Acutely prepared hippocampal slices (400 µm) were obtained from anesthetized (Isoflurane) adult (2–4 months of age) mice. Transverse brain slices were dissected in ice cold, oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (aCSF) containing (mM): NaCl (125), KCL (2.5), KH2PO4 (1.25), MgSO4 (1.2), CaCl2 (2), NaHCO3 (25), and dextrose (10). The CA3 region was surgically removed from all slices (Fig. 1A). Slices were then transferred to a submerged recording chamber, which continuously superfused aCSF (saturated with 95% O2/5% CO2) at a rate of 1.5 ml/minute, with temperature maintained at 30°C. The slices were allowed to recover from dissection for at least an hour in the recording chamber before experiments were begun.
Extracellular recording electrodes (borosilicate glass, ~1 µm tip) filled with aCSF were placed in the stratum radiatum of CA1. We chose to assess field excitatory post-synaptic potentials (fEPSPs) because they provide a reliable and stable measure of excitatory synaptic transmission. Dendritic fEPSP responses were evoked with a bipolar tungsten stimulating electrode (Rhodes, Inc) placed on either the CA3 or the subicular side of the recording electrode in the stratum radiatum. Responses were amplified using the Axoclamp-2A amplifier (Molecular Devices, Inc). The electrical stimulus consisted of a single square waveform of 0.3 msec duration given at intensities of 10–130 µA generated by a Grass S88 stimulator equipped with stimulus isolation unit PSIU6.
Data acquisition and analysis
Data were acquired with Clampex 10 and analyzed with Clampfit 10 software (Molecular Devices). The initial slope of the fEPSP (which provides a measure of the strength of excitatory synaptic transmission) was measured by fitting a straight line to the first millisecond of the fEPSP immediately following the fiber volley, and was monitored in real-time in every experiment. A stimulus-response curve was then determined using stimulation intensities between 10–130 µA. Baseline stimulation parameters were selected to evoke a response of 40–60% of the maximum slope. Baseline stimulation was then commenced at a frequency of 0.033 Hz for the entire length of the experiment. The paired-pulse protocol consisted of two pulses at baseline intensity separated by 50 milliseconds. Control synaptic responses were normalized by dividing all slopes by the average of the 10 fEPSP slopes 5 minutes pre-tetanus. LTP was quantified as the normalized fEPSP response at 55–60 minutes post-tetanus.