Briefly, a poly-l-lysine (PLL)-coated pattern was made on the surface of a square glass chip by microcontact printing (see the areas in in Fig

Briefly, a poly-l-lysine (PLL)-coated pattern was made on the surface of a square glass chip by microcontact printing (see the areas in in Fig. and the rise in Ca2+, resulting from Ca2+ influxes through calcium-permeable AMPA receptors, voltage-gated Ca2+ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of Rabbit polyclonal to IL1R2 brain-derived neurotrophic factor (BDNF) on HAMNO the local protein synthesis in cortical axons were also studied. The results indicate that Ca2+ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca2+ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain. (37) was used here. On the chip surface (Fig. 1schematic presentation of the chip used here. chip (1.4 1.4 cm) contains a PLL-coated micropattern (region enclosed by the in the at higher magnification. Fifteen to sixteen days after plating neurons (experimental procedures for metabolically labeling cultured cortical neurons with AHA and for assaying incorporated AHA moieties. Cells on chips are incubated with methionine-free DMEM for 45 min and then with methionine-free DMEM supplemented with AHA for 2 h. The axons connecting regions 1 and 2 are severed at the position as indicated by the in just before the addition of AHA. in the indicate the periods when glutamate or BDNF is present in different experiments. Cells on chips are then subjected to washes and fixation, followed by alkyne-Alexa Fluor 647 tagging and fluorescence immunostaining. images obtained from an experiment wherein neurons on the chip surface are assayed by the procedures shown in and is the merge of the and 100 m. Experimental Procedures Reagents and Antibodies Pregnant Sprague-Dawley rats were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). The culture medium, including minimum Eagle’s medium, neurobasal (NB), B27, DMEM, and methionine-free DMEM, were obtained from Gibco. Azidohomoalanine (AHA) was purchased from AnaSpec; alkyne-Alexa Fluor 647 (A10278), Click-iT cell reaction buffer kit (“type”:”entrez-nucleotide”,”attrs”:”text”:”C10269″,”term_id”:”1535340″,”term_text”:”C10269″C10269), alkyne-biotin (“type”:”entrez-nucleotide”,”attrs”:”text”:”C33372″,”term_id”:”2365168″,”term_text”:”C33372″C33372), and HRP (horseradish peroxidase)-streptavidin (43C4323) were obtained from Invitrogen. Glutamic acid, BDNF, cycloheximide, GdCl3, and EGTA HAMNO were purchased from Sigma. The following were obtained from Tocris: -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective agonist of AMPA receptors; (37). Briefly, a poly-l-lysine (PLL)-coated pattern was made on the surface of a square glass chip by microcontact printing (see the areas in in Fig. 1(DIV) 1, the stencil was lifted off, and the medium was replaced by neurobasal medium supplemented with 2% B27, 0.5 mm glutamine, and 25 m glutamate. On DIV 3, neurons were treated with 5 m cytosine–d-arabinofuranoside for 24 h to curtail the growth of glial cells. Afterward, ? of the medium over the chip was replaced by fresh NB-B27 supplemented with 0.5 mm glutamine every 3C4 days. On DIV 8C9, axons extending from neurons in region 1 and migrating on PLL-coated lines started entering region 2; region 2 was fully occupied by axons at DIV HAMNO 15C16 (indicated by areas in in Fig. 1right before the addition of AHA. Cells were then incubated at 37 C and in 5% CO2 for another 2 h. During this period, drugs were added to the medium at different time points (Fig. 1for 20 min at 4 C to remove cell debris and nuclei. The supernatant was collected and reacted with alkyne-biotin according to the manufacturer’s instructions. Proteins were then heated at 95 C for 10 min in SDS-PAGE sample buffer (62.5 mm Tris-HCl at pH 6.8 containing 2.5% SDS, 5% -mercaptoethanol, and 10% glycerol) and subjected to SDS-PAGE analysis with 12% polyacrylamide gels. After electrophoresis, proteins on the gels were electrotransferred to a PVDF membrane (Millipore). The resultant blots were incubated in the Tris-buffered saline (20 mm Tris-HCl at pH 7.4 and 50 mm NaCl) containing 0.1% Tween 20, 5% nondairy creamer, and 3% BSA overnight and then probed with HRP-streptavidin for 2 h at room temperature. After reacting with ECL Western blot detection reagent (Amersham Biosciences), HRP-labeled proteins on blots were detected by using ImageQuantTM LAS 4000 mini system (GE Healthcare) and quantified by using ImageJ software (National Institutes of Health). Fluorescence Immunocytochemistry After conjugating the metabolically incorporated AHA moieties in nascent proteins with alkyne-Alexa Fluor 647, cells on coverslips or chips were washed with PBS three times and then incubated with mouse anti-III-tubulin antibody and, in some experiments, with rabbit anti-p-4E-BP1 antibody at 37 C for 2 h. Cells were washed with PBS three times and incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG (Cy3-conjugated goat anti-rabbit IgG.