KRAP tethers IP3 receptors to actin and licenses them to evoke cytosolic Ca2+ signals

Regulation of IP3 receptors (IP3Rs) by IP3 and Ca2+ allows regenerative Ca2+ signals, the smallest being Ca2+ puffs, which arise from coordinated openings of a few clustered IP3Rs. Cells express thousands of mostly mobile IP3Rs, yet Ca2+ puffs occur at a few immobile IP3R clusters. By imaging cells with endogenous IP3Rs tagged with EGFP, we show that KRas-induced actin-interacting protein (KRAP) tethers IP3Rs to actin beneath the plasma membrane. Loss of KRAP abolishes Ca2+ puffs and the global increases in cytosolic Ca2+ concentration evoked by more intense stimulation. Over-expressing KRAP immobilizes additional IP3R clusters and results in more Ca2+ puffs and larger global Ca2+ signals. Endogenous KRAP determines which IP3Rs will respond: it tethers IP3R clusters to actin alongside sites where store-operated Ca2+ entry occurs, licenses IP3Rs to evoke Ca2+ puffs and global cytosolic Ca2+ signals, implicates the actin cytoskeleton in IP3R regulation and may allow local activation of Ca2+ entry.

A positive feedback loop between Flower and PI(4,5)P2 at periactive zones controls bulk endocytosis in Drosophila

Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) compartmentalization governs control of these processes in Drosophila. Strong stimuli trigger PI(4,5)P2 microdomain formation at periactive zones. Upon exocytosis, Flower translocates from SVs to periactive zones, where it increases PI(4,5)P2 levels via Ca2+ influxes. Remarkably, PI(4,5)P2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PI(4,5)P2 microdomain compartmentalization. PI(4,5)P2 microdomains drive ADBE and SV reformation from bulk endosomes. PI(4,5)P2 further retrieves Flower to bulk endosomes, terminating endocytosis. We propose that the interplay between Flower and PI(4,5)P2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation.

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The “handshake” that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca2+chelators. Using hair bundles from the rat’s cochlea and the bullfrog’s sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca2+chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca2+chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca2+concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.

Comparative effects of cantharidin and endothall on gene expression and activity of antioxidant enzymes in Cichorium intybus L.

<p>Cantharidin and its analog, endothall, are known to have phytotoxic effects and their mechanism of action involves the inhibition of phosphatases. Enzymes and antioxidant compounds act as barriers against phytotoxic compounds. Catalase and peroxidase are among the most important antioxidant enzymes. <em>Cichorium intybus </em>L. has traditionally been used for its medicinal properties and contains various phytochemical and enzymatic compounds. The present study aimed to investigate the comparative effects of cantharidin and endothall with concentration of 2.5, 5.5 and 10 µg ml^-1 on the changes in the gene expression of catalase and glutathione peroxidase. Furthermore, we assess the activities of these enzymes in the shoots and roots of <em>Cichorium intybus</em> L.. According to the findings, the expression of catalase and glutathione peroxidase increased in the samples treated with cantharidin more than endothall compared to the controls in both shoot (the most significant is in cantharidin with 2.5 µg ml^-1concentration) and root samples (the most significant is in cantharidin with 5.5 µg ml^-1concentration). In addition, the activity of catalase and concentrations of cantharidin (2.5 µg ml^-1) in shoot samples led to the more intense stimulation of catalase and glutathione peroxidase compared to root samples. We suggest that cantharidin and endothall have negative effect on expression and absorption of antioxidant enzymes.</p>

SAY NO to mild ovarian stimulation for all poor responders: it is time to realize that not all poor responders are the same

ABSTRACT Over the last 25 years, a vast body of literature has been published evaluating different treatment modalities for the management of poor ovarian responders. Despite the evidence that maximizing ovarian response can improve the chances of live born babies in poor responders, there are still voices suggesting that all poor responders are the same, irrespective of their age and their actual ovarian reserve. This has resulted in the suggestion of adopting a mild ovarian stimulation approach for all poor responders, based on the results of several trials which failed to identity differences when comparing mild and more intense stimulation in predicted poor responders. The current article analyzes in detail these studies and discusses the shortcomings in terms of type of population included, outcomes and settings performed, which may actually be responsible for the belief that only mild stimulation should be used. In the era of individualization in medicine, it must be realized that there are subgroups of predicted poor responders who will benefit from an individual rather than ‘one fits all’ mild stimulation approach and thus we should provide the same standard of treatment for all our poor responder patients.

