Yoshiko Takagishi

Different presynaptic roles of synapsins at excitatory and inhibitory synapses.

The functions of synapsins were examined by characterizing the phenotype of mice in which all three synapsin genes were knocked out. Although these triple knock-out mice were viable and had normal brain anatomy, they exhibited a number of behavioral defects. Synaptic transmission was altered in cultured neurons from the hippocampus of knock-out mice. At excitatory synapses, loss of synapsins did not affect basal transmission evoked by single stimuli but caused a threefold increase in the rate of synaptic depression during trains of stimuli. This suggests that synapsins regulate the reserve pool of synaptic vesicles. This possibility was examined further by measuring synaptic vesicle density in living neurons transfected with green fluorescent protein-tagged synaptobrevin 2, a marker of synaptic vesicles. The relative amount of fluorescent synaptobrevin was substantially lower at synapses of knock-out neurons than of wild-type neurons. Electron microscopy also revealed a parallel reduction in the number of vesicles in the reserve pool of vesicles >150 nm away from the active zone at excitatory synapses. Thus, synapsins are required for maintaining vesicles in the reserve pool at excitatory synapses. In contrast, basal transmission at inhibitory synapses was reduced by loss of synapsins, but the kinetics of synaptic depression were unaffected. In these terminals, there was a mild reduction in the total number of synaptic vesicles, but this was not restricted to the reserve pool of vesicles. Thus, synapsins maintain the reserve pool of glutamatergic vesicles but regulate the size of the readily releasable pool of GABAergic vesicles.

Local Calcium Release in Dendritic Spines Required for Long-Term Synaptic Depression

We have used rats and mice with mutations in myosin-Va to evaluate the range and function of IP3-mediated Ca2+ signaling in dendritic spines. In these mutants, the endoplasmic reticulum and its attendant IP3 receptors do not enter the postsynaptic spines of parallel fiber synapses on cerebellar Purkinje cells. Long-term synaptic depression (LTD) is absent at the parallel fiber synapses of the mutants, even though the structure and function of these synapses otherwise appear normal. This loss of LTD is associated with selective changes in IP3-mediated Ca2+ signaling in spines and can be rescued by photolysis of a caged Ca2+ compound. Our results reveal that IP3 must release Ca2+ locally in the dendritic spines to produce LTD and indicate that one function of dendritic spines is to target IP3-mediated Ca2+ release to the proper subcellular domain.

The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice ☆

The absence of smooth endoplasmic reticulum (SER) in the dendritic spine of Purkinje cells was found in dilute-lethal (dl) mouse cerebella as detected by immunohistochemistry using anti-inositol 1,4,5-triphosphate receptor antibody and electron microscopy. Since SER in the spine has been suggested to play a crucial role for synaptic regulation as an intracellular Ca2+ store (for reviews, see [Miller, R.J., Prog. Neurobiol., 37 (1991) 255-285: Simpson, P.B., Challiss, R.A.J. and Nahorski, S.R., Trends Neurosci., 18 (1995) 299-306]), a neurological defect, characterized by clonic convulsions with opisthotonus and ataxia, in the dilute-lethal mouse with homozygous trait may be attributable to the absence of SER in the dendritic spine of Purkinje cells.

Connexin45, a Major Connexin of the Rabbit Sinoatrial Node, Is Co-expressed with Connexin43 in a Restricted Zone at the Nodal-Crista Terminalis Border

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

Computer Three-Dimensional Reconstruction of the Sinoatrial Node

Background—There is an effort to build an anatomically and biophysically detailed virtual heart, and, although there are models for the atria and ventricles, there is no model for the sinoatrial node (SAN). For the SAN to show pacemaking and drive atrial muscle, theoretically, there should be a gradient in electrical coupling from the center to the periphery of the SAN and an interdigitation of SAN and atrial cells at the periphery. Any model should include such features. Methods and Results—Staining of rabbit SAN preparations for histology, middle neurofilament, atrial natriuretic peptide, and connexin (Cx) 43 revealed multiple cell types within and around the SAN (SAN and atrial cells, fibroblasts, and adipocytes). In contrast to atrial cells, all SAN cells expressed middle neurofilament (but not atrial natriuretic peptide) mRNA and protein. However, 2 distinct SAN cell types were observed: cells in the center (leading pacemaker site) were small, were organized in a mesh, and did not express Cx43. In contrast, cells in the periphery (exit pathway from the SAN) were large, were arranged predominantly in parallel, often expressed Cx43, and were mixed with atrial cells. An ≈2.5-million-element array model of the SAN and surrounding atrium, incorporating all cell types, was constructed. Conclusions—For the first time, a 3D anatomically detailed mathematical model of the SAN has been constructed, and this shows the presence of a specialized interface between the SAN and atrial muscle.

