Another possible beneficial effect of cytosolic alkalinization on terminal function is an increase in the rate of glycolysis, mediated by the pronounced pH sensitivity of the rate-limiting glycolytic enzyme

phosphofructokinase (Trivedi and Danforth, 1966 and Mellergard and Siesjo, 1998). In summary, measurements of stimulation-induced pH changes in motor nerve terminals made by monitoring changes in the fluorescence of transgenically expressed YFP demonstrate a prominent, prolonged, spatially heterogeneous alkalinization phase. Properties of this phase suggest that it results from the activity of vATPase pumps inserted into the plasma membrane during exocytosis and subsequently retrieved by endocytosis. Evidence suggests that this cytosolic alkalinization facilitates endocytotic retrieval of vesicular contents. click here The decay of this alkalinization offers a way to measure the time course of an aspect of endocytosis. Mice used in this study expressed YFP (a red-shifted, enhanced variant of Green Fluorescent Protein) in the cytosol of certain neurons, including motor neurons and their axons and terminals

[Feng et al., 2000; bred from B6.Cg-Tg(Thy 1-YFP)16Jrs/J (stock #3709) from Jackson Labs, Bar Harbor, ME]. Animal use protocols were approved 3-deazaneplanocin A order by the Animal Care and Use Committee of the University of Miami Miller School of Medicine. Experiments used the levator auris longus (Angaut-Petit et al., 1987) and epitrochleoanconeus next (Bradley et al., 1989) nerve-muscle preparations; these thin muscles, isolated from the head and forelimb (respectively), permit easy visualization of YFP-filled motor terminals. Preparations were pinned flat in a chamber with silicon walls constructed atop a glass coverslip. Action potentials were evoked by stimulating the motor nerve with brief, suprathreshold depolarizing pulses via a suction electrode; unless otherwise noted, stimulus trains were 50 Hz for 20 s. Muscle contractions were blocked using d-tubocurarine (15 μM). Preparations in Figure 1, Figure 2, Figure 3, Figure 4,

Figure 5 and Figure 6 were perfused with a standard physiological saline, composed of the following (in mmol/l): 128 NaCl, 24 NaHCO3, 4 KCl, 1.8 CaCl2, 1.1 MgCl2, 11.2 glucose, and 0.33 NaH2PO4, in an atmosphere containing 5% CO2 /95% O2 (pH 7.3), heated to 28°C–30°C. For experiments in Figure 7A, HEPES buffer (11.5 mM) was substituted for HCO3−, and the preparation was gassed with 100% O2. Changes in cytosolic pH were measured from images of YFP fluorescence obtained with a Retiga EXI camera (Qimaging, Surrey, Canada) mounted on an inverted Nikon TE2000E microscope (Nikon, Melville, NY). Images were obtained using a 60× water immersion lens (NA 1.2, Olympus, Melville, NY). YFP was excited at 488 nm from a Xenon lamp-equipped monochromator (PTI, Birmingham, NJ), using a dichroic mirror (505 nm) with a 535 nm emission filter (40 nm bandwidth, Chroma, Rockingham, VT). Images were acquired at 1 Hz using 0.8 s exposures (IPLAB v. 3.