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. 2016 Apr 4;17(5):682–694. doi: 10.15252/embr.201541569

Figure 3. Shh inhibits glutamate transporter activities and increases the extracellular glutamate levels.

Figure 3

  • A
    Glutamate levels assayed by HPLC in the medium of neurons incubated with the indicated agents. n = 4–6.
  • B
    The paired‐pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13–14 slices, six rats). Insets: representative traces recorded in response to paired‐pulse stimuli with different intervals.
  • C, D
    Quantification of 3H‐glutamate uptake by neurons treated with the indicated agents (C, n = 10) or transfected with two lentivirus‐based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n = 3–6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative Western blots for EAAC1.
  • E, F
    Aspartate (Asp)‐evoked currents at −70 mV from hippocampal neurons transfected with RNAi or Non (E, n = 17–23) or treated with the indicated agents (F, n = 16–18).
  • G
    Upper panel: representative Western blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower panel: 3H‐glutamate uptake by cells transfected with the indicated vectors. n = 9.
  • H, I
    Shh effects on 3H‐glutamate uptake (H, n = 6) or Asp‐evoked currents (I, n = 17–23) of neurons with or without pertussis toxin (PTX) pretreatment.
Data information: The upper panels in (E, F, I) show representative traces. Shh: 500 ng/ml. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. Ctrl or Non (Ctrl) with Student's t‐test.