the UT cohort (p=0.002). Histological analyses of the skeletal muscle (gastroc, tibialis anterior, quadriceps and triceps) showed remodeling of muscle, a switch to fatigue resistant oxidative fibers in both cohorts along with fiber size increase. In-house toxicology studies in the HD cohort revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. AAV.CAPN3 gene therapy improved the LGMD2A-related phenotype in the CAPN3-KO mouse model in both low and high doses tested, independent of age at the time of vector-administration. Improvements shown were supported with the absence of cardiotoxicity, enhancing the efficacy and safety of AAV.CAPN3 vector as a potential gene therapy for LGMD2A.

Risk assessment of neuromuscular stimulation by energy-based transurethral resection devices: an ex vivo test standard

BackgroundDuring transurethral resection of bladder tumours (TURB), radio-frequency (RF) currents can lead to adverse neuromuscular stimulation (NMS). Here we present a novel ex vivo method to determine the risk of RF generators and their bipolar TURB modes to cause NMS. We aimed to develop an experimental platform for safety evaluation of new RF generators and their modes with a newly established test standard, suitable for replacement or reduction of animal testing.MethodsWe tested four contemporary RF generators with their bipolar modes for TURB in saline. A two-stage ex vivo approach was pursued: First, we recorded voltages at possible positions of the obturator nerve behind a porcine bladder wall in a TURB model using 18 RF applications per generator. Second, these voltage records were used as stimuli to evoke nerve compound action potentials (CAPs) in isolated porcine axillary nerves. The NMS potential was defined as the ratio between the observed area under the CAPs and the theoretical CAP area at maximum response and a firing rate of 250 Hz, which would reliably induce tetanic muscle responses in most human subjects. The measurement protocol was tailored to optimise reproducibility of the obtained NMS potentials and longevity of the nerve specimens.ResultsAs prerequisite for the clinical translation of our results, the robustness of our test method and reproducibility of the NMS potential are demonstrated with an excellent correlation (r = 0.93) between two sets of identical stimuli (n = 72 each) obtained from 16 nerve segments with similar diameters (4.2 ± 0.37 mm) in the nerve model. The RF generators differed significantly (p < 0.0001) regarding NMS potential (medians: 0–3%).ConclusionsOur test method is suitable for quantifying the NMS potential of different electrosurgical systems ex vivo with high selectivity at a reasonable degree of standardization and with justifiable effort. Our results suggest that the clinical incidence of NMS is considerably influenced by the type of RF generator. Future generations of RF generators take advantage from the proposed test standard through higher safety and less animal testing. Health professionals and treated patients will benefit most from improved RF surgery using generators with a low NMS risk.

Neurotoxicity of Micrurus lemniscatus lemniscatus (South American coralsnake) venom in vertebrate neuromuscular preparations in vitro and neutralization by antivenom.

We investigated the effect of South American coralsnake (Micrurus lemniscatus lemniscatus) venom on neurotransmission in vertebrate nerve-muscle preparations in vitro. The venom (0.1-30µg/ml) showed calcium-dependent PLA2 activity and caused irreversible neuromuscular blockade in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, contractures to exogenous acetylcholine and carbachol (CCh), but not KCl, were abolished by venom concentrations ≥ 0.3µg/ml; in PND preparations, the amplitude of the tetanic response was progressively attenuated, but with little tetanic fade. In low Ca2+ physiological solution, venom (10µg/ml) caused neuromuscular blockade in PND preparations within ~ 10min that was reversible by washing; the addition of Ca2+ immediately after the blockade temporarily restored the twitch responses, but did not prevent the progression to irreversible blockade. Venom (10µg/ml) did not depolarize diaphragm muscle, prevent depolarization by CCh, or cause muscle contracture or histological damage. Venom (3µg/ml) had a biphasic effect on the frequency of miniature end-plate potentials, but did not affect their amplitude; there was a progressive decrease in the amplitude of evoked end-plate potentials. The amplitude of compound action potentials in mouse sciatic nerve was unaffected by venom (10µg/ml). Pre-incubation of venom with coralsnake antivenom (Instituto Butantan) at the recommended antivenom:venom ratio did not neutralize the neuromuscular blockade in PND preparations, but total neutralization was achieved with a tenfold greater volume of antivenom. The addition of antivenom after 50% and 80% blockade restored the twitch responses. These results show that M. lemniscatus lemniscatus venom causes potent, irreversible neuromuscular blockade, without myonecrosis. This blockade is apparently mediated by pre- and postsynaptic neurotoxins and can be reversed by coralsnake antivenom.

