Itraconazole decreases left ventricular contractility in isolated rabbit heart: mechanism of action.

Itraconazole (ITZ) is an approved antifungal agent that carries a "black box warning" in its label regarding a risk of negative cardiac inotropy based on clinical findings. Since the mechanism of the negative inotropic effect is unknown, we performed a variety of preclinical and mechanistic studies to explore the pharmacological profile of ITZ and understand the negative inotropic mechanism. ITZ was evaluated in: (1) an isolated rabbit heart (IRH) preparation using Langendorff retrograde perfusion; (2) ion channel studies; (3) a rat heart mitochondrial function profiling screen; (4) a mitochondrial membrane potential (MMP) assay; (5) in vitro pharmacology profiling assays (148 receptors, ion channels, transporters, and enzymes); and (6) a kinase selectivity panel (451 kinases). In the IRH, ITZ decreased cardiac contractility (>30%) at 0.3µM, with increasing effect at higher concentrations, which indicated a direct negative inotropic effect upon the heart. It also decreased heart rate and coronary flow (≥1µM) and prolonged PR/QRS intervals (3µM). In mechanistic studies, ITZ inhibited the cardiac NaV channel (IC50: 4.2µM) and was devoid of any functional inhibitory effect at the remaining pharmacological targets. Lastly, ITZ did not affect MMP, nor interfere with mitochondrial enzymes or processes involved with fuel substrate utilization or energy formation. Overall, the cardiovascular and mechanistic data suggest that ITZ-induced negative inotropy is a direct effect on the heart, in addition, the potential involvement of mitochondria function and L-type Ca(2+) channels are eliminated. The exact mechanism underlying the negative inotropy is uncertain, and requires further study.

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

Assessment of sarcomere shortening and calcium transient in primary human and dog ventricular myocytes.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca2+ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca2+-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca2+-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (ß-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca2+sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

BeKm-1, a peptide inhibitor of human ether-a-go-go-related gene potassium currents, prolongs QTc intervals in isolated rabbit heart.

Drug-induced cardiac arrhythmia, specifically Torsades de pointes, is associated with QT/QTc interval prolongation, thus prolongation of the QT interval is considered as a biomarker for Torsades de pointes risk (N Engl J Med 350:1013-1022, 2004). Specific inhibition of human ether-a-go-go-related gene (hERG) potassium channels has been recognized as the main mechanism for QT prolongation (Cardiovasc Res 58:32-45, 2003). This mechanism has been demonstrated for a variety of small-molecule agents, which access the inner pore of the hERG channel preferentially from inside the cell. Peptide inhibitors of hERG, such as BeKm-1, interact with the extracellular amino acid residues close to the external pore region of the channel. In this study, the isolated rabbit heart was used to assess whether BeKm-1 could induce QTc prolongation like dofetilide and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide (E-4031). Five hearts were perfused with 10 and 100 nM BeKm-1 sequentially. ECG parameters and left ventricular contractility were measured with spontaneously beating hearts. Both concentrations of BeKm-1 prolonged QTc intervals significantly and concentration-dependently (4.7 and 16.3% at 10 and 100 nM, respectively). When evaluated for their inhibitory effect in a hERG functional assay, BeKm-1, dofetilide, and E-4031 caused QTc prolongation at concentrations that caused significant hERG channel inhibition. Lastly, two polyclonal anti-hERG antibodies were also assessed in the hERG channel assay and found to be devoid of any inhibitory effect. These results indicated that the isolated rabbit heart assay can be used to measure QTc changes caused by specific hERG inhibition by peptides that specifically block the external pore region of the channel.

Validation of using cardioplegic solutions for preserving cardiac function in isolated rabbit heart assays.

