Cellular identity and Ca2+ signaling activity of the non-reproductive GnRH system in the Ciona intestinalis type A (Ciona robusta) larva.

Tunicate larvae have a non-reproductive gonadotropin-releasing hormone (GnRH) system with multiple ligands and receptor heterodimerization enabling complex regulation. In Ciona intestinalis type A larvae, one of the gnrh genes, gnrh2, is conspicuously expressed in the motor ganglion and nerve cord, which are homologous structures to the hindbrain and spinal cord, respectively, of vertebrates. The gnrh2 gene is also expressed in the proto-placodal sensory neurons, which are the proposed homologue of vertebrate olfactory neurons. Tunicate larvae occupy a non-reproductive dispersal stage, yet the role of their GnRH system remains elusive. In this study, we investigated neuronal types of gnrh2-expressing cells in Ciona larvae and visualized the activity of these cells by fluorescence imaging using a calcium sensor protein. Some cholinergic neurons and dopaminergic cells express gnrh2, suggesting that GnRH plays a role in controlling swimming behavior. However, none of the gnrh2-expressing cells overlap with glycinergic or GABAergic neurons. A role in motor control is also suggested by a relationship between the activity of gnrh2-expressing cells and tail movements. Interestingly, gnrh2-positive ependymal cells in the nerve cord, known as a kind of glia cells, actively produced Ca2+ transients, suggesting that active intercellular signaling occurs in the glia cells of the nerve cord.

Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.

Induced pluripotent stem cells (iPSCs) constitute a potential source of autologous patient-specific cardiomyocytes for cardiac repair, providing a major benefit over other sources of cells in terms of immune rejection. However, autologous transplantation has substantial challenges related to manufacturing and regulation. Although major histocompatibility complex (MHC)-matched allogeneic transplantation is a promising alternative strategy, few immunological studies have been carried out with iPSCs. Here we describe an allogeneic transplantation model established using the cynomolgus monkey (Macaca fascicularis), the MHC structure of which is identical to that of humans. Fibroblast-derived iPSCs were generated from a MHC haplotype (HT4) homozygous animal and subsequently differentiated into cardiomyocytes (iPSC-CMs). Five HT4 heterozygous monkeys were subjected to myocardial infarction followed by direct intra-myocardial injection of iPSC-CMs. The grafted cardiomyocytes survived for 12 weeks with no evidence of immune rejection in monkeys treated with clinically relevant doses of methylprednisolone and tacrolimus, and showed electrical coupling with host cardiomyocytes as assessed by use of the fluorescent calcium indicator G-CaMP7.09. Additionally, transplantation of the iPSC-CMs improved cardiac contractile function at 4 and 12 weeks after transplantation; however, the incidence of ventricular tachycardia was transiently, but significantly, increased when compared to vehicle-treated controls. Collectively, our data demonstrate that allogeneic iPSC-CM transplantation is sufficient to regenerate the infarcted non-human primate heart; however, further research to control post-transplant arrhythmias is necessary.

LIS1-dependent retrograde translocation of excitatory synapses in developing interneuron dendrites.

Synaptic remodelling coordinated with dendritic growth is essential for proper development of neural connections. After establishment of synaptic contacts, synaptic junctions are thought to become stationary and provide fixed anchoring points for further dendritic growth. However, the possibility of active translocation of synapses along dendritic protrusions, to guide the proper arrangement of synaptic distribution, has not yet been fully investigated. Here we show that immature dendrites of γ-aminobutyric acid-positive interneurons form long protrusions and that these protrusions serve as conduits for retrograde translocation of synaptic contacts to the parental dendrites. This translocation process is dependent on microtubules and the activity of LIS1, an essential regulator of dynein-mediated motility. Suppression of this retrograde translocation results in disorganized synaptic patterns on interneuron dendrites. Taken together, these findings suggest the existence of an active microtubule-dependent mechanism for synaptic translocation that helps in the establishment of proper synaptic distribution on dendrites.

Both 5-hydroxytryptamine 5-HT2A and 5-HT1B receptors are involved in the vasoconstrictor response to 5-HT in the human isolated internal thoracic artery.

1. The 5-hydroxytryptamine (5-HT, serotonin) receptor subtypes that mediate vasoconstriction in the human internal thoracic artery (ITA), which is frequently used as an arterial graft, remain unclear. The aim of the present study was to elucidate the 5-HT receptor subtypes responsible for 5-HT-induced contraction of the human ITA. 2. The contractile responses to 5-HT of endothelium-denuded human ITA obtained from patients undergoing coronary bypass surgery were examined. In addition, we investigated the effects of sarpogrelate and SB224289, antagonists of 5-HT(2A) and 5-HT(1B) receptors, respectively, on the 5-HT-induced vasoconstriction. Finally, 5-HT(2A) and 5-HT(1B) receptors in the human ITA were immunolabelled. 3. 5-Hydroxytryptamine (1 nmol/L-10 micromol/L) caused vasoconstriction in a concentration-dependent manner. Both sarpogrelate (1 micromol/L) and SB224289 (1 micromol/L) significantly, but not completely, inhibited 5-HT-induced vasoconstriction. 4. Conversely, simultaneous pretreatment with supramaximum concentrations (1 micromol/L for both) of sarpogrelate and SB224289 almost completely inhibited the 5-HT-induced vasoconstriction. 5. Immunopositive staining for 5-HT(2A) and 5-HT(1B) receptors was detected in smooth muscle cells of the human ITA. 6. These results demonstrate that, in human ITA, 5-HT-induced vasoconstriction is mediated by activation of both 5-HT(2A) and 5-HT(1B) receptors. Thus, when the human ITA is used as an arterial graft, a combination of 5-HT(2A) and 5-HT(1B) receptor antagonists would appear to be most useful to prevent 5-HT-induced vasospasm.

Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor.

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent "junctional" sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca(2+) signaling complexes in many cell types. We examined the possibility that some Ca(2+) signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca(2+) sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the alpha1 or alpha2 isoform of the Na(+) pump catalytic (alpha) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that alpha2(f)GCaMP2, like native Na(+) pumps with alpha2-subunits, sorted to PM domains that colocalized with subjacent S/ER; alpha1(f)GCaMP2, like Na(+) pumps with alpha1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca(2+) signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca(2+) channel-mediated Ca(2+) entry after S/ER unloading with cyclopiazonic acid (in Ca(2+)-free medium). When cytosolic Ca(2+) diffusion was markedly restricted with EGTA, however, only alpha2(f)GCaMP2 detected the local, store-operated Ca(2+) channel-mediated Ca(2+) entry signal. Thus, alpha1 Na(+) pumps are apparently excluded from the PM microdomains occupied by alpha2 Na(2+) pumps. The jS/ER and adjacent PM may communicate by Ca(2+) signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca(2+) signals in other subPM microdomains and signals associated with other organelles.