Effects of resveratrol, catechin and epicatechin on rat phrenic nerve hemi-diaphragm

Aim: Resveratrol, catechin and epicatechin are known for their preventive effects of skeletal muscle atrophy, anti-fatique effects and enhancement of exercise capacity. In this study, we investigate the effects these antioxidants on muscle contraction by using rat phrenic nerve hemi-diaphragm preparations. Methods: Twitch responses are investigated by giving direct and indirect and tetanic stimulation to rat phrenic nerve hemi-diaphragm preparations. Results: Resveratrol and catechin facilitated twitch responses to the indirect stimulus in short duration and suppressed in long duration. The decrease in the responses to the direct stimulation by applying epicatechin (p=0.010) was significantly higher than the responses by giving indirect stimulation. Epicatechin significantly suppressed tetanic responses (p=0.005), but not resveratrol and catechin. Conclusion: The results of the study show that these antioxidants have presynaptic and postsynaptic effects on neuromuscular junction. Resveratrol effective on muscle contraction via direct stimulus, catechin and epicatechin effects muscle contraction by direct and indirect stimulus.

Prolonged Low‐frequency Force Depression is Underestimated When Assessed with Doublets Compared to Trains

Unaccustomed eccentric exercise induces muscle damage, which leads to an impairment of force or torque that can last for several days. A common indirect measure of muscle damage is a greater depression of the force response to low‐frequency (e.g., 10Hz) compared to high‐frequency (e.g., 100Hz) stimulation; i.e., a post‐exercise reduction in the 10:100Hz ratio. This phenomenon is termed, prolonged low‐frequency force depression (PLFFD). Historically, trains of stimuli (e.g., 1s) have been used to evoke tetanic responses for the ratio. More recently, some authors have used only paired stimuli to evoke doublets. However, it is unknown if the PLFFD indicated by doublet responses is equivalent to the value derived from the traditional tetanic responses. Hence, the purpose of this study was to compare the magnitude of PLFFD determined by doublet vs. tetanic responses. We hypothesized that the doublet ratio (DR) would indicate significantly less PLFFD than the train ratio (TR). Eight participants (four females) performed 200 eccentric maximal voluntary contractions (4 sets of 50 repetitions, 1s rest between repetitions and 1 min rest between sets) of the dorsiflexors to induce muscle damage. Eccentric contractions were performed at 60°/s, started at a neutral ankle position, and finished at 30° of plantar flexion. Before the fatiguing protocol, baseline neuromuscular function was measured by a sequence of isometric contractions evoked by supramaximal electrical stimulation of the fibular nerve (paired stimuli at 10 and 100Hz, and 1s trains at 10 and 100Hz, each separated by 1s rest). This neuromuscular function test was repeated three times prior to fatigue and 2 min, 3 min, 5 min, 10 min and 48h after the fatiguing protocol. Using the peak torque of each response, 10:100Hz ratios were calculated at all time points; post‐exercise values were expressed as a percentage of the respective baseline ratio. At baseline, the DR was greater than TR (0.81 ± 0.04 vs. 0.48 ± 0.09; P &lt; 0.01). PLFFD was observed by 2 min for TR (73 ± 12%) and 3 min for DR (83 ± 9%), continued to develop over the acute recovery phase (at 10 min, DR = 73 ± 9%; TR = 51 ± 11%), and recovered by 48h (DR = 98 ± 6%; TR = 91 ± 12%). The TR was reduced more than the DR at all acute recovery time points (P &lt; 0.05), but not 48h. With DR and TR revealing different levels of PLFFD following damaging eccentric contractions, our results indicate that these techniques should not be used interchangeably. Whenever possible, trains of stimuli should be used to indicate the true extent of PLFFD. In cases where the reproducibility of the tetanic force response may be problematic (e.g., femoral nerve stimulation) or a train is deemed prohibitively uncomfortable, it should be acknowledged that paired stimuli will underestimate PLFFD.Support or Funding InformationSupported by NSERC, CFI and BCKDFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transformation of individual contractile responses during tetanus in rat fast and slow skeletal muscles

