Acute Brain Slice Electrophysiology

The electrophysiological studies were conducted with C57BL/6 mice 30–60 days after birth. Acute brain slices were prepared as described previously [17]. All of the experimental procedures were approved by the Local Ethics Committee. Recordings were made in artificial cerebrospinal fluid (aCSF) that consisted of the following: 125 mM NaCl, 25 mM NaHCO₃, 2.6 mM KCl, 1.25 mM NaH₂PO₄, 2.5 mM CaCl₂, and 20 mM glucose, pH 7.4, at a temperature of 31 °C. Schaffer collateral (SCH) axons were stimulated with a concentric bipolar electrode (0.1 Hz, 0.25 ms). fEPSPs were recorded with glass micropipettes that were filled with aCSF (1-3 MΩ resistance) in the stratum radiatum of the CA1 region (150–200 μm from the stratum pyramidale). Population spikes were simultaneously recorded with another electrode that was placed in the stratum pyramidale below on the same axis (Fig. 1a). NMDAR-mediated signals were isolated with the AMPA/kainate receptor antagonist DNQX (20 μM) and l-type calcium channel blocker nifedipine (20 μM) in Mg²⁺-free solutions, as described previously [17]. At the end of each recording, the NMDAR antagonist APV (50 μM) was used to confirm the origin of the recorded fEPSP_NMDA. We used the following MMP inhibitors: MMP-3 inhibitors NNGH (10 μM) and UK356618 (2 μM) and MMP-2/9 inhibitor SB3CT (10 μM). All of the drugs were obtained from Sigma-Aldrich (Poland), Tocris (UK), and Merck/Calbiochem (USA). Linear peptide GRGDSP was purchased from Proteogenix (France). The electrophysiology data were analyzed using pClamp10.3 software (Molecular Devices, USA) and AxoGraphX software (developed by John Clements) as described previously [17].

Temporal dependence of hippocampal E-S potentiation on NMDARs and MMP-3 activity. a Recording scheme. Two recording (REC) electrodes simultaneously monitored fEPSPs and population spikes in the CA1 region in response to Schaffer collateral stimulation (STIM). b Left, Time-course of maximal fEPSP slopes normalized to baseline values in control slices (black circles) and when bath-applied with the NMDAR antagonist APV (50 μM) at varying time points relative to HFS: before HFS (red triangles), 15 min post-HFS (green squares), or 60 min post-HFS (blue diamonds). The top shows exemplary traces of fEPSPs before HFS (1) and 90 min after HFS (colors match figure legend). Scale bar = 0.5 mV, 20 ms. Right, Time-course of population spike (PS) amplitudes normalized to baseline values in control slices (black circles) and when bath-applied with the NMDAR antagonist APV (50 μM) at varying time points relative to HFS: before HFS (red triangles), 15 min post-HFS (green squares), or 60 min post-HFS (blue diamonds). The top shows exemplary and normalized traces of PS before HFS (1) and 90 min after HFS (colors match figure legend). Scale bar = 0.5 mV, 10 ms. c Statistics of average PS/fEPSP ratio presented in b at 90 min post-HFS. The asterisk indicates a significant difference vs. slices in which HFS was applied in the absence of APV. Notice that APV that was applied up to 30 min post-HFS (green) significantly attenuated the PS/fEPSP upregulation following HFS. d Impact of APV on E-S coupling before HFS (circles) and 90 min after HFS (triangles). Relationships between stimulus strength and the PS/fEPSP slope ratio are shown. Notice that APV that was applied up to 30 min post-HFS (yellow) significantly attenuated the PS/fEPSP upregulation following HFS that was observed in CTR (black). e Effect of MMP-3 inhibitor NNGH (10 μM) on E-S potentiation. Middle and right, fEPSP slope and population spike amplitude time-course, respectively, as in b, except instead of APV, an MMP-3 inhibitor was applied at varying time points with regards to HFS. Left, Exemplary traces of fEPSPs (top) and population spikes (bottom) before and 90 min after HFS in the presence of NNGH (colors match figure legends). Scale bars as in b. f Statistics of average PS/fEPSP ratio presented in e at 90 min post-HFS. The asterisk indicates a significant difference vs. slices in which HFS was applied in the absence of NNGH. Notice that NNGH that was applied up to 15 min post-HFS (green) significantly attenuated the PS/fEPSP upregulation following HFS. g Impact of NNGH on E-S coupling before HFS (circles) and 90 min after HFS (triangles). Relationships between stimulus strength and the PS/fEPSP slope ratio are shown. Notice that NNGH that was applied up to 15 min post-HFS (green) significantly attenuated the PS/fEPSP upregulation following HFS that was observed in CTR (black). The zero value on the time bars represents the moment of tetanization (HFS, 4 × 100 Hz). The horizontal colored bars represent drug application. The numbers on the graphs refer to the number of experiments. *p < 0.05