Extracellular electrophysiology

After eight systemic injections of the indicated human plasma versus vehicle (PBS) in either young or aged NSG mice or rTIMP2 (50 μg kg⁻¹) versus vehicle in aged WT mice, deep anaesthesia was induced with isoflurane before mice were euthanized. Brains were removed and placed in ice-chilled artificial cerebrospinal fluid (in mM: NaCl 124.0, KCl 2.5, KH₂PO₄ 1.2, CaCl₂ 2.4, MgSO₄ 1.3, NaHCO₃ 26.0 and glucose 10.0 (pH 7.4)) that was continuously bubbled with 5% CO₂/95% O₂. Hippocampal slices (400 μm) were prepared using a tissue chopper (Stoelting) and incubated in continuously oxygenated artificial cerebrospinal fluid for at least 1 h before recording with continuous 5% CO₂/95% O₂ bubbling. Data were collected using an Axopatch-2B amplifier and pClamp 10.4 software (Axon Instruments). Slices were in submerged mode at room temperature, continuously perfused with oxygenated artificial cerebrospinal fluid at a flow rate of 2 ml min⁻¹ using a peristaltic pump (Haake Buchler). Population spikes were recorded in the granule cell layer of the dentate gyrus with glass microelectrodes (resistance 1–3 MΩ). Stimulation of the perforant path with biphasic current pulses (0.2 ms duration for one phase, 0.4 ms in total) was delivered in intervals of 10 s through a bipolar, concentric stimulating electrode (FHC). Using this frequency stimulation protocol, no obvious synaptic depression or facilitation was observed. Slices were recorded within 8 h of dissection. Signals were digitized at 20 kHz, and a Gaussian low-pass filter was applied as needed to reduce noise. Input–output curves were constructed for each slice using stimulus intensity from threshold to 1.0 mA. Test pulse intensities were adjusted to evoke field potential to approximately 35–50% of maximal input–output response. Tetanic stimulation was executed with two trains of 100 pulses (0.4 ms pulse duration, 100 Hz) delivered with an inter-train interval of 5 s. Amplitudes of population spikes were measured from the initial phase of the negative wave with Clampfit 10.4. LTP was plotted as the percentage of baseline just before tetanic stimulation (mean ± s.e.m.) and the graph scaled to reflect effects in the maintenance phase (first one or two points after baseline could be outside scale). The maintenance phase was quantified over the final 6 min (39.5–45.5 min) of the LTP experiment. Paired-pulse ratios were measured with various inter-pulse intervals (20 ms, 50 ms, 100 ms). Paired-pulse ratio was calculated before or after LTP as the percentage of the amplitude of the second response minus the first response divided by the value of the first response. In rare cases, values were not obtained at all time points owing to experimental or equipment error. Data were processed and analysed with pClamp 10.4, Microsoft Excel, and StatView 5.0.

For experiments in which isolated slices from young WT (C57Bl/6J) or TIMP2 KO mice were assessed, or for those in which slices were incubated with vehicle (PBS), control IgG (R&D) or anti-TIMP2 IgG (R&D, AF971), LTP was performed in essentially the same fashion but with minor differences. Briefly, tetanic stimulation consisted of one train of 100 pulses (0.4 ms pulse duration, 100 Hz) since preliminary experiments revealed that two trains of high-frequency stimulation in younger slices tended to create short-term depression of field potentials in dentate gyrus, lasting approximately 10 min. For experiments involving bath incubation of the isolated slices, we adapted a previously described method for antibody blocking experiments in the context of LTP^{37, 38}. Slices were incubated with fresh control IgG (R&D Systems) or anti-TIMP2 IgG (R&D Systems) at 4 μg ml⁻¹in a clean chamber for ~2 h (1.8 ± 0.2 h, IgG control; and 1.9 ± 0.3 h, anti-TIMP2 IgG) at room temperature before transferring to recording chamber. Antibodies or vehicle were perfused continuously through a closed loop during recording through silicon tubing (VWR) (with the exception of a 5-cm portion connected to the pump). BSA was omitted from perfusion to minimize bubbling; efforts were taken to minimize bubbling of artificial cerebrospinal fluid in the perfusion system that would perturb oxygen and pH consistency.