The last contractile responses (LCRN), where N is the number of individual contractile responses within tetanus, were separated from the integral tetanic responses of fast, m. Extensor digitorum longus (m. EDL), and slow, m. Soleus, rat muscles using a computer-graphic technique. The average amplitude of LCR5 in m. Soleus at a 20 Hz stimulation rate decreased to 64 ± 9 % re the amplitude of a single contraction. As N was increasing, a restoration of LCRN was observed with their subsequent rise to values almost twofold exceeding the initial single contractile responses of that muscle (up to 211 ± 10 % for LCR50). Simultaneously, against the background of rise of individual contractile responses of these muscles, a considerable shortening of their half-life time (to ≈ 50%) and formation of a stationary plateau within LCRN were observed. In m. EDL at a 50 Hz stimulation rate only single-phase rise of LCRN was observed (up to 165 ± 18% for LCR50) without change of their half-life time and plateau formation. After the end of tetanic responses in muscles of both types a prolonged (up to 30 s) "hyper-relaxation effect was shown to develop manifested as a decrease of muscle tension with its subsequent restoration to the initial values. Possible mechanisms of these effects are discussed. It is supposed that transformation of individual contractile responses in skeletal muscles may be executed at the expense of specialized microdomains in muscle fibers regulating accumulation and extrusion levels of Ca2+ ions during tetanic activity. The possible involvement of an additional, Ca(2+)-induced Ca2+ release (CICR), in the basic, depolarization-induced Ca2+ release (DICR), is analyzed.

Effect of insulin on characteristics of contractile responses of fast and slow skeletal muscles of rats with acute streptozotocin-induced diabetes

Comparison of amplitude-time characteristics of fast (m. EDL) muscles in rats with acute model of streptozotocin diabetes (SD) (12 and 30 days after treatment with streptozotocin) did not reveal significant changes in strength of single normalized contractile responses as compared with control. In slow (m. Soleus) muscles of rats with the 30-day SD there were observed essential changes in amplitude-time characteristics of such contractile responses: a decrease of their amplitude and an increase of duration. In diabetic rats treated with a course of insulin, resistance of skeletal muscles to both types of exogenous insulin is developing. Both in control and in diabetic animals the fatiguing stimulation of m. EDL by firing from 5 impulses did not reveal significant differences at early (up to 3 min) terms of development of fatigue. Under similar conditions, fatigue of m. Soleus in rats of both diabetic groups developed significantly faster as compared with control (as early as 30 s after the beginning of stimulation). Insulin at a concentration of 5-1 nM produced a dose-dependent decrease of amplitude of single contractile responses in fast and slow muscles of rats with the acute SD model (the negative inotropic action). The same effect of insulin, but at the higher concentrations, we demonstrated earlier in health rats. Insulin at a concentration of 10 nM did not produce essential effect on the time course of depression of responses in the course of development of fatigue at tetanic stimulation of m. EDL and m. Soleus both in control and in diabetic rats, but affected essentially the time course of change in duration of the half-decline (T(hd)) of their tetanic responses. The presence of insulin in the bath solution led to stabilization of the muscle relaxation period in the course of development fatigue in all studied animal groups.

Temperature Effects on the Myoplasmic Free Ca2+ Transients in FDB Muscle Fibers of the Mouse

