Hippocampus slice electrophysiology

Electrophysiological field potential recordings from Schaffer collateral-CA1 synapses in in vitro hippocampal slices were performed using standard methods as described previously^52,53. Mice were decapitated under deep isoflurane anesthesia, the brains quickly removed, hemisected, and cut with a vibratome (Leica model VT1200S) at a thickness of 350 µm. The tissue block was glued with cyanoacrylate adhesive to a stage immersed in ice-cold sucrose-based artificial cerebrospinal fluid (aCSF), in mM: 87 NaCl, 25 NaHCO₃, 25 glucose, 75 sucrose, 2.5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂ and 7 MgCl₂ (equilibrated with 95% O₂/5% CO₂), then placed in a chamber containing the same high-sucrose, low-magnesium aCSF composition at 32 °C for 30 min. After equilibrating for 30 min, the slices were transferred to another holding chamber in room temperature aCSF (in mM: 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 1.3 MgCl, 2.5 CaCl₂, 26 NaHCO₃, 10 glucose) saturated with 95%O₂/5%CO₂. Once transferred to the recording chamber, slices were continuously perfused with aCSF maintained at 32 °C. Borosilicate-glass recording electrodes (1–2 Mohms; A-M Systems) were pulled with a Sutter micropipette puller (Model P-97, Sutter Instrument, Novato, CA), and inserted in the stratum radiatum of hippocampal field CA1 to record field excitatory post-synaptic potentials (fEPSPs). To elicit evoked responses, current pulses applied with stimulus intensity adjusted to evoke ~ 50% of maximal fEPSPs (50 pA to 100 pA; 100 µs duration) at 30 s intervals was delivered using a bipolar stainless-steel stimulating electrode that was placed in the Schaffer collateral/commissural fibers. Electrical stimulation was delivered by an ISO-Flex isolator controlled by a Master eight pulse generator (AMPI, Jerusalem, Israel), triggered and recorded with a Multiclamp 700B amplifier (Molecular Devices, San Jose, CA). The slopes of fEPSPs were measured by linear interpolation from 20 to 80% of maximum negative deflection. Raw values of fEPSPs at half-maximal stimulus intensity (SI) during 15 min baseline recording were averaged to compare baseline excitability across groups. Data were analyzed with SciWorks (DataWave Technologies, Parsippany, NJ). The high-frequency theta burst stimulus (TBS) paradigm for induction of LTP consisted of 2 theta burst trains separated by 3 min, 10 bursts each, 5 pulses per burst, a burst frequency of 100 Hz and inter-burst interval of 200 ms. cAMP-dependent chemical LTP was induced by bath application of forskolin (10 μM) and rolipram (10 μM) for 20 min.

For analysis of paired-pulse inhibition/facilitation of population spikes in CA1 neurons, the stimulating electrode was placed in the stratum radiatum to stimulate Schaffer collaterals, and the recording electrode in CA1 stratum pyramidale. Population spike (PS) magnitudes were measured as the amplitude of the negative spike, extrapolated by drawing a tangent between the peak of the EPSP and the peak of the PS, and then taking the vertical distance from the negative peak of the PS to the tangent line. Population spikes were evoked at a stimulus intensity that elicited 50% of maximal amplitude in the first pulse spike. A series of inter-pulse intervals (IPIs), 10–1000 ms in duration, was used to study synaptic properties of facilitation or depression. Four sweeps of population spikes were recorded for each IPI per slice, and the responses averaged. The ratio of the second evoked population spike amplitude (PS2) to the first (PS1) was used to determine depression or facilitation, with a ratio > 1 corresponding to facilitation, and a ratio < 1 indicating depression.