Hippocampal field LTP recordings in acute brain slices

For local field potential recordings at hippocampal CA3-to-CA1 synapses we used transverse, acute brain slices^35,103–106. In brief:

Slice preparation. Adult mice (2–4 months, 3–5 mice, per genotype and experiment) were sacrificed with Suprane (Baxter) and the brains were gently removed from the skull. Transverse slices (400 μm) were cut from the middle and dorsal portion of each hippocampus (Supplementary Data: Video 3) with a vibroslicer (Campden Instruments NVSLM1) in cold aCSF (4 °C, bubbled with 95% O₂–5% CO₂) containing (in mM): 124 NaCl, 2 KCl, 1.25 KH₂PO₄, 2 MgSO₄, 1 CaCl₂, 26 NaHCO₃, and 12 glucose. Slices were placed in an interface chamber exposed to humidified gas at 28–32 °C and perfused with aCSF (pH 7.3) containing 2 mM CaCl₂ for at least 1 h prior to the experiments. In some of the experiments, dl-2-amino-5-phosphopentanoic acid (AP5, 50 μM; Sigma-Aldrich) was added to the aCSF in order to block NMDAR-mediated synaptic plasticity or a 10 µM concentration of GluN2B-specific antagonist CP101,606 (CP) (Pfizer) was added to the perfusion media.

Synaptic excitability. Orthodromic synaptic stimuli (<300 µA, 0.1 Hz) were delivered through tungsten electrodes placed in either stratum radiatum proximal or stratum oriens distal of the hippocampal CA1 region. The presynaptic volley and the fEPSP were recorded by a glass electrode (filled with aCSF) placed in the corresponding synaptic layer while another electrode placed in the pyramidal cell body layer (stratum pyramidale) monitored the population spike. Following a period of at least 10–15 min with stable responses, we stimulated the afferent fibers with increasing strength. A similar approach was used to elicit paired-pulse responses (50 ms interstimulus interval, the two stimuli being equal in strength). To assess synaptic transmission, we measured the amplitudes of the presynaptic volley and the fEPSP at different stimulation strengths. In order to pool data from the paired-pulse experiments, we selected responses to a stimulation strength just below the threshold for eliciting a population spike on the second fEPSP.

LTP of synaptic transmission. Orthodromic synaptic stimuli (50 μs, <300 μA) were delivered alternately through two tungsten electrodes, one located in the stratum radiatum and another one in the stratum oriens of the hippocampal CA1 region. Extracellular synaptic potentials were monitored by two glass electrodes (filled with aCSF) placed in the corresponding synaptic layers. After obtaining stable synaptic responses in both pathways (0.1 Hz stimulation) for at least 10–15 min, one of the pathways was tetanized (either with a single 100 Hz tetanization for 1 s or repeated four times at 5 min intervals). To standardize the procedure, the stimulation strength used for tetanization was just above the threshold for generating a population spike in response to a single test shock. Synaptic efficacy was assessed by measuring the slope of the fEPSP in the middle third of its rising phase. Six consecutive responses (1 min) were averaged and normalized to the mean value recorded 1–4 min prior to tetanization. Data of the same experimental group were pooled across animals and are presented as mean ± SEM (see also ref. ³⁵). Statistical significance was evaluated by using a linear mixed model analysis (SAS 9.1) with p < 0.05 being designated as statistically significant.

Hippocampal field LTP recordings in freely moving mice: Local field potential at hippocampal CA3-to-CA1 synapses in freely moving mice were recorded from implanted electrodes in freely moving mice¹⁰⁷. Briefly, 2-month-old mice were deeply anesthetized with a mixture of ketamine (65 mg/kg) and xylazine (14 mg/kg). In a stereotaxic frame, two mini-screws were fixed above the cerebellum serving as reference and ground electrodes respectively. Then, a bipolar stimulation electrode (two insulated tungsten wires glued together, each 52 µm in diameter, California Fine Wire) and a recording electrode (single wire, same material with stimulation electrode) were positioned in stratum radiatum by a motorized manipulator (Luigs & Neumann)¹⁰⁷. Electrodes were permanently fixed with dental acrylic and surgical wounds were sutured. Singly-housed mice were allowed to recover with access to food and water ad libitum for at least 1 week before recording.

Mice were put in the recording chamber (50 cm diameter round arena, 50 cm high) for environmental acclimation overnight. A miniature headstage (1 g, npi electronic GmbH, Tamm) was connected to electrodes/pins in the presence of 95% O₂ containing 4% isoflurane for stress relief. Evoked LFPs were filtered with a band width of 0.3–500 Hz, amplified with the miniature headstage (EXT-02F, npi electronic GmbH), and stored at 10 kHz (ITC-16, HEKA Elektronik) after 50 Hz noise filtration by a Hum Bug Noise Eliminator (AutoMate Scientific, Inc.). Extracellular stimulation was generated with an isolated stimulator (A365, WPI). The slopes of evoked LFPs were analyzed based on the middle one-third of the rising phase (Fitmaster, HEKA Elektronik). At the beginning of each recording, two IO curves per mouse were generated, applying stimulation voltages with both polarities. To evaluate changes in synaptic efficacy, a stimulus strength eliciting 35–40% of the maximum slope was used as the test pulse and was given every 30 s. For LTP induction, two trains of high-frequency stimulation (50 × 100 Hz, 100 μs pulse width, same intensity as test pulse) separated by 5 min were used.

After recordings, mice were deeply anesthetized and electrical lesions were induced twice (20 µA, 10 s) for each single tungsten wire. Subsequently, mice were perfused with phosphate-buffered saline (PBS) followed by 4%  Paraformaldehyde (PFA, 16005 Sigma Aldrich). The mouse brains were sectioned at 80 µm thickness and classic Nissl staining was performed to verify the locations of electrodes.