The fast, low affinity, Ca2+ dye Mag-Fluo-4 loaded in its AM moiety, in mouse FDB fast-twitch fibers has allowed to study, with high temporal resolution, the decay phase of single or tetanic (350 ms-100 Hz), Ca2+ transients (Calderon et al. JMRCM 2009;30). At 23°C, a double exponential function with time constants (always expressed in ms), τ1=1.4±0.1, τ2=15.3±1.3 (n=20), and τ1=23.4±2.5, τ2=594±102.4 (n=13), respectively, describe the decay phase of single and tetanic transients. These time constants have been associated with Ca2+ binding to parvalbumin, and SERCA, respectively. We tested for differential effect of tetanic Ca2+ loads and temperature on the putative Ca2+ removal mechanisms. Increasing temperature, from 23 to 33°C, reversibly diminishes the single transient amplitude (ΔF/F), by 31%, and increases the base line by 32% (n=25). At 33°C, in 15 out of 20 fibers, the decay of single transients followed a single exponential, τ=4.7±0.7, while in the other 5 fibers, two exponentials provided a better fit, τ1=0.6± 0.1 and τ2=5.1±0.4; two exponentials τ1=10.1±1.8 and τ2=394.9±51.2 (n=13), provided the best fit for tetanic decay. The results show that the two time constants, that describe Ca2+ single and tetanic responses decay, are differentially affected by temperature and by tetani imposed Ca2+ loads. The fast decay components of single and tetanic responses, represented by τ1, and associated with parvalbumin Ca2+ binding, are affected similarly by Ca2+ load (17x increase) or by temperature (2.3x increase for 10°C increase). The slower components of the single and tetanic responses, represented by τ2, and associated with SERCA operation, are differentially affected by a 10°C temperature raise (3.1x vs 1.5x) and by the tetanic Ca2+ load (39x vs 77x) respectively.

Effect of repeat acute injury on contractile function of the external anal sphincter in an animal model

Our aim was to estimate the physiologic effects of early repeat transection and repair on the contractile properties of the external anal sphincter (EAS) in a rat model. Eighty young female rats underwent anal sphincter transection and repair. After 7 days, they were randomized to repeat sphincter transection (injury-injury, n = 40) or sham operation (injury-sham, n = 40). Thereafter, the anal sphincter complex was dissected, mounted, and analyzed for contractile function 7 days, 21 days, 3 months, or 6 months after the second operation. Contractile function was also determined in 40 age-matched unoperated controls (n = 10 for each time point). Statistical analysis was performed using analysis of variance (ANOVA) with Tukey-Kramer adjustment for multiple testing. P ≤ 0.05 was considered significant. Although single injury (injury-sham) resulted in modest compromise of sphincter function, repeat injury (injury-injury) resulted in profound impairment of twitch tension, maximal tetanic responses, and maximal electrical-field stimulation (EFS) induced-force generation at 7 days. After single injury, parameters of contractile function returned to baseline uninjured levels by 21 days. In contrast, sphincter function remained reduced 21 days after repeat injury. Contractile function of sphincters from both injury-sham and injury-injury animals were no longer impaired at 3 and 6 months. In this animal model, repeat injury and repair of the EAS 7 days after the initial injury resulted in prolonged compromise of EAS function compared with single injury. Nevertheless, contractile function of the double-injured sphincter fully recovered with time, resulting in no long-term impairment.

The electrophysiological study of changes in the activities of locus coeruleus neurons under conditions of parathyroidectomy and parathyroid hormone administration

The evoked activity of single neurons in the locus coeruleus (LC) in rats was studied in the norm, after parathyroidectomy (PTX), and after the administration of parathyroid hormone (PTH) during high frequency-stimulation of the supraoptic nucleus of the hypothalamus (SON). Spike activity was analyzed using mathematical software. The excitatory poststimulus neuronal activity in the LC with PTH decreases the average frequency of activity compared to the norm, but it approaches the norm after the introduction of PTH. The poststimulus inhibitory responses of neurons in the LC with PTH were lowered, as compared to the norm; they recovered to the normal level after the introduction of PTH. Calculation of the ratio between the sum of spikes in excitatory poststimulus responses with PTX and the sum in the norm showed a twofold decrease in the responses; after administration of PTH, this sum returned back to normal. However, after PTX we observed an extreme reduction of neurons that responded by excitation as compared to the norm; after PTH administration we found a twofold increase in the number of these neurons as compared to the norm. For the sum of spikes in depression tetanic responses, the introduction of PTH was accompanied by their identical decrease compared to the norm and by an increase in the number of responding neurons after treatment with PTH as compared to both the norm and PTX. Our results led us to conclude that the greater intensity of tetanic responses of poststimulus inhibitory activity apparently results from an activation of the protective inhibitory GABAergic mechanism in intact animals, a sharp deficit of tetanic excitatory effects under conditions of PTX, and the return of these effects to the norm in the presence of PTH.

