Different mechanisms of synapsin-induced vesicle clustering at inhibitory and excitatory synapses.

Synapsins cluster synaptic vesicles (SVs) to provide a reserve pool (RP) of SVs that maintains synaptic transmission during sustained activity. However, it is unclear how synapsins cluster SVs. Here we show that either liquid-liquid phase separation (LLPS) or tetramerization-dependent cross-linking can cluster SVs, depending on whether a synapse is excitatory or inhibitory. Cell-free reconstitution reveals that both mechanisms can cluster SVs, with tetramerization being more effective. At inhibitory synapses, perturbing synapsin-dependent LLPS impairs SV clustering and synchronization of gamma-aminobutyric acid (GABA) release, while preventing synapsin tetramerization does not. At glutamatergic synapses, the opposite is true: synapsin tetramerization enhances clustering of glutamatergic SVs and mobilization of these SVs from the RP, while synapsin LLPS does not. Comparison of inhibitory and excitatory transmission during prolonged synaptic activity reveals that synapsin LLPS serves as a brake to limit GABA release, while synapsin tetramerization enables rapid mobilization of SVs from the RP to sustain glutamate release.

Novel luciferase-opsin combinations for improved luminopsins.

Previous work has demonstrated that fusion of a luciferase to an opsin, to create a luminescent opsin or luminopsin, provides a genetically encoded means of manipulating neuronal activity via both chemogenetic and optogenetic approaches. Here we have expanded and refined the versatility of luminopsin tools by fusing an alternative luciferase variant with high light emission, Gaussia luciferase mutant GLucM23, to depolarizing and hyperpolarizing channelrhodopsins with increased light sensitivity. The combination of GLucM23 with Volvox channelrhodopsin-1 produced LMO4, while combining GLucM23 with the anion channelrhodopsin iChloC yielded iLMO4. We found efficient activation of these channelrhodopsins in the presence of the luciferase substrate, as indicated by responses measured in both single neurons and in neuronal populations of mice and rats, as well as by changes in male rat behavior during amphetamine-induced rotations. We conclude that these new luminopsins will be useful for bimodal opto- and chemogenetic analyses of brain function.

Author Correction: Structural basis for delta cell paracrine regulation in pancreatic islets.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis for delta cell paracrine regulation in pancreatic islets.

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.

Src-mediated phosphorylation of ßPix-b regulates dendritic spine morphogenesis.

PAK-interacting guanine nucleotide exchange factor (ßPix; also known as Arhgef7) has been implicated in many actin-based cellular processes, including spine morphogenesis in neurons. However, the molecular mechanisms by which ßPix controls spine morphology remain elusive. Previously, we have reported the expression of several alternative spliced ßPix isoforms in the brain. Here, we report a novel finding that the b isoform of ßPix (ßPix-b) mediates the regulation of spine and synapse formation. We found that ßPix-b, which is mainly expressed in neurons, enhances spine and synapse formation through preferential localization at spines. In neurons, glutamate treatment efficiently stimulates Rac1 GEF activity of ßPix-b. The glutamate stimulation also promotes Src-mediated phosphorylation of ßPix-b in both an AMPA receptor- and NMDA receptor-dependent manner. Tyrosine 598 (Y598) of ßPix-b is identified as the major Src-mediated phosphorylation site. Finally, Y598 phosphorylation of ßPix-b enhances its Rac1 GEF activity that is critical for spine and synapse formation. In conclusion, we provide a novel mechanism by which ßPix-b regulates activity-dependent spinogenesis and synaptogenesis via Src-mediated phosphorylation.

Molecular Mechanisms of Short-Term Plasticity: Role of Synapsin Phosphorylation in Augmentation and Potentiation of Spontaneous Glutamate Release.

