Monitoring synaptic function at the neuromuscular junction of a mouse expressing synaptopHluorin

We monitored presynaptic exocytosis and vesicle recycling at neuromuscular junctions of transgenic mice expressing synaptopHluorin (spH), using simultaneous optical and electrophysiological recordings. Synaptic transmission was indistinguishable from that in wildtype controls. Fluorescence rose during and decayed monotonically after stimulus trains to the nerve, with amplitudes and decay times increasing with the amount of stimulation. The relatively large size of synaptic terminals allowed us to examine the spatial profile of fluorescence changes. We identified hot spots of exocytosis, which were stable with repeated trains. Photobleach experiments showed that spH freshly exposed by nerve stimulation was not preferentially retrieved by compensatory endocytosis; instead, most retrieved spH preexisted in the surface membrane. Finally, we compared fluorescence and electrical [summed end-plate potentials (EPPs)] estimates of exocytosis, which diverged during repeated trains, as fluorescence exceeded summed EPPs, although the average amplitude of miniature EPPs was unchanged. This might reflect exocytosis of spH-containing, acetylcholine-free (“empty”) vesicles or other organelles during intense stimulation

Essential Role forSonic hedgehogduring Hair Follicle Morphogenesis

AbstractThe hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control andShh−/− embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression ofGli1andPtc1was reduced inShh−/− dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected inShh−/− skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development

Identification of Novel Inhibitors of Dietary Lipid Absorption Using Zebrafish

Pharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes

Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals

The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t1/2 9 min vs 10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation

Active zones and the readily releasable pool of synaptic vesicles at the neuromuscular junction of the mouse

Synchronous neurotransmitter releaseis a highly regulated processthattakes place at specializations atthe presynapticmembrane called active zones (AZs). The relationships between AZs, quantal release, and vesicle replenishment are not well understood in a mature synapse. We have measured the number, distribution, and other properties of AZs in mouse motor nerve terminals and combined these observations with electrophysiological estimates of the size of the readily releasable pool (RRP) of synaptic vesicles. On average, we counted 850 AZs per terminal. Assuming two primary docked vesicles per AZ, we predict a total of 1700 vesicles optimally positioned for exocytosis. Electrophysiological estimates of the size of the RRP, using a simple kinetic model that assumes exponential depletion of the initial pool and refilling by recruitment, gave an average value of 1730 quanta during 100 Hz stimulation, in satisfying agreement with the morphology. At lower stimulus frequencies, however, the model revealed that the estimated RRP size is smaller, suggesting that not all AZs participate in release at low stimulation frequencies

The spatial pattern of exocytosis and post-exocytic mobility of synaptopHluorin in mouse motor nerve terminals

We monitored the spatial distribution of exo- and endocytosis at 37°C in mouse motor nerve terminals expressing synaptopHluorin (spH), confirming and extending earlier work at room temperature, which had revealed fluorescent ‘hot spots’ appearing in repeatable locations during tetanic stimulation. We also tested whether hot spots appeared during mild stimulation. Averaged responses from single shocks showed a clear fluorescence jump, but revealed no sign of hot spots; instead, fluorescence rose uniformly across the terminal. Only after 5–25 stimuli given at high frequency did hot spots appear, suggesting a novel initiation mechanism. Experiments showed that about half of the surface spH molecules were mobile, and that spH movement occurred out of hot spots, demonstrating their origin as exocytic sources, not endocytic sinks. Taken together, our results suggest that synaptic vesicles exocytose equally throughout the terminal with mild stimulation, but preferentially exocytose at specific, repeatable locations during tetanic stimulation

<i>c-kit</i>^{<i>IRES-Cre</i>} mice allow for MLI targeting in mature mice.

<p><b>(A)</b> Schematic of <i>c-kit</i>^{<i>IRES-Cre</i>} mouse generation. ML, molecular layer; PCL, Purkinje cell layer; GL, granule cell layer. <b>(B)</b> Reporter protein expression in a <i>c-kit</i>^{<i>IRES-Cre</i>}:Ai14 (<i>lox</i>-P flanked, Rosa26-tdTomato) mouse perfused at PND 38. This cross will report the entire history of Cre recombinase activity including during embryogenesis. <b>(C)</b> The cerebella from <i>c-kit</i>^{<i>IRES-Cre</i>} mice injected with Cre-reporter virus at PND 0 or PND 64. Tissue was prepared from animals 27 and 14 days after injection, respectively. Arrow points to a labeled PC in PND 0-injected cerebellum. Note the absence of Cre activity in PCs in mature animals. Scale bar in lower right applies to all images.</p

Chemogenetic suppression of molecular layer inhibition using DREADDs.

<p><b>(A)</b> MLIs in a <i>c-kit</i>^{<i>IRES-Cre</i>} mouse transduced with the DREADD hM4Di by Cre-dependent AAV. <b>(B)</b> Cartoon depicting the PC recording configuration. MLIs in a region of high hM4Di expression were induced to fire by extracellular electrical stimulation in the molecular layer. <b>(C)</b> Average IPSCs recorded in a PC in control and after application of CNO. Summary plot on the right shows the effect of DREADD activation on the peak IPSC amplitude in PCs (*P = 0.035, paired t-test).<b>(D)</b> MLIs in a <i>c-kit</i>^{<i>IRES-Cre</i>} mouse transduced with a bicistronic, Cre-dependent AAV containing both ChR2 and hM4Di. <b>(E)</b> Cartoon depicting the PC recording configuration. MLIs were stimulated using blue light directed towards the molecular layer. <b>(F)</b> Similar to panel C but using optogenetic (3.3 mW/mm²) rather than electrical stimulation to drive MLI inhibition onto PCs (*P = 0.005, paired t-test).</p