The Glycolysis Inhibitor 2-Deoxy-d-Glucose Exerts Different Neuronal Effects at Circuit and Cellular Levels, Partially Reverses Behavioral Alterations and does not Prevent NADPH Diaphorase Activity Reduction in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy

Animals

This study was carried out on 64 adult male NMRI mice (weighing 30–35 g; Pasteur institute, Tehran). The animals were housed with free access to standard pellet diet and tap drinking water ad libitum. They were kept in a temperature-controlled (23 ± 2 °C) animal house free from any source of chemical or noise pollution under the 12:12 h light: dark cycle. All animals received human care and gentle handling throughout the study, as it has been shown that proper techniques and frequency of handling were used to reduce stress and anxiety [24, 25]. Mice were single housed after the surgery; although social housing is deemed to be the optimal way of housing, previous studies showed that single housing does not significantly affect behavioral tests in mice [26]. Hence, we single-housed the mice in order not to arouse aggression, especially in epileptic animals. All experimental procedures and animal care were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Biomedical Research Ethics Committee of the National Institute for Medical Research Development (Approval ID: IR.NIMAD.REC.1399.259) and the Ethics Committee of Shahid Beheshti University of Medical Sciences (Authorization Code: IR.SBMU.MSP.REC.1400.630).

Study Design

The present study intended to investigate the behavioral, electrophysiological and histochemical consequences of glycolysis inhibition on the intrahippocampal kainic acid model of temporal lobe epilepsy. Three separate groups of experiments were conducted to assess the effects of glycolysis inhibition by 2-DG on: (1) Kainic acid-induced hyperexcitability in CA1 pyramidal neurons using patch-clamp recording, (2) Local field potential (LFP) recordings to measure epileptiform activity, and (3) Behavioral tests to assess the locomotor activity, anxiety and depression behaviors. To assess histological alterations, however, the animals were randomly chosen from the animals which had undergone behavioral tests.

Kainic acid was stereotaxically microinjected into the dorsal hippocampus of the left hemisphere (day 0, for more details, see the epilepsy induction section) and 2-DG (300 mg/kg, i.p) was injected 3 weeks after IHKA injection for 7 days (Fig. 1) and the last injection was given 90 min before starting the experiments. This time was chosen based on the study done by Koenig et al., 2019, who reported that 90 min after 2-DG injection, induction of ketosis is observed [27].

Fig. 1figure 1

Time line indicating the study design. IHKA Intrahippocampal injection, 2-DG 2-Deoxy d-glucose, LFP Local field potentials, WCP Whole-cell patch-clamp

The dose of 2-DG was chosen based on previous studies that report the anticonvulsant and antiepileptic action of 2-DG [5, 9].

The animals randomly were divided into the following five groups: Control group, a group in which the mice underwent stereotaxic surgery and received intrahippocampal saline (40 nL) injection ( as a solvent for kainic acid) on the 0th day; Control + 2-DG group, a group that mice received intrahippocampal saline (40 nL) on the 0th day and i.p injection of 300 mg/kg 2-DG from the 20th to 28th day, once a day; Epileptic group, a group in which the mice received an intrahippocampal injection of kainic acid on the 0th day; Epileptic + 2-DG group: a group in which epileptic mice treated i.p with 2-DG at the dose of 300 mg/kg from the 20th to 28th day, once a day; epileptic + saline group: a group that epileptic mice received 300 mg/kg i.p saline from the 21st to 27th day, once a day.

The order of the behavioral tests was as follows: zero maze, open field and sucrose preference test on the 20th, 21st, and 22nd days respectively in control and epileptic groups; in epileptic + 2-DG and control + 2-DG groups, however, the tests were performed on the 26th, 27th, and 28th days respectively (Fig. 1A). Since in the behavioral study, we did not find a significant difference between control groups and control + 2-DG groups (for more details see results), and according to the animal ethics guidelines to minimize the number of animals used, this group was omitted in LFP and patch-clamp recordings. Due to the fact that saline and handling did not lead to a significant alteration in LFP groups, we omitted epileptic + saline groups from patch-clamp recordings (Fig. 1C). It should be noted that according to previous studies in our laboratory, saline and surgery do not lead to considerable variation between the groups; hence, the study does not contain intact and sham groups [28].

