The functional network of human induced pluripotent stem cell (hiPSC)-derived neurons is a potentially powerful in vitro model for evaluating disease mechanisms and drug responses. However, the culture time required for the full functional maturation of individual neurons and networks is uncertain. We investigated the development of spontaneous electrophysiological activity and pharmacological responses for over 1 year in culture using multi-electrode arrays (MEAs). The complete maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists required 20–30 weeks. At this stage, neural networks also demonstrated epileptiform synchronized burst firing (SBF) in response to pro-convulsants and SBF suppression using clinical anti-epilepsy drugs. Our results reveal the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for mechanistic analyses and drug screening. However, developmental changes in electrophysiological and pharmacological properties indicate the necessity for the international standardization of culture and evaluation procedures.

The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes.

A new inbred strain JF1 (Japanese Fancy Mouse 1) was established from a strain of fancy mouse. Morphological and genetical analysis indicated that the mouse originated from the Japanese wild mouse, Mus musculus molossinus. JF1 has characteristic coat color, black spots on the white coat, with black eyes. The mutation appeared to be linked to an old mutation piebald (s). Characterization of the causative gene for piebald, endothelin receptor type B (ednrb), demonstrated that the allele in JF1 is same as that of classic piebald allele, suggesting an identical origin of these two mutants. Possibly, classic piebald mutation was introduced from the Japanese tame mouse, which was already reported at the end of the 1700s. We showed that JF1 is a useful strain for mapping of mutant genes on laboratory strains owing to a high level of polymorphisms in microsatellite markers between JF1 and laboratory strains. The clarified genotypes of JF1 for coat color are "aa BB CC DD ss".

]m reduction. Cytosolic Na ϩ concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal ⌬⌿ m and 7.6 mM at ⌬⌿m dissipation. We conclude that 1) the mitochondrial Ca 2ϩ uniporter accumulates Ca 2ϩ in a manner that is dependent on ⌬⌿m at the physiological range of [Ca 2ϩ ]c; 2) ⌬⌿m dissipation opens the mPTP and results in Ca 2ϩ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca 2ϩ from the matrix even after ⌬⌿m dissipation. permeability transition pore; Na ϩ /Ca 2ϩ exchange; depolarization; ischemia-reperfusion injury ACCUMULATING EVIDENCE REVEALS that mitochondria play primary roles in fatal cell damage during ischemia-reperfusion (33). Key events that occur during ischemia include cytosolic Ca 2ϩ elevation, ATP depletion, high P i concentration, depolarized membrane potential, and acidotic pH. On reperfusion and recovery of normal pH, a burst of reactive oxygen species occurs, mitochondrial Ca 2ϩ overload ensues, and these lead to opening of the mitochondrial permeability transition pore (mPTP; Refs. 8,12,34). Opening of the mPTP allows water and solutes Յ1,500 Da in size to enter the matrix and cause mitochondrial swelling, rupture of the outer mitochondrial membrane, and release of cytochrome c or apoptosis-inducing factor, which initiates apoptotic programmed cell death (12,24,34). Because previous studies (8,12,13) (10,11). Furthermore, recent studies (4,8,10,11,18) also suggest a possible contribution by the mPTP to Ca 2ϩ homeostasis in both the cytosol and mitochondria. Despite the considerable attention given to the pathophysiological significance of mitochondrial Ca 2ϩ , the regulation and/or modulation of mitochondrial Ca 2ϩ during pathophysiological conditions such as ischemia-reperfusion injury are unclear. In previous studies, information about mitochondrial Ca 2ϩ was obtained using isolated mitochondria, whereby the structural and functional properties of organelles were seriously affected, and other cellular architectures were separated from the mitochondria. In this study, we measured [Ca 2ϩ ] m in saponin-permeabilized rat ventricular myocytes and investigated how ⌬⌿ m depolarization affects [Ca 2ϩ ] m and mitochondrial Ca 2ϩ transport systems such as the Ca 2ϩ uniporter, the mPTP, and mitoNCX.