Reproductive endocrine disorders are prominent comorbidities of temporal lobe epilepsy (TLE) in both men and women. The neural mechanisms underlying these comorbidities remain unclear, but hypothalamic gonadotropin-releasing hormone (GnRH) neurons may be involved. Here, we report the first direct demonstrations of aberrant GnRH neuron function in an animal model of epilepsy. Recordings of GnRH neuron firing and excitability were made in acute mouse brain slices prepared two months after intrahippocampal injection of kainate (KA) or control saline, a well-established TLE model in which most females develop comorbid estrous cycle disruption. GnRH neurons from control females showed elevated firing and excitability on estrus compared with diestrus. By contrast, cells from KA-injected females that developed prolonged, disrupted estrous cycles (KA-long) showed the reverse pattern. Firing rates of cells from KA-injected females that maintained regular cycles (KA-regular) were not different from controls on diestrus, but were reduced on estrus. In KA-injected males, only GnRH neurons in the medial septum displayed elevated firing. In contrast to the diestrus versus estrus and sex-specific changes in firing, GnRH neuron intrinsic excitability was elevated in all KA-injected groups, indicating a role for afferent synaptic and neuromodulatory inputs in shaping overall changes in firing activity. Furthermore, KA-injected females showed cycle-stage-specific changes in circulating sex steroids on diestrus and estrus that also differed between KA-long and KA-regular groups. Together, these findings reveal that the effects of epilepsy on the neural control of reproduction are dynamic across the estrous cycle, distinct in association with comorbid estrous cycle disruption severity, and sex-specific.

Summary The precise spatiotemporal characteristics of subcellular calcium (Ca 2+ ) transients are critical for the physiological processes. Here we report a green Ca 2+ sensor called “G-CatchER + ” using a protein design to report rapid local ER Ca 2+ dynamics with significantly improved folding properties. G-CatchER + exhibits a superior Ca 2+ on rate to G-CEPIA1er and has a Ca 2+ -induced fluorescence lifetimes increase. G-CatchER + also reports agonist/antagonist triggered Ca 2+ dynamics in several cell types including primary neurons that are orchestrated by IP 3 Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER + -JP45), we report a rapid local Ca 2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER + in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca 2+ dynamics. G-CatchER + will be an invaluable tool to examine local ER/SR Ca 2+ dynamics and facilitate drug development associated with ER dysfunction.

Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.

Multi-scale calcium (Ca 2+ )d ynamics,e xhibiting wide-ranging temporal kinetics,constitutes aubiquitous mode of signal transduction. We report an ovel endoplasmicreticulum (ER)-targeted Ca 2+ indicator,R -CatchER,w hich showed superior kinetics in vitro (k off ! 2 10 3 s À1 ,k on ! 7 10 6 M À1 s À1 )a nd in multiple cell types.R -CatchER captured spatiotemporal ER Ca 2+ dynamics in neurons and hotspots at dendritic branchpoints,e nabled the first report of ER Ca 2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca 2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose ap rinciple to rationally design genetically encoded Ca 2+ indicators with as ingle Ca 2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore.T he design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range.Our work may facilitate protein design, visualizing Ca 2+ dynamics, and drug discovery.

This research demonstrates that decreases in nutrient value and antioxidant capacity can result from processing, but that regeneration of these losses can be seen corresponding to the apparent breakdown of pulp structure. © 2015 Society of Chemical Industry.