Epilepsy Induction

Temporal lobe epilepsy was induced as previously described by Sada et al. [2]. Briefly, mice were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) and fixed in the stereotaxic frame. Then, 0.8 nmol kainic acid was dissolved in 40 nL normal saline and directly injected into the left dorsal hippocampus (− 1.6 mm to the Bregma, 1.6 from the midline, and 1.2 mm deep from the dura mater) according to the atlas of Paxinos and Franklin (2001). Due to the non-convulsive status epilepticus, verification of model induction was endorsed by frequent interictal epileptiform activity (sharp-wave complexes) (see below for details) as well as severe cell loss in the dorsal CA1 pyramidal cell layer (see Nissl Staining) (Fig. 2C, D). After the experiments, the anesthetized animals were decapitated and the brains were dissected out for injection site verification (Fig. 2A).

Fig. 2figure 2

Verification of injection site, electrode site, and cell loss following kainic acid injection. A the injection site of kainic acid or saline. B electrode location in LFP group animals. C normal dorsal hippocampus (control); D severe cell loss in CA1 and, to less extent, in CA3 as well as swollen dentate gyrus in kainic acid-treated hippocampus compared to the control hippocampus. Scale bar: 300 µm

Behavioral TestsOpen Field Test

To measure the locomotor activity, animals were placed in the center of an open field (35 × 35 × 35 cm) after half an hour of habituation to the experimental room. In the following ten minutes, moved distance and velocity were analyzed by EthoVision XT 11 software. To assess thigmotaxis, which is the tendency of an animal to stay near the walls of the open field, a center (15 × 15 × 15) was defined and time spent in the center was analyzed. The arena was cleaned with ethanol 85% between the trials.

Zero-Maze Test

Anxiety behavior was evaluated by using zero-maze apparatus [29]. The apparatus (60 cm in diameter, 5 cm wide circular corridor, 16 cm high walls and 60 cm high from the floor) was made of wood and painted dark black. After half an hour of habituation to the experiment room, each animal was placed in an open arm-closed arm intersection, facing the closed arm. During the following 5 min, the animal was videotaped and analyzed offline afterwards. Five parameters were assessed, as described before by Shepherd [30] including the time spent in the open arms, the number of entries to the open arm, the latency of the first open arm entry, head dipping frequency, and body stretching frequency. Between the trials, the apparatus was cleaned using 70% ethanol.

Sucrose Preference Test

Rodents are shown to prefer sweet water rather than tap water. Suffering from depression, however, they tend to consume less sweet water in comparison with tap water in normal conditions [21]. To perform the test, the animals were given access to two tap water bottles for 24 h as habituation in their home cage. During the next 24 h, both bottles were taken and replaced with two new bottles, one containing 3% sucrose solution while the other filled with tap water. As the diameter of the drinking hole had been noted to influence on the amount of water consumed by the animal [31], the holes were all equalized in size using a 2 mm drill. The proportion of sweet/tap water consumption was calculated afterwards.