Modulation of contractile apparatus Ca2+ sensitivity and disruption of excitation–contraction coupling by S‐nitrosoglutathione in rat muscle fibres

S-Nitrosoglutathione (GSNO) is generated in muscle and may S-glutathionylate and/or S-nitrosylate various proteins involved in excitation–contraction (EC) coupling, such as Na+-K+-ATPases, voltage-sensors (VSs) and Ca2+ release channels (ryanodine receptors,RyRs), possibly changing their properties. Using mechanically skinned fibres from rat extensor digitorum longus muscle, we sought to identify which EC coupling processes are most susceptible to GSNO-modulated changes and whether these changes could be important in muscle function and fatigue. For comparison, we examined the effect of other oxidation, nitrosylation, or glutathionylation treatments (S-nitroso-N-acetyl-penicillamine (SNAP), hydrogen peroxide,2,2-dithiodipyridine and reduced glutathione) on twitch and tetanic force, action potential (AP) repriming, sarcoplasmic reticulum (SR) Ca2+ loading and leakage, and contractile apparatus properties. None of the treatments detectably altered AP repriming, indicating that t-system excitability was relatively insensitive to such oxidative modification. Importantly, the overall effect on twitch and tetanic force of a given treatment was determined primarily by its action on Ca2+ sensitivity of the contractile apparatus. For example, S-nitrosylation with the NO• donor,SNAP, caused matching decreases in the contractile Ca2+ sensitivity and twitch response, and GSNO applied ∼10 min after preparation had very similar effects. The only exception was when GSNO was applied immediately after preparation, which resulted in irreversible decreases in twitch and tetanic responses even though it concomitantly increased Ca2+ sensitivity by∼0.1 pCaunits, the latter evidently due to S-glutathionylation of the contractile apparatus. This decrease in AP-mediated force responses was due to impaired VS–RyR coupling and was accompanied by increased Ca2+ leakage through RyRs. Such oxidation-related impairment of coupling could be responsible for prolonged low frequency fatigue in certain circumstances.

GABAA receptor-mediated modulation of neuronal activity propagation upon tetanic stimulation in rat hippocampal slices

Tetanic stimulation (100 Hz), which can induce long-term potentiation in synaptic connections in the hippocampal CA1 region, causes γ-aminobutyric acid (GABA)(A) receptor-mediated long-lasting depolarization of postsynaptic neurons. However, it is not clear how this stimulation modulates neuronal activity propagation. We studied tetanic burst-induced neuronal responses in the hippocampal CA1 region by using optical-recording methods employing a voltage-sensitive dye and focused on GABA(A) receptor-mediated modulation. We observed that burst stimulation induced long-lasting depolarization and progressive decrease in individual excitatory postsynaptic potentials (EPSPs). Both these effects were suppressed by picrotoxin, a GABA(A) receptor antagonist. Under whole-cell voltage-clamp conditions, we observed a long-lasting inhibitory current (IPSC) and a prominent progressive decrease in the amplitude of the excitatory postsynaptic current (EPSC). Further, picrotoxin inhibited the IPSC and the progressive decrease in EPSC. The optically recorded long-lasting depolarization and progressive decrease of EPSPs were strongly dependent on the distance between the recording electrode and the stimulation site. Optical recordings performed across a wide swatch of CA1 revealed that the decrease in activity propagation was followed by facilitation of propagation after recovery and that this facilitation also depended on GABA(A) receptors. Intense activation of GABA(A) receptors is a key factor shaping the spatiotemporal patterns of high-frequency stimulation-induced responses in the CA1 region.