Remodeling of Gap Junctional Coupling in Hypertrophied Right Ventricles of Rats With Monocrotaline-Induced Pulmonary Hypertension

The present study investigates the remodeling of gap junctional organization in relation to changes in anisotropic conduction properties in hypertrophied right ventricles (RVs) of rats with monocrotaline (MCT)-induced pulmonary hypertension. In contrast to controls that showed immunolocalization of connexin43 (Cx43) labeling largely confined to the intercalated disks, RV myocytes from MCT-treated rats showed dispersion of Cx43 labeling over the entire cell surface. The disorganization of Cx43 labeling became more pronounced with the progression of hypertrophy. Desmoplakin remained localized to the intercalated disks, as in controls. In RV tissues, the proportion of Cx43 label at the intercalated disk progressively decreased. Quantitative analysis of en face views of intercalated disks revealed a significant decrease in the disk gap junctional density in RV tissues of MCT-treated rats (control, 0.18 versus MCT-treated, 0.14 at 2 weeks; control, 0.16 versus MCT-treated, 0.11 at 4 weeks). Conduction velocity in RVs parallel to the fiber orientation was significantly lower (30.2% [n=9]) in MCT-treated rats at 4 weeks than in control rats, whereas there was no significant difference observed in the conduction velocity across the fiber orientation between control and MCT-treated rats. The anisotropic ratio of MCT-treated rats (1.38+/-0.10) was significantly lower than that of control rats (1.98+/-0.12). These results suggest that RV hypertrophy induced by pressure overload is associated with both disorganization of gap junction distribution and alteration of anisotropic conduction properties.

Endoplasmic reticulum is missing in dendritic spines of Purkinje cells of the ataxic mutant rat.

Dilute-opisthotonus (dop) is a spontaneous ataxic mutation in the rat, regulated by an autosomal recessive gene. Immunohistochemical staining with anti-inositol 1,4,5-trisphosphate receptor antibody and electron microscopic examinations revealed that the endoplasmic reticulum in dendritic spines of Purkinje cell was missing in the ataxic rat. This could impair the intracellular signal transduction in the parallel fiber-Purkinje cell synapse, and be a cause of the severe ataxic movement.

Distribution of the muscarinic K+ channel proteins Kir3.1 and Kir3.4 in the ventricle, atrium, and sinoatrial node of heart.

The functionally important effects on the heart of ACh released from vagal nerves are principally mediated by the muscarinic K+ channel. The aim of this study was to determine the abundance and cellular location of the muscarinic K+ channel subunits Kir3.1 and Kir3.4 in different regions of heart. Western blotting showed a very low abundance of Kir3.1 in rat ventricle, although Kir3.1 was undetectable in guinea pig and ferret ventricle. Although immunofluorescence on tissue sections showed no labeling of Kir3.1 in rat, guinea pig, and ferret ventricle and Kir3.4 in rat ventricle, immunofluorescence on single ventricular cells from rat showed labeling in t-tubules of both Kir3.1 and Kir3.4. Kir3.1 was abundant in the atrium of the three species, as shown by Western blotting and immunofluorescence, and Kir3.4 was abundant in the atrium of rat, as shown by immunofluorescence. Immunofluorescence showed Kir3.1 expression in SA node from the three species and Kir3.4 expression in the SA node from rat. The muscarinic K+ channel is activated by ACh via the m2 muscarinic receptor and, in atrium and SA node from ferret, Kir3.1 labeling was co-localized with m2 muscarinic receptor labeling throughout the outer cell membrane.

Heterogeneous Expression of Ca2+ Handling Proteins in Rabbit Sinoatrial Node

We investigated the densities of the L-type Ca2+ current, iCa,L, and various Ca2+ handling proteins in rabbit sinoatrial (SA) node. The density of iCa,L, recorded with the whole-cell patch-clamp technique, varied widely in sinoatrial node cells. The density of iCa,L was significantly (p<0.001) correlated with cell capacitance (measure of cell size) and the density was greater in larger cells (likely to be from the periphery of the SA node) than in smaller cells (likely to be from the center of the SA node). Immunocytochemical labeling of the L-type Ca2+ channel, Na+-Ca2+ exchanger, sarcoplasmic reticulum Ca2+ release channel (RYR2), and sarcoplasmic reticulum Ca2+ pump (SERCA2) also varied widely in SA node cells. In all cases there was significantly (p<0.05) denser labeling of cells from the periphery of the SA node than of cells from the center. In contrast, immunocytochemical labeling of the Na+-K+ pump was similar in peripheral and central cells. We conclude that Ca2+ handling proteins are sparse and poorly organized in the center of the SA node (normally the leading pacemaker site), whereas they are more abundant in the periphery (at the border of the SA node with the surrounding atrial muscle).

Localization of myosin II and V isoforms in cultured rat sympathetic neurones and their potential involvement in presynaptic function

While vesicle transport is one of the principal functions of myosin motors in neurones, the role played by specific myosin subtypes in discrete vesicle trafficking is poorly understood. We conducted electrophysiological and morphological experiments to determine whether myosin isoforms II and V might be involved in the transport of small synaptic vesicles in presynaptic nerve terminals of a model cholinergic synapse. Electron microscopy revealed the presence of normal synaptic architecture and synaptic vesicle density in presynaptic terminals of cultured superior cervical ganglion neurones (SCGNs) from myosin Va null rats (dilute‐opisthotonus, dop). Similarly, electrophysiological analyses of synaptic transmission and synaptic vesicle cycling at paired SCGN synapses failed to uncover any significant differences in synaptic development and function between normal and dop rats. Immunocytochemistry and in situ localization of green fluorescent protein (GFP)‐fusion proteins in wild‐type synapses revealed that myosins IIB and Va were distributed throughout the cell soma and processes of SCGNs, while myosins IIA and Vb were not detected in SCGNs. Myosin Va was conspicuously absent in presynaptic nerve terminals, but myosin IIB alone was found to be expressed. Furthermore, synaptic transmission was inhibited by introduction of myosin IIB heavy chain fragments into presynaptic terminals of SCGNs. Together these results suggest that only myosin IIB isoform participates in vesicle trafficking in presynaptic nerve terminals of cultured SCGNs.