Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking solution made up of 4% normal goat serum and 0.04% Triton X-100 in PBS. of the same terminals with an antibody directed against the vesicular glutamate transporter 1 (vGluT-1), a stain designed to label all glutamate synapses regardless of activation status. We find that depolarizing stimuli induce presynaptic Rabbit Polyclonal to PEX10 silencing. The population of synapses that is silent under baseline conditions can be activated by prolonged electrical silencing or by activation of cAMP signaling pathways. Open in a separate window Click here to view.(61M, flv) Protocol Culture preparation Prepare dissociated cell cultures of rat or mouse hippocampal cells from postnatal day 0 to 3 animals1. Our neurons adhere to an underlying astrocyte monolayer, which in turn adheres to a collagen layer spread on a number 0 thickness coverslip. Plate the neurons at a density of approximately 500 cells per square centimeter. Allow the cultures to grow for 10-14 days to allow synaptic development and maturation. Treat the cultures at day in vitro 4 with the antimitotic AraC to arrest further glial growth. Feed them at day in vitro 5 with a half medium exchange with Neurobasal medium plus B27 product to enhance neuronal survival. During the culture period, expose treatments designed to alter the number of G907 functionally silenced synapses. We find that one convenient way to silence a large percentage (~80%) of glutamate synapses is usually a 4-hour treatment with 30 mM potassium. Longer incubations with weaker depolarizing stimuli induce comparable silencing2. 4-hour activation with G907 50 M forskolin, an activator of adenylyl cyclases, can be used to awaken the population of silent terminals at G907 baseline3. Visualizing silent synapses Cells should have a relatively low density with well-separated neuritic processes at day in vitro 10-14 so as to facilitate visualization of discrete synaptic terminals using fluorescence microscopy. Prepare a stock answer of 5 mM FM1-43FX in distilled water. The stock should be maintained in the dark at 4C. All experiments using FM1-43FX should also be performed in the dark. Challenge the cultures for 2 moments with 45 mM potassium chloride in a HEPES-buffered saline answer made up of 100 mM sodium chloride, 2 mM calcium chloride, 1 mM magnesium chloride, 10 mM glucose, and 10 M FM1-43FX. This answer should also contain 1 M NBQX and 25 M D-APV to block postsynaptic receptors and prevent recurrent signaling. This challenge will label synaptic terminals that are capable of exocytosis and endocytosis. The challenge time and strength are designed to cause at least one round of exocytosis of all available vesicles in the recycling pool4. However, the challenge is not sufficient to cause additional silencing of terminals. Wash the culture for 3 to 5 5 seconds only, using the same HEPES-buffered saline without potassium chloride, but with 500 M Advasep-7 to remove nonspecific dye5. Note that the timing of this step is critical as prolonged application of Advasep-7 will result in the loss of all FM1-43FX labeling. Next wash in HEPES-buffered saline without Advasep-7 for five 2-minute intervals. Fix cells with 4% paraformaldehyde and 0.2% glutaraldehyde in PBS, pH 7.4 for 10 minutes. Wash the cells once briefly with PBS, then incubate for 15 minutes in blocking answer containing 4% normal goat serum and 0.04% Triton X-100 in PBS. Note that FM1-43FX is also very sensitive to the amount of Triton X-100 used here and in later steps. Generally speaking, FM1-43FX labeling is best in the absence of any Triton X-100, but some detergent is necessary to permeabilize cells for subsequent immunostaining. We have decided empirically that 0.04% Triton X-100 is optimal for examination of presynaptically silent synapses. Dilute the primary vGluT-1 antibody in blocking answer at a concentration of 1 1:2500. Incubate the cells with gentle shaking/rotation in the diluted vGluT-1 antibody for 3 hours at room temperature. Be sure to cover your culture dishes with foil to prevent bleaching of the FM1-43FX dye. Wash the cells twice in PBS and then incubate the cells with an anti-guinea pig antibody conjugated to a fluorophore with different spectral characteristics from FM1-43FX to distinguish the two staining. For our.