INTRODUCTION: Cardioplegic solutions were first developed to preserve heart function during cardiac surgeries and heart transplants but have application in the nonclinical setting. Due to lack of lab space in the vivarium, cardioplegic solution was used to conserve cardiac function for ex-vivo studies performed in a separate building. All studies in this report were conducted with isolated female rabbit hearts (IRHs) via retrograde perfusion using the Langendorff apparatus to investigate if cardioplegia usage affects cardiac function. METHODS: Cardioplegia was achieved with a hyperkalemia (27 mM KCL) solution kept at 4 °C. Cardiac function was assessed by measuring ECG parameters, left ventricular contractility, and coronary flow under constant perfusion pressure. IRHs were cannulated with Krebs Henseleit buffer (KH) either fresh or after cardioplegic solution storage (C-IRH). Three comparisons were performed with and without cardioplegia; (i) direct side-by side studies of cardiac function; (ii) pharmacological responses to typical ion channels blockers, dofetilide, flecainide, and diltiazem; (iii) retrospective evaluation of cardiac functions in a large sample of hearts. RESULTS: In the side-by-side comparisons, cardioplegia-stored IRHs (C-IRH; storage time 90 min) had similar electrocardiographic (ECG) and hemodynamic parameters to fresh-cannulated hearts with KH buffer (KH-IRH). In addition, responses to dofetilide, flecainide, and diltiazem, were similar for C-IRH and KH-IRH hearts. Over the years (2006-2011), baseline data was collected from 79 hearts without cardioplegia and 100 hearts with cardioplegia (C-IRH; storage time 15 min), which showed no meaningful differences in a retrospective analysis. DISCUSSION: Cardiac function was preserved after cardioplegic treatment, however, coronary flow rates were decreased (-19.3%) in C-IRH hearts which indicated an altered coronary vascular tone. In conclusion, storage in cardioplegic solution preserves rabbit cardiac function, a practice that enables heart tissues to be collected at one site (e.g., vivarium) and transported to a laboratory in a separate location.

Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk.

Omecamtiv mecarbil (OM) is a myosin activator (myotrope), developed as a potential therapeutic agent for heart failure with reduced ejection fraction. To characterize the potential pro-arrhythmic risk of this novel sarcomere activator, we evaluated OM in a series of International Conference on Harmonization S7B core and follow-up assays, including an in silico action potential (AP) model. OM was tested in: (i) hERG, Nav1.5 peak, and Cav1.2 channel assays; (ii) in silico computation in a human ventricular AP (hVAP) population model; (iii) AP recordings in canine cardiac Purkinje fibers (PF); and (iv) electrocardiography analysis in isolated rabbit hearts (IRHs). OM had low potency in the hERG (half-maximal inhibitory concentration [IC50 ] = 125.5 µM) and Nav1.5 and Cav1.2 assays (IC50  > 300 µM). These potency values were used as inputs to investigate the occurrence of repolarization abnormalities (biomarkers of pro-arrhythmia) in an hVAP model over a wide range of OM concentrations. The outcome of hVAP analysis indicated low pro-arrhythmia risk at OM concentration up to 30 µM (100-fold the effective free therapeutic plasma concentration). In the isolated canine PF assay, OM shortened AP duration (APD)60 and APD90 significantly from 3 to 30 µM. In perfused IRH, ventricular repolarization (corrected QT and corrected JT intervals) was decreased significantly at greater than or equal to 1 µM OM. In summary, the comprehensive proarrhythmic assessment in human and non-rodent cardiac models provided data indicative that OM did not delay ventricular repolarization at therapeutic relevant concentrations, consistent with clinical findings.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura-2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

Decreased contractility and altered responses to inotropic agents in myocytes from tachypacing-induced heart failure canines.

Contractility measurements using primary isolated cardiac myocytes (CM) have commonly been used in understanding the physiology and pharmacology of cellular mechanics. In the majority of studies, CM from healthy animals were used, and fewer studies were performed with CM from diseased hearts. To better understand the translational value of contractility on the cellular level of a diseased animal model, myocytes were isolated from left ventricles of a tachypacing-induced heart failure (HF) canine model, and their contractility was measured by recording sarcomere shortening using an image-based IonOptix video system. A side-by-side comparison study was performed in myocytes isolated from 13 normal and 5 tachypacing-induced HF canines by evaluating both basal contractility and pharmacological responses to inotropic agents with different mechanisms, including dobutamine, isoproterenol, milrinone, levosimendan, pimobendan, diltiazem, and flecainide. Myocytes isolated from HF canines exhibited compromised contractility at the sarcomere level in comparison to normal myocytes, specifically, HF myocytes have smaller sarcomere contraction amplitude, longer resting sarcomere length, slower velocity of contraction and relaxation. In addition, they have altered pharmacological responses compared to that of normal canines, with much less potent effects observed in the application of classic inotropic agents, such as isoproterenol, dobutamine, and milrinone. These results indicate that myocytes isolated from tachy-paced HF canines have altered physiological and pharmacological properties, which could be utilized for understanding pathophysiology and developing pharmacological interventions for HF.

Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists.

