Protein synthesis inhibition blocks the induction of mossy fiber long-term potentiation in vivo

Abstract

Protein synthesis inhibitors block the maintenance of NMDA receptor-dependent long-term potentiation (LTP) both in vivo and in vitro. Protein synthesis inhibitors block mossy fiber(MF) LTP maintenance in vitro, but little is known about the effect of protein synthesis inhibitors on either induction or maintenance in MF-LTP in vivo. Here we study the role of protein synthesis in the induction of long-term potentiation at the mossy fiber-CA3 hippocampal synapse in vivo in anesthetized rats. The protein synthesis inhibitor anisomycin was administered at different doses (0.04, 10, or 40 nmol) into area CA3 15 min before delivering high-frequency stimulation (two times at 100 Hz, 1 sec). Anisomycin blocked MF-LTP induction in a dose-dependent manner; both 40 and 10 nmol blocked MF-LTP induction, but a lower dose of 0.04 nmol was without effect. The inhibitory effect of anisomycin on protein synthesis was determined by measuring the incorporation of [(35)S]methionine into the newly synthesized proteins. Percentages of protein synthesis inhibition were determined by comparing [(35)S] incorporation of anisomycin-treated samples with vehicle controls. Doses of 0.04, 10, or 40 nmol of anisomycin produced 21, 82, or 83% inhibition of [(35)S]methionine incorporation, respectively. The effect of anisomycin was verified using a single dose of the protein synthesis inhibitor cycloheximide (40 nmol). Cycloheximide also blocked MF-LTP induction. These results suggest that protein synthesis plays an important role in the induction of mossy fiber long-term potentiation in vivo.

Fig. 1.

Inhibition of protein synthesis by anisomycin blocks the induction of MF-LTP. A, Induction of MF-LTP after the injection of Ringer's solution (n = 5).B, A low dose of anisomycin (0.04 nmol) does not block the induction of MF-LTP (n = 7), whereas higher doses of 10 (n = 5) or 40 (n = 7) nmol block its induction (C, D). Representative traces for each panel were taken 1 min before and 60 min after high-frequency stimulation. Calibration: 0.5 mV, 5 msec.

Fig. 2.

The effect of anisomycin is localized ipsilateral to the injected side. A, Administration of anisomycin into the CA3 region of the contralateral hippocampus had no effect on MF-LTP (n = 4). B, MF-LTP is induced in animals that received the NMDA receptor antagonist CPP (10 mg/kg, i.p.). C, Injection of the protein synthesis inhibitor cycloheximide (40 nmol) also blocked the induction of MF-LTP (n = 5). D, Anisomycin does not affect low-frequency evoked responses. Representativetraces for each panel were taken 1 min before and 60 or 120 (CPP group) min after high-frequency stimulation. Calibration: 0.5 mV, 5 msec.

Fig. 3.

Anisomycin does not affect voltage-gated calcium entry. A, Micrograph of a CA3 pyramidal neuron. The patch electrode filled with fura-2 is visible at thetop. The three squares represent regions of interest for measuring changes in fluorescence at the soma, ∼100 and 200 μm from the soma. Scale bar, 100 μm. B, Time course of somatic ΔF/F before and during the application of 100 μm anisomycin.C, Calcium transients at the soma, 100 and 200 μm from the soma in response to 10 action potentials before (C₁) and after (C₂) exposure to anisomycin.