We used genetic and pharmacological approaches to identify the signaling pathways involved in augmentation and potentiation, two forms of activity dependent, short-term synaptic plasticity that enhance neurotransmitter release. Trains of presynaptic action potentials produced a robust increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). Following the end of the stimulus, mEPSC frequency followed a bi-exponential decay back to basal levels. The time constants of decay identified these two exponential components as the decay of augmentation and potentiation, respectively. Augmentation increased mEPSC frequency by 9.3-fold, while potentiation increased mEPSC frequency by 2.4-fold. In synapsin triple-knockout (TKO) neurons, augmentation was reduced by 83% and potentiation was reduced by 74%, suggesting that synapsins are key signaling elements in both forms of plasticity. To examine the synapsin isoforms involved, we expressed individual synapsin isoforms in TKO neurons. While synapsin IIIa rescued both augmentation and potentiation, none of the other synapsin isoforms produced statistically significant amounts of rescue. To determine the involvement of protein kinases in these two forms of short-term plasticity, we examined the effects of inhibitors of protein kinases A (PKA) and C (PKC). While inhibition of PKC had little effect, PKA inhibition reduced augmentation by 76% and potentiation by 60%. Further, elevation of intracellular cAMP concentration, by either forskolin or IBMX, greatly increased mEPSC frequency and occluded the amount of augmentation and potentiation evoked by electrical stimulation. Finally, mutating a PKA phosphorylation site to non-phosphorylatable alanine largely abolished the ability of synapsin IIIa to rescue both augmentation and potentiation. Together, these results indicate that PKA activation is required for both augmentation and potentiation of spontaneous neurotransmitter release and that PKA-mediated phosphorylation of synapsin IIIa underlies both forms of presynaptic short-term plasticity.

Calcium-Dependent and Synapsin-Dependent Pathways for the Presynaptic Actions of BDNF.

We used cultured hippocampal neurons to determine the signaling pathways mediating brain-derived neurotrophic factor (BDNF) regulation of spontaneous glutamate and GABA release. BDNF treatment elevated calcium concentration in presynaptic terminals; this calcium signal reached a peak within 1 min and declined in the sustained presence of BDNF. This BDNF-induced transient rise in presynaptic calcium was reduced by SKF96365, indicating that BDNF causes presynaptic calcium influx via TRPC channels. BDNF treatment increased the frequency of miniature excitatory postsynaptic currents (mEPSCs). This response consisted of two components: a transient component that peaked within 1 min of initiating BDNF application and a second component that was sustained, at a lower mEPSC frequency, for the duration of BDNF application. The initial transient component was greatly reduced by removing external calcium or by treatment with SKF96365, as well as by Pyr3, a selective blocker of TRPC3 channels. In contrast, the sustained component was unaffected in these conditions but was eliminated by U0126, an inhibitor of the MAP kinase (MAPK) pathway, as well as by genetic deletion of synapsins in neurons from a synapsin triple knock-out (TKO) mouse. Thus, two pathways mediate the ability of BDNF to enhance spontaneous glutamate release: the transient component arises from calcium influx through TRPC3 channels, while the sustained component is mediated by MAPK phosphorylation of synapsins. We also examined the ability of these two BDNF-dependent pathways to regulate spontaneous release of the inhibitory neurotransmitter, GABA. BDNF had no effect on the frequency of spontaneous miniature inhibitory postsynaptic currents (mIPSCs) in neurons from wild-type (WT) mice, but surprisingly did increase mIPSC frequency in synapsin TKO mice. This covert BDNF response was blocked by removal of external calcium or by treatment with SKF96365 or Pyr3, indicating that it results from calcium influx mediated by TRPC3 channels. Thus, the BDNF-activated calcium signaling pathway can also enhance spontaneous GABA release, though this effect is suppressed by synapsins under normal physiological conditions.

Synapsin Isoforms Regulating GABA Release from Hippocampal Interneurons.