Local Field Potentials (LFP) Recordings

Mice underwent stereotaxic surgery to record local field potentials. They were anesthetized with an intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The ear bars were placed delicately prior to muzzle fixation. Lidocaine 2% was injected under the scalp skin 5 min before making an approximately 2 cm incision in the skin. Following Bregma-Lambda adjustment to a plane level, three holes were made by a fine drill. To prepare electrodes, two stainless steel wires (127 μm in diameter, A.M. system Inc., USA) were intertwined to give the electrode suitable strength and flexibility. The electrode, then, was soldered to a connector and placed in the dorsal hippocampus (− 2.1 mm AP, 1.5 mm ML, 1.2 mm DV). Six screws (one as the reference electrode above the cerebellum) were screwed to the scalp. Lastly, dental cement was used to fix the electrodes. The LFP signals were continuously recorded for 13 h at 1 kHz sample rate and low-pass filtered at 250 Hz while the animals were freely moving. Interictal epileptiform discharges were defined as sharp-waves, having more than twofold amplitude compared with baseline, as well as having a frequency between 1 and 20 Hz. The discharges were detected and analyzed by MATLAB 2016 software. At the end of the experiments, brains were removed to verify the proper placement of the electrode (Fig. 2B).

Patch-Clamp Recording

To investigate the possible cellular-level effects of epilepsy induction by kainic acid on the electrophysiological properties of hippocampal CA1 pyramidal neurons, and whether 2-DG can reverse these possible alterations, whole-cell patch-clamp recording was performed as follows. Briefly, the animals were deeply anaesthetized with ether and then decapitated. The brains were removed immediately and placed in ice-cold artificial CSF (ACSF) containing (in mM): 206 sucrose, 2.8 KCl, 1 CaCl2, 1 MgCl2, 2 MgSO4, 1.25 NaH2PO4, 26 NaHCO3, 10 d-glucose, and saturated with 95% O2 and 5% CO2 (pH 7.3–7.4; 300 mOsm). Transverse slices (300 μm) were cut by using a vibroslicer (7000 smz-2, Campden Instruments Ltd, UK). Slices were placed in a holding chamber containing ACSF composed of (in mM) 125 NaCl, 2.5 KCl, 1.5 CaCl2, 1.25 NaH2PO4, 25NaHCO3, 10 d-glucose, pH 7.4, 300 mOsm for at least 60 min at 32–35 °C. The slices were kept at room temperature (23–25 °C) before transferring to the recording chamber. After incubation for at least 1 h, each slice was individually transferred to a submerged recording chamber on the stage of an upright microscope (BX51WI, Olympus); they were continuously superfused with oxygenated ACSF at a rate of 2–3 ml/min at 23–25 °C afterwards. Patch pipettes [borosilicate glass capillary (1.5 mm O.D., 0.86 mm I.D)] were pulled with a PC10 two-stage vertical puller (Narishige, Japan). The pipettes’ resistance was 3–6 MΩ when filled with an internal solution containing (in mM): 135 potassium gluconate, 10 KCl, 10 HEPES, 1 MgCl2, 2 Na2ATP and 0.4 Na2GTP. The pH of the internal solution was set to 7.3 by KOH, and the osmolarity was adjusted to 290 mOsm. Whole-cell patch-clamp recordings were performed using Multiclamp 700B amplifier equipped with Digidata 1322A data acquisition board, and pClamp nine software (Axon, Molecular Devices, CA, USA). All recordings were done from CA1 pyramidal neurons in current-clamp mode. The recordings were filtered at 5 kHz, sampled at 10 kHz and stored on a personal computer for offline analysis. The data were analyzed offline by using clampfit version 11.2 (Molecular devices) and MATLAB 2016 software.

The passive electrical properties of the CA1 pyramidal neurons were measured by applying hyperpolarizing current pulses (− 50 to − 400 pA, 800 ms). The resting membrane potential was recorded after the initial break-in of the cell membrane. To obtain input resistance, the current–voltage curve was drawn and its slope was measured as the resistance using the first four sweeps (0 to − 150 pA). The membrane time constant (tau) was evaluated by the exponential fitting of capacitive voltage relaxation. Further, membrane capacitance was obtained by dividing the time constant by the input resistance.

