Role for Rapid Dendritic Protein Synthesis in Hippocampal mGluR-Dependent Long-Term Depression

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Hippocampal slices from postnatal day 21 to 30 Long-Evans rats (Charles River Cambridge MA) were prepared as described (27). Slices were allowed to recover for 1 hour at 30°C and three additional hours at room temperature in ACSF containing (in mM) NaCl (124) KCl (5) NaH 2 PO 4 (1.25) NaHCO 3 (26) MgCl 2 (1) CaCl 2 (2) and dextrose (10) saturated in 95% O 2 5% CO 2 . For recording slices were placed in a submersion recording chamber maintained at 30°C and perfused with ACSF at a rate of 2 ml/min. FPs were evoked recorded and analyzed as described (27). Average values (±SEM) are expressed as the percentage of initial baseline response measured 55 to 60 min after DHPG or conditioning stimulation unless otherwise indicated.

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For voltage-clamp experiments picrotoxin (25 μM) was added to the ACSF to isolate EPSCs. Whole-cell pipettes (3 to 7 megohm) were fabricated from thick-wall (outer diameter 1.5 mm; inner diameter 0.86 mm; Sutter Instruments) borosilicate glass and filled with (in mM) K-gluconate (134) KCl (6) NaCl (4) Hepes (10) EGTA (0.2) MgATP (4) tris-GTP (0.3) and phosphocreatine (14). The pH of the internal solution was adjusted to 7.25 with KOH and the osmolarity was adjusted to 300 mOsm with H 2 O or sucrose. Series resistance was monitored throughout the experiment by measuring the peak of the fast-current transient (filtered at 30 kHz; electrode capacitance was compensated for) in response to a 5-mV step. Average series resistance was 36 ± 3 megohm for cap cells and 28 ± 3 megohm for controls. Average input resistance was 153 ± 10 megohm for cap cells and 134 ± 11 megohm for controls. Only experiments in which there was less than a 15% change in series resistance were included in the analysis. Cap analog experiments were performed in two series one with 100 μM m 7 GpppG ( n = 5) and the second with 250 μM m 7 GpppG ( n = 6). Both concentrations produced comparable inhibition of LTD relative to control cells (300 μM GTP; n = 5 and 6 respectively) so the data were combined. To ensure a reliable and robust LTD in all cases we used 100 μM DHPG to induce LTD.

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For experiments on isolated CA1 dendrites an incision was made at the border of the stratum pyramidale and stratum radiatum with a microdissection knife visualized under a dissection microscope (20). Slices were left to recover from the dendritic isolation procedure for at least 2 hours at 30°C. For recording slices were placed in a submersion recording chamber maintained at 30°C and perfused with ACSF at a rate of 2 ml/min. Higher stimulation intensities (20 to 60 μA) were required to elicit FPs in isolated dendrites. Slices were used only if a slope of at least 0.1 mV/ms was obtained with a stimulation intensity of 100 μA. In these experiments the absence of a population spike in response to high-intensity stimulation (150 μA) confirmed that the CA1 dendrites were isolated from their somata.

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Special thanks to J. Fallon and J. Richter for providing essential advice to W. C. Abraham for helpful discussions and to P. Ornstein (Eli Lilly Company) for the gift of LY341495. Thanks to S. Meagher and E. Sklar for assistance. K.M.H. was supported by a grant from the National Eye Institute.