UNLABELLED: Although synapsins regulate GABA release, it is unclear which synapsin isoforms are involved. We identified the synapsin isoforms that regulate GABA release via rescue experiments in cultured hippocampal neurons from synapsin I, II, and III triple knock-out (TKO) mice. In situ hybridization indicated that five different synapsin isoforms are expressed in hippocampal interneurons. Evoked IPSC amplitude was reduced in TKO neurons compared with triple wild-type neurons and was rescued by introducing any of the five synapsin isoforms. This contrasts with hippocampal glutamatergic terminals, where only synapsin IIa rescues the TKO phenotype. Deconvolution analysis indicated that the duration of GABA release was prolonged in TKO neurons and this defect in release kinetics was rescued by each synapsin isoform, aside from synapsin IIIa. Because release kinetics remained slow, whereas peak release rate was rescued, there was a 2-fold increase in GABA release in TKO neurons expressing synapsin IIIa. TKO neurons expressing individual synapsin isoforms showed normal depression kinetics aside from more rapid depression in neurons expressing synapsin IIIa. Measurements of the cumulative amount of GABA released during repetitive stimulation revealed that the rate of mobilization of vesicles from the reserve pool to the readily releasable pool and the size of the readily releasable pool of GABAergic vesicles were unaffected by synapsins. Instead, synapsins regulate release of GABA from the readily releasable pool, with all isoforms aside from synapsin IIIa controlling release synchrony. These results indicate that synapsins play fundamentally distinct roles at different types of presynaptic terminals. SIGNIFICANCE STATEMENT: Synapsins are a family of proteins that regulate synaptic vesicle (SV) trafficking within nerve terminals. Here, we demonstrate that release of the inhibitory neurotransmitter GABA is supported by many different synapsin types. This contrasts with the release of other neurotransmitters, which typically is supported by only one type of synapsin. We also found that synapsins serve to synchronize the release of GABA in response to presynaptic action potentials, which is different from the synapsin-dependent trafficking of SVs in other nerve terminals. Our results establish that different synapsins play fundamentally different roles at nerve terminals releasing different types of neurotransmitters. This is an important clue to understanding how neurons release their neurotransmitters, a process essential for normal brain function.

Synapsin Isoforms and Synaptic Vesicle Trafficking.

Synapsins were the first presynaptic proteins identified and have served as the flagship of the presynaptic protein field. Here we review recent studies demonstrating that different members of the synapsin family play different roles at presynaptic terminals employing different types of synaptic vesicles. The structural underpinnings for these functions are just beginning to be understood and should provide a focus for future efforts.

Anti-HIV protease activity from rosa family plant extracts and rosamultin from Rosa rugosa.

To identify substances with anti-human immunodeficiency virus (HIV) activity from plant sources, 12 extracts of Rosa family plants were screened for their inhibitory effects against HIV-1 protease. Of the extracts tested, the strongest inhibitory effects were observed in the root of Rosa rugosa and the leaves of Prunus sargentii, at a concentration of 100 microg/mL. Rosamultin isolated from the root of R. rugosa inhibited HIV-1 protease by 53% at a concentration of 100 microM.

The topographical guidance of neurons cultured on holographic photo-responsive polymer.

Neuronal cells to respond to submicron-scale groove structure. On the grooved structure of particular dimension, it has been reported that neuronal cells grew perpendicular to the groove direction. We used holographic photo-responsive polymer to form a submicron-scale surface relief grating structure. A sinusoidal groove pattern is built up by holographic interference of 488 nm Ar ion laser beams. The primary hippocampal neurons cultured on the surface of the polymer film grew extending their neurites in a perpendicular orientation to the groove direction. This suggests that laser holography can be used to control the neurites orientation and growth. The holographic grating and photo-responsive polymer will raise the possibility of controlling neural network formation between living cells by light.

Solitary Senescent Osteochondroma of the Sacrum Producing Sciatica: A Case Report / 대한정형외과학회잡지

Benign solitary osteochondroma is uncommon in the vertebra (2%). Vertebral ostoechondroma arises predominantly in the lumbar and cervical regions, and rarely in the sacrum. We describe a case of a sacral solitary senescent osteochondroma compressing the sciatic nerve, producing sciatica. The tumor was removed by posterior paramedian incision. The excised mass was cylindrical, measuring 3.5x1x1 cm in size and consisting of lamellar bone with Haversian system similar to the architecture of normal cortical bone and trabecular bone.