Slice electrophysiology

Hippocampal slices were prepared from postnatal day 15 (P15) male Long-Evans rats. Animals were decapitated, and their left hippocampi were dissected free and cut at 400 μm in the transverse plane on a tissue chopper (Stoelting Co., Wood Dale, IL). Slices were cut at room temperature and transferred within 5 minutes of decapitation to a 34°C static-pool interface chamber in ACSF (117 mM NaCl, 5.3 mM KCl, 2.5 mM CaCl₂, 1.3 mM MgSO₄, 1 mM NaH₂PO₄, 26 mM NaHCO₃, 10 mM glucose, and 95% O₂/5% CO₂ at pH 7.4). After one hour of recovery, two concentric bipolar stimulating electrodes (100 μm outer diameter; FHC, Bowdoin, ME) were placed 600–800 μm apart in CA1 stratum radiatum with a 120 mM NaCl-filled glass recording pipette halfway between them, approximately 150–200 μm from the cell body layer (Figure 1a). Responses were amplified, filtered at 2 kHz, and recorded using customized IGOR software (WaveMetrics Inc., Lake Oswego, OR). An initial input-output curve was used to set the stimulus intensity of each electrode to 75% of the population spike threshold for the remainder of the experiment. Test pulses were given every 120 s, with a 30 s interval between the two electrodes. Responses were measured as the maximal slope over a 400 μs time frame of the initial field excitatory postsynaptic potential (fEPSP). After a minimum of 40 minutes of stable baseline recordings, LTP was induced at one of the two inputs by theta-burst stimulation (TBS), consisting of 8 trains at 30 s intervals of 10 bursts at 5 Hz of 4 200 μs biphasic pulses at 100 Hz. Test pulse stimulation resumed for the remainder of the experiment. The TBS and control electrodes were counterbalanced between the subicular and CA3 sides of the slices.

Experimental design and physiology outcomes. a) Drawing of hippocampal slice showing placement of recording pipet (rec) in CA1 between two stimulating electrodes (S1 and S2). DG=dentate gyrus, SUB=subiculum. b) Average fEPSP slope as % of pre-TBS baseline for all three time points. For clarity, only the post-TBS time points immediately before each fixation time point are shown. Inset: average waveforms from the 30 minute time point. c) Drawing of area around electrodes showing location of sampling for electron microscopy (gray cubes).