Spontaneous activity was recorded and analyzed in a 60 s epoch. The firing regularity was quantified by the coefficient of variation (CV) of the ISI (inter-spike interval) which was calculated as the ratio of the standard deviation to the mean of ISI. The amplitude of AHP was measured from the threshold to the peak of the hyperpolarization following the action potential. To investigate the impact of kainic acid and 2-DG injection on rebound APs, a hyperpolarizing ramp current (1000 ms, − 300 pA with a slope of 0.345 pA/ms) followed by a depolarizing current pulse (100 pA for 300 ms) was applied. Burst activity was assessed in 125 s epochs.

Histochemical AssessmentNissl Staining

To show the brain injury induced by intrahippocampal injection of kainic acid, Nissl staining was performed. Following anesthesia (100 mg/kg ketamine and 10 mg/kg xylazine), transcardial perfusion was performed with saline and 4% paraformaldehyde, 1.33% picric acid in 0.1 M phosphate buffer (pH 7.4). Then, the mice were decapitated and brains were removed and post-fixed in the same fixative. To verify injection and electrode sites, the brains were cryoprotected in 20% sucrose buffer at 4 °C overnight. Coronal Sects. (20 μm) containing the hippocampus were serially cut using a cryostat (Leica CM1850, Germany). However, to evaluate cell loss, the brain blocks were processed and embedded in paraffin and 8 µm sections were obtained using rotary microtome apparatus (Cut5062, Germany) and mounted on gelatin-coated slides. Nissl staining (0.1% Cresyl violet) was performed afterwards. To assess morphological properties of the CA1 pyramidal neurons (diameter of the soma), the long axis length of the soma was measured in the neurons containing a visible nucleus, nucleolus, and primary dendritic cone (from the neck of the dendritic cone to the opposite pole of the soma) using a computer-based image analysis system (Olympus BX60, DP12, Olysia Soft Imaging System, Japan).

NADPH Diaphorase Staining

The mice were anesthetized (100 mg/kg ketamine and 10 mg/kg xylazine, i.p) on the day 21st (epileptic and control groups) or 29th (epileptic + 2-DG group) (Fig. 1A) and were perfused transcardially with a cold fixative containing 4% paraformaldehyde and 1.33% picric acid in 0.1 M phosphate buffer (PB, pH 7.4) following 0.9% saline perfusion. The brains were then dissected out from the skull, post-fixed overnight in the same fixative at 4 °C and cryoprotected by being immersed in 20% sucrose until they sank. The brains were freeze-sectioned coronally at 50 µm thickness, between the AP 1.2 and 2.4 mm posterior to the Bregma (Paxinos and Franklin, 2001) using a cryostat (Leica CM1850, Germany). NADPH-d staining was performed by incubating free-floating sections in the light-protected 0.1 M PB (pH 7.4) solution, containing 1 mg/ml nicotinamide adenine dinucleotide phosphate diaphorase (β-NADPH-d), 0.1 mg/ml nitroblue tetrazolium (NBT), and 0.3% Triton X-100 (all reagents were obtained from Sigma, St. Louis, MO, USA) at 37 °C for 1–2 h. The sections, then, were mounted on the gelatin-coated slides and cover-slipped with Entellan. Seven sections from the anterior–posterior axis of hippocampal CA1 area per animal were examined under light microscopy to localize NADPH-d+ neurons. The NADPH reactive cells were photomicrographed by the same Olympus microscope as mentioned above and manually counted.

Statistical Analysis

SPSS 26 (IBM SPSS Statistics. Armonk, NY: IBM Corp) and GraphPad Prism 8 software (GraphPad, La Jolla, CA, USA, respectively) were employed to compare the data between groups and significance levels. One-way ANOVA and student’s t-test were used to make a comparison between independent variables while the ANCOVA test was utilized to mask the effect of locomotion on anxiety behavior (see results and discussion). Pearson’s test (or Spearman’s test when a non-parametric test was needed) was employed to assess the correlation between the variables. Numerical data were expressed as mean ± standard error of the mean (SEM) and a value of P < 0.05 was considered statistically significant.

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