Tissue slice preparation. Mice were anaesthetized with isoflurane and decapitated at ZT4 or ZT8. The brain was rapidly removed from the skull and placed in the ice-cold ACSF as above. The ACSF solution consisted of the following: NaCl (126 mM), KCl (3.50 mM), NaH2PO4 (1.25 mM), NaHCO3 (25 mM), CaCl2 (2.00 mM), MgCl2 (1.30 mM), and dextrose (5 mM) (pH 7.4). ACSF was aerated with 95% O2/5% CO2 gas mixture. Slices of ventral hippocampus (350 μm) were cut, as described (62), using a tissue slicer (Leica VT 1200 S, Leica Microsystem, Germany). The ice-cold (<6°C) cutting solution consisted of the following: K-gluconate (140 mM), Hepes (10 mM), Na-gluconate (15 mM), EGTA (0.2 mM), and NaCl (4 mM), pH adjusted to 7.2 with KOH. Slices were then immediately transferred to a multisection, dual-side perfusion holding chamber with constantly circulating ACSF and allowed to recover for 2 hours at room temperature (22° to 24°C). Slices were then transferred to a recording chamber continuously superfused with ACSF (flow rate of 7 ml/min, warmed at 30° to 31°C) with access to both slice sides.

Synaptic stimulation and field potential recordings. Schaffer collaterals were stimulated using a DS2A isolated stimulator (Digitimer Ltd., UK) with a bipolar metal electrode. Stimulus current was adjusted using single pulses (100 to 250 μA, 200 μs, 0.15 Hz) to induce a local field potential (LFP) of about 60% of the maximal amplitude. LFPs were recorded using glass microelectrodes filled with ASCF, placed in stratum pyramidale, and connected to an EXT-02F/2 amplifier (NPI Electronic GmbH, Germany). Synaptic stimulation, consisting of a stimulus train (200-μs pulses) at 10 Hz lasting 30 s, was used to trigger metabolic response.

NAD(P)H fluorescence imaging and extracellular glucose/lactate measurement. NADPH and reduced form of nicotinamide adenine dinucleotide (NAD+) (NADH) have similar optical properties; therefore, it is expected that NADPH may contribute to the total autofluorescence signal. Because the cellular NADP+/ NADPH pool is about one order of magnitude lower than the NAD/NADH pool, in the present study, we assume that short-term variations of experimentally detected fluorescent change responses account for variations in NADH [see discussion in (71)]. Here, for the fluorescent signal, we use the term NAD(P)H. Changes in NAD(P)H fluorescence in hippocampal slices were monitored using a 290- to 370-nm excitation filter and a 420-nm long-pass filter for the emission (Omega Optical, Brattleboro, VT). The light source was the pE-2 illuminator (CoolLed, UK) equipped with 365-nm light-emitting diode. Slices were epi-illuminated and imaged through a Nikon upright microscope (FN1, Eclipse) with 4×/0.10 Nikon Plan objective. Images were acquired using a 16-bit Pixelfly CCD (charge-coupled device) camera (PCO AG, Germany). Because of a low level of fluorescence emission, NAD(P)H images were acquired every 600 to 800 ms as 4 × 4 binned images (effective spatial resolution of 348 × 260 pixels). The exposure time was adjusted to obtain baseline fluorescence intensity between 30 and 40% of the CCD dynamic range. Fluorescence intensity changes in the stratum radiatum near the site of the LFP recording were averaged from three regions of interest >500-μm distant from the stimulation electrode tip using the ImageJ software [National Institutes of Health (NIH), USA]. Data were expressed as the percentage changes in fluorescence over a baseline [(∆F/F) • 100]. Signal analysis was performed using the Igor Pro software (WaveMetrics Inc., OR, USA).

Tissue glucose and lactate concentrations were measured with enzymatic microelectrodes (tip diameter of 25 μm; Sarissa Biomedical, Coventry, UK), polarized at 0.5 V, and driven with free radical analyzer TBR4100 (WPI, USA). Calibration was performed before and after each slice recording, and the recorded data were corrected for eventual changes in electrode sensitivity. To avoid an interaction between enzymatic electrodes, glucose and lactate measurements were performed in separate sets of experiments.

Statistical analysis and signal processing. NAD(P)H dip and overshoot amplitudes and maximal changes in extracellular glucose/lactate concentrations were expressed as means ± SEM. Normality was rejected by Shapiro-Wilk normality test, and we used nonparametric Wilcoxon rank sum test. The level of significance was set at P < 0.05.

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