Functional anatomy of the vagina muscles in the adult western conifer seed bug, Leptoglossus occidentalis (Heteroptera: Coreidae), and its implication for the egg laying behaviour in insects

The anatomy of the female reproductive tract and the nerve-evoked contractions of the vagina muscles and their association with the ovipositor in the western conifer seed bug, Leptoglossus occidentalis (Heidemann) are investigated for the first time. The reproductive tract consists of a set of paired telotrophic ovaries, each containing seven ovarioles, located in the anterior lateral regions of the abdomen. Each ovary is attached to a lateral oviduct which spans most of the abdomen to attach to a relatively short common oviduct that joins the vagina near the rear of the animal. The vagina is associated with a pair of bilaterally symmetrical muscles attached at their posterior ends to lateral extensions of sternite VIII, the valvifer of the Type II ovipositor. From this attachment site, the muscles fan out medially and anteriorly to converge along the dorsal midline of the vagina up to the base of the common oviduct. Vagina muscles respond to a single stimulation of their motor nerves by producing a smooth contraction lasting approximately 1 s. With increasing frequencies of stimulation, the muscle contractions summate to create a tetanic response. The muscles are fatigue resistant being able to maintain the same degree of tension for up to 10 min at 10 Hz stimulation. Visual observation shows that other muscles associated with the valves of the ovipositor behave in a similar fashion to that of the vagina muscles from which the tension recordings were obtained. Fatigue-resistant vagina muscles are discussed in relation to copulation, sperm transport and this insect's ability to deposit a series of eggs directly onto the surface of a conifer needle in a manner by which eight or more blunt-ended eggs are packed end-to-end in a single row.

Ca2+ Transients and Myosin Heavy Chain (MHC) Composition in Murine Enzymatically Dissociated Fibers

Single and tetanic Ca2+ transients reported with MagFluo4-AM were obtained together with MHC electrophoretic patterns in enzymatically dissociated fibres from adult mice soleus and extensor digitorum longus (EDL) muscles. Kinetics of transient rise (Ca2+ release) and decay (Ca2+ clearance) of both twitch and tetanic responses showed a continuum from the slowest records obtained in fibers type I, to the fastest obtained in fibers IIX/D and IIB. Fibers IIA were fast regarding Ca2+ release but slow regarding Ca2+ clearance.

Chronic stability and selectivity of four-contact spiral nerve-cuff electrodes in stimulating the human femoral nerve

This study describes the stability and selectivity of four-contact spiral nerve-cuff electrodes implanted bilaterally on distal branches of the femoral nerves of a human volunteer with spinal cord injury as part of a neuroprosthesis for standing and transfers. Stimulation charge threshold, the minimum charge required to elicit a visible muscle contraction, was consistent and low (mean threshold charge at 63 weeks post-implantation: 23.3 ± 8.5 nC) for all nerve-cuff electrode contacts over 63 weeks after implantation, indicating a stable interface with the peripheral nervous system. The ability of individual nerve-cuff electrode contacts to selectively stimulate separate components of the femoral nerve to activate individual heads of the quadriceps was assessed with fine-wire intramuscular electromyography while measuring isometric twitch knee extension moment. Six of eight electrode contacts could selectively activate one head of the quadriceps while selectively excluding others to produce maximum twitch responses of between 3.8 and 8.1 N m. The relationship between isometric twitch and tetanic knee extension moment was quantified, and selective twitch muscle responses scaled to between 15 and 35 N m in tetanic response to pulse trains with similar stimulation parameters. These results suggest that this nerve-cuff electrode can be an effective and chronically stable tool for selectively stimulating distal nerve branches in the lower extremities for neuroprosthetic applications.

Mouse Latissimus Dorsi as a model system for evaluating tissue engineered skeletal muscle

The inability to engineer clinically relevant functional muscle tissues in vitro remains a major obstacle to the development of successful procedures for skeletal muscle (SKM) replacement in vivo. The goal of these studies is to further develop the technologies required for the creation of skeletal muscle biomimetics that can be used clinically for restoration of muscle function in vivo. As a first step in this direction we have examined the rodent latissimus dorsi (LD) as a model system for testing functional replacement of native SKM with tissue engineered SKM constructs (TE‐SKM). Porcine bladder acellular matrix was prepared and rat muscle precursor cells were seeded onto the matrix and preconditioned in a custom designed, computer controlled cyclic strain bioreactor for one week prior to subcutaneous implantation in nu/nu mice onto LD for 1 month. The size of the TE‐SKM was 15 mm X 3mm X 0.35 mm; dimensions similar to that of the native mouse LD. TE‐SKM isolated after one month implantation showed a maximal isometric tetanic response of 42.9 mN/mm2 which was 26 % of that of native LD isolated from contra lateral side, but 50‐ and 3500‐fold greater than that previously observed for TE‐SKM. To our knowledge, this is the largest specific force yet recorded for TE‐SKM on a decellularized matrix. In short, the LD appears to be an attractive model for further studies of functional SKM replacement with TE‐SKM constructs.