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents. Here, we have characterized CHO cells expressing human and rat TRPA1 driven by an inducible promoter. As reported previously, both human and rat TRPA1 are activated by AITC and inhibited by ruthenium red. We have also characterized noxious cold response of these cell lines and show that noxious cold activates both human and rat TRPA1. Further, we have used CHO cells expressing human TRPA1 to screen a small molecule compound library and discovered that 'trichloro(sulfanyl)ethyl benzamides' (AMG2504, AMG5445, AMG7160 and AMG9090) act as potent antagonists of human TRPA1 activated by AITC and noxious cold. However, trichloro(sulfanyl)ethyl benzamides' (TCEB compounds) displayed differential pharmacology at rat TRPA1. AMG2504 and AMG7160 marginally inhibited rat TRPA1 activation by AITC, whereas AMG5445 and AMG9090 acted as partial agonists. In summary, we conclude that both human and rat TRPA1 channels show similar AITC and noxious cold activation profiles, but TCEB compounds display species-specific differential pharmacology at TRPA1.

Oxytocin does not directly alter cardiac repolarization in rabbit or human cardiac myocytes.

Oxytocin, a nine amino acid peptide, is highly conserved in placental mammals, including humans. Oxytocin has a physiological role in parturition and parenteral administration of the synthetic peptide is used to induce labor and control postpartum hemorrhage. Endogenous levels of oxytocin before labor are ∼20 pg/mL, but pharmacological administration of the peptide can achieve levels of 110 pg/mL (0.1 nmol/L) following intravenous administration. Cardiac arrhythmia and premature ventricular contractions have been associated with oxytocin administration in addition to QTc interval prolongation. In the conscious rabbit model, intravenous oxytocin produced QT and QTc prolongation. The mechanism of oxytocin-induced QTc prolongation is uncertain but could be the result of indirect changes in autonomic nervous tone, or a direct effect on the duration of cardiomyocyte repolarization. The purpose of this study was to examine the ability of oxytocin to alter cardiac repolarization directly. Two conventional models were used: QTc interval evaluation in the isolated rabbit heart (IRH) and assessment of action potential duration (APD) in human ventricular myocytes (HVM). Oxytocin did not prolong QTc intervals in IRH or APD in HVM when tested at suprapharmacological concentrations, for example, up to 1 µmol/L. The results indicate that oxytocin has very low risk for eliciting QTc and APD prolongation directly, and infer that the QTc changes observed in vivo may be attributed to an indirect mechanism.

Human embryonic stem cell derived cardiac myocytes detect hERG-mediated repolarization effects, but not Nav1.5 induced depolarization delay.

INTRODUCTION: Cardiac safety is of paramount importance in contemporary drug development. Efficient and sensitive evaluation of cardiac safety in the research and development of new molecular agents begins with preclinical in-vitro models. A new model that is currently under evaluation is the human embryonic stem-cell derived cardiac myocytes (hESC-CM) (Peng, Lacerda, Kirsch, Brown, & Bruening-Wright, 2010). METHODS: hESC-CM were exposed in-vitro to 15 test compounds, and action potentials (AP) recorded with perforated patch-clamp technique to assess changes in AP duration (APD90) and upstroke velocity (Vmax). The test compounds included: 10 hERG channel, 4 Na⁺ channel, and 1 IKs channel inhibitors. For comparison purposes, the test compounds were evaluated in the isolated rabbit heart assay (IRH) to determine changes in conduction (QRS) and repolarization (QTc). Potency at hERG, NaV1.5 and IKs channel was also determined. RESULTS: For 7 of 10 hERG channel inhibitors, the potency values across the three functional assays were similar (≤5-fold). Three compounds (dofetilide, sertindole, and terfenadine) showed >10-fold discrepancy between hERG potency and inhibitory concentrations in the hESC-CM and IRH assays. Of the four Na⁺ channel inhibitors, only mexiletine exhibited similar potency values across the three assays (~3-fold); the others exhibited marked variation (>10-fold) in inhibitory potency. No effect on repolarization was observed in hESC-CM treated with a potent IKs blocker, but QTc prolongation was evident in the IRH. DISCUSSION: The functional data indicate that hESC-CM are sensitive for detecting repolarization delay induced by hERG channel blockade, and AP prolongation correlated with potency in the hERG channel and IRH assays. However, hESC-CM were less sensitive for detecting depolarizing delay by Na⁺ channel blockers, and unable to detect delayed repolarization caused by IKs blockade.

Pharmacological effects of nonselective and subtype-selective nicotinic acetylcholine receptor agonists in animal models of persistent pain.

Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain. Nonselective nAChR agonists ABT-594 and varenicline were effective analgesics. By contrast, the selective alpha4beta2 agonist ispronicline and a novel alpha4beta2-selective potentiator did not appear to produce analgesia in either model. alpha7-selective agonists reduced the pain-related endpoint, but the effect could be ascribed to nonspecific reduction of movement rather than to analgesia. Neither selective nor nonselective alpha7 nicotinic agonists affected the release of pro-inflammatory cytokines in response to antigen challenge. Electrophysiological recordings from spinal cord slice showed a strong nicotine-induced increase in inhibitory synaptic transmission that was mediated partially by alpha4beta2 and only minimally by alpha7 subtypes. Taken with previous studies, the results suggest that agonism of alpha4beta2 nAChRs is necessary but not sufficient to produce analgesia, and that the spinal cord is a key site where the molecular action of nAChRs produces analgesia.