Flower initiates a positive feedback loop upon PIP2 enrichment at periactive zones to control bulk endocytosis

Synaptic vesicle (SV) endocytosis is coupled to exocytosis to maintain SV pool size and thus neurotransmitter release. Intense stimulation induces activity-dependent bulk endocytosis (ADBE) to recapture large quantities of SV constituents in large endosomes from which SVs reform. How these consecutive processes are spatiotemporally coordinated remains unknown. Here, we show that the Flower Ca2+ channel-dependent phosphatidylinositol 4,5-bisphosphate (PIP2) compartmentalization governs such control. Strong stimuli trigger PIP2 microdomain formation at periactive zones. Upon exocytosis Flower translocates from SVs to periactive zones, where it increases PIP2 levels via Ca2+ influxes. Remarkably, PIP2 directly enhances Flower channel activity, thereby establishing a positive feedback loop for PIP2 microdomain compartmentalization. The PIP2 microdomains drive ADBE and SV reformation from bulk endosomes. PIP2 further sorts Flower to bulk endosomes, thereby terminating endocytosis. Hence, we propose that the interplay between Flower and PIP2 is the crucial spatiotemporal cue that couples exocytosis to ADBE and subsequent SV reformation.

Can transcranial electric stimulation with multiple electrodes reach deep targets?

To reach a deep target in the brain with transcranial electric stimulation (TES), currents have to pass also through the cortical surface. Thus, it is generally thought that TES cannot achieve focal deep brain stimulation. Recent efforts with interfering waveforms and pulsed stimulation have argued that one can achieve deeper or more intense stimulation in the brain. Here we argue that conventional transcranial stimulation with multiple current sources is just as effective as these new approaches. The conventional multi-electrode approach can be numerically optimized to maximize intensity or focality at a desired target location. Using such optimal electrode configurations we find in a detailed and realistic head model that deep targets may in fact be strongly stimulated, with cerebrospinal fluid guiding currents deep into the brain.

A Three-Pool Model Dissecting Readily Releasable Pool Replenishment at the Calyx of Held

Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP) and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

Acute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation

Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutant (A53T) with a properly folded NTD phenocopied the synaptic vesicle recycling defects observed with wild type. Interestingly, the vesicle recycling defects were not observed when the stimulation frequency was reduced (5 Hz). Thus excess α-synuclein impairs synaptic vesicle recycling evoked during intense stimulation via a mechanism that requires a properly folded N-terminal α-helix.

Abstract 18368: Evidence for Activation of Cerebral Autonomic Center in Response to Cardiac Electrical Stimulation in Humans -A New Finding of Cardio-Cerebral Connection

Introduction: Anterior cingulate cortex (ACC) is one of the cortical receptive fields of afferent nerves innervating the visceral organs and modulates autonomic nervous system in collaboration with hypothalamus and brainstem. Cardiac afferent nerves are associated with not only chest pain but also sympathetic arousal in heart failure, in which ACC could be involved. However, the cortical receptive fields of cardiac afferent nerves still remain to be elucidated. Hypothesis: We thus investigated whether ACC is activated in response to cardiac electrical stimulation in humans. Methods: We studied 10 patients (74.7±1.9 yrs, M/F 9/1) with cardiac pacemaker implantation. Before the measurement, mode of cardiac pacemaker was changed to VVI 80-90 bpm with 1.5V intensity. Cerebral blood flow (CBF) was measured using [15O]H2O positron emission tomography (PET) during sham stimulation (1.5V) and intense stimulation (7.5-8V). The CBF images of intense stimulation were compared with those of sham stimulation using SPM8, a common analysis tool for neuroimages. Cardiac sensation was evaluated with an ordinate scale after CBF measurements. Blood samples were obtained from the cubital vein before and after CBF measurements for plasma catecholamine levels (one patient was excluded for the analysis due to urination before the last blood sampling). Results: Intense stimulation did not induce cardiac sensation but significantly increased plasma adrenaline levels as compared with sham stimulation (intense, 6.1±1.8 vs. sham, 0.1±3.0 pg/ml, P=0.031, n=9 each). CBF in ACC was significantly increased in response to intense stimulation as compared with sham stimulation (intense, 64.6±0.7 vs. sham, 61.0±1.3 ml/100g/min, P=0.008, n=10 each) ( Figure, yellow arrowhead ). Conclusions: This study demonstrates for the first time that ACC could be the cortical receptive field of cardiac afferent nerves associated with sympathetic arousal in humans.