Functional Identification Of Fiber Types In Enzymatically Dissociated Murine Flexor Digitorum Brevis (FDB) And Soleus Muscles

Enzymatically dissociated FDB and soleus fibers from mouse were used to compare the kinetics of electrically elicited Ca2+ transients of slow and fast skeletal muscle fibers, using the fast Ca2+ dye MagFluo4-AM, at 20-22°C. For the case of FDB fibers we found two different morphologies for both single-twitch and tetanic Ca2+ transients named morphology type I (MT-I) and morphology type II (MT-II). The kinetic parameters (mean±s.e.m) of MT-I (11 fibers, 19%) and MT-II (47 fibers, 81%) single-twitch transients were: amplitude (ΔF/F): 0.36±0.03 vs. 0.69±0.03; rise time (ms): 1.44±0.15 vs. 1.01±0.02; half-amplitude width (ms): 10.25±0.92 vs. 3.87±0.12; decay time (ms): 46.15±1.99 vs. 21.08±0.89; and time constants of decay (τ1 and τ2, ms): 2.57±0.19 and 33.71±2.29 vs. 1.51±0.05 and 13.19±0.63, respectively; all differences being statistically significant (p 0.1) from those of MT-I FDB fibers. Tetanic responses (100 Hz) of MT-I FDB and soleus fibers showed a staircase shape while the time course of decay followed a single exponential (τ, ms): 73.36±6.82 for FDB and 74.59±6.24 for soleus (both n=8). In MT-II FDB tetani no staircase was present, the first peak was larger than the others, and the time course of decay was bi-exponential (Capote et al, J Physiol 2005;564:451). Histochemical and biochemical characterization of both muscles suggest that signals assigned MT-I correspond to slow type I and fast IIA fibers while those assigned MT-II correspond to fast IIX/D fibers. The results point to the importance of Ca2+ signaling for characterization of muscle fibers, but also to its possible role in determining fiber function. (FONACIT G-2001000637 and G-2005000372). JCC was supported by Universidad de Antioquia, Colombia.

The Absence of Utrophin Does Not Further the Impairment of Ca2+ Release Displayed by mdx Muscle

The double mutant utr-/-/mdx mouse has been postulated to be a better model of Duchenne Muscular Dystrophy than mdx mouse because it displays a progression of pathological features comparable to that in humans. We previously demonstrated that FDB fibers from mdx mice show limitations in action potential (AP) elicited Ca2+ release. Here we investigated the properties of the APs and the ability to release Ca2+ (30 mM [EGTA], 20°C) in response to single and trains of APs (20 pulses, at 33Hz and 100Hz) in FDB fibers isolated from control, mdx and utr-/-/mdx mice. Single APs of normal amplitude but longer duration were recorded in utr-/-/mdx fibers, whereas the amplitude of the Ca2+ release was ∼37% smaller than in normal fibers, but comparable with that found in mdx fibers. Fibers from the three strains sustained trains of Ca release at 33 Hz in which the amplitude of individual Ca2+ release transients decayed exponentially towards a sustained release amplitude with two time constants (τ1=10ms, τ2=200ms). In response to 100Hz trains, the amplitude of Ca2+ release in normal fibers decayed still with a double exponential (τ1=3.5ms, τ2=41ms) in which the amplitude of the 2nd and 20th transient along the train were ∼50% and ∼35% that of the first one, respectively. In contrast, fibers isolated from both mdx and utr-/-/mdx mice could be divided in two groups according to their tetanic response to 100Hz trains: approximately 75% of mdx and 33% of utr-/-/mdx fibers showed a behavior similar to that observed in normal fibers; in the remaining 25% of mdx and 67% of utr-/-/mdx fibers, respectively, the amplitude of the 2nd Ca2+ release transient was ∼20% of the first one, and this amplitude was sustained throughout the train.