Ethanol dual modulatory actions on spontaneous postsynaptic currents in spinal motoneurons.

Recently we have shown that acute ethanol (EtOH) exposure suppresses dorsal root-evoked synaptic potentials in spinal motoneurons. To examine the synaptic mechanisms underlying the reduced excitatory activity, EtOH actions on properties of action potential-independent miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were studied in spinal motoneurons of newborn rats. Properties of mEPSCs generated by activation of N-methyl-D-aspartate receptors (NMDARs) and non-NMDA receptors and of mIPSCs mediated by glycine and gamma-aminobutyric acid-A receptors (GlyR and GABA(A)R) were examined during acute exposure to 70 and 200 mM EtOH. In the presence of 70 mM EtOH, the frequency of NMDAR- and non-NMDAR-mediated mEPSCs decreased to 53 +/- 5 and 45 +/- 7% (means +/- SE) of control values, respectively. In contrast, the frequency of GlyR- and GABA(A)R-mediated mIPSCs increased to 138 +/- 15 and 167 +/- 23% of control, respectively. Based on the quantal theory of transmitter release, changes in the frequency of miniature currents are correlated with changes in transmitter release, suggesting that EtOH decreased presynaptic glutamate release and increased the release of both glycine and GABA. EtOH did not change the amplitude or rise and decay times of either mEPSCs or mIPSCs, indicating that the presynaptic changes were not associated with changes in the properties of postsynaptic receptors/channels. Acute exposure to 200 mM EtOH increased mIPSC frequency two- to threefold, significantly higher than the increase induced by 70 mM EtOH. However, the decrease in mEPSC frequency was similar to that observed in 70 mM EtOH. Those findings implied that the regulatory effect of EtOH on glycine and GABA release was dose-dependent. Exposure to the higher EtOH concentration had opposite actions on mEPSC and mIPSC amplitudes: it attenuated the amplitude of NMDAR- and non-NMDAR-mediated mEPSCs to ~80% of control and increased GlyR- and GABA(A)R-mediated mIPSC amplitude by ~20%. EtOH-induced changes in the amplitude of postsynaptic currents were not associated with changes in their basic kinetic properties. Our data suggested that in spinal networks of newborn rats, EtOH was more effective in modulating the release of excitatory and inhibitory neurotransmitters than changing the properties of their receptors/channels.

Interactions between multiple rhythm generators produce complex patterns of oscillation in the developing rat spinal cord.

Neural networks capable of generating coordinated rhythmic activity form at early stages of development in the spinal cord. In this study, voltage-imaging techniques were used to examine the spatiotemporal pattern of rhythmic activity in transverse slices of lumbar spinal cord from embryonic and neonatal rats. Real-time images were recorded in slices stained with the voltage-sensitive fluorescent dye RH414 using a 464-element photodiode array. Fluorescence signals showed spontaneous voltage oscillations with a frequency of 3 Hz. Simultaneous recordings of fluorescence and extracellular field potential demonstrated that the two signals oscillated with the same frequency and had a distinct phase relationship, indicating that the fluorescence changes represented changes in transmembrane potentials. The oscillations were reversibly blocked by cobalt (1 mM), indicating a dependence on Ca(2+) influx through voltage-gated Ca(2+) channels. Extracellular field potentials revealed oscillations with the same frequency in both stained and unstained slices. Oscillations were apparent throughout a slice, although their amplitudes varied in different regions. The largest amplitude oscillations were produced in the lateral regions. To examine the spatial organization of rhythm-generating networks, slices were cut into halves and quarters. Each fragment continued to oscillate with the same frequency as intact slices but with smaller amplitudes. This finding suggested that rhythm-generating networks were widely distributed and did not depend on long-range projections. In slices from neonatal rats, the oscillations exhibited a complex spatiotemporal pattern, with depolarizations alternating between mirror locations in the right and left sides of the cord. Furthermore, within each side depolarizations alternated between the lateral and medial regions. This medial-lateral pattern was preserved in hemisected slices, indicating that pathways intrinsic to each side coordinated this activity. A different pattern of oscillation was observed in slices from embryos with synchronous 3-Hz oscillations occurring in limited regions. Our study demonstrated that rhythm generators were distributed throughout transverse sections of the lumbar spinal cord and exhibited a complex spatiotemporal pattern of coordinated rhythmic activity.