Excitatory Amino Acid Antagonists

Data from Mehta et al. [41 (link)] were used to estimate the magnitude of differences between litters on a number of outcome variables. This study was chosen because it included animals from fourteen litters (five saline, nine VPA) and therefore it was possible to get a reasonable estimate of the litter-to-litter variation. In addition, the study mentioned using randomisation and blind assessment of outcomes. Half of the animals in each condition were also given MPEP (2-methyl-6-phenylethyl-pyrididine), a metabotropic glutamate receptor 5 antagonist. To assess the magnitude of the litter effects, the effect of VPA, MPEP, and sex (if relevant) were removed, and the remaining variability in the data that could be attributed to differences between litters was estimated. More specifically, models with and without a random effect of litter were compared with a likelihood ratio test. This analysis is testing whether the variance between litters is zero, and it is known that p-values will be too large because of “testing on the boundary”, and therefore the simple method of dividing the resulting p-values by two was used as recommended by Zuur et al[42 ]. The exact specification of the models is provided as R code in Additional file 1 and the data are provided in Additional file 2.

All experiments were designed equal sizes (n = 6) per groups. All values were expressed as mean ± SD. A p value less than 0.05 was considered statistically significant. The microdialysis study were compared by linear mixed effects model (LME) using SPSS for Windows (ver 25, IBM, Armonk, NY, USA) followed by Tukey’s post hoc test using BellCurve for Excel (Social Survey Research Information Co., Tokyo, Japan), when the F-value of LME was significant. To represent the statistical significance of drug factor compared with LME and Tukey’s post hoc test, the data (levels of L-glutamate) was expressed as the area under the curve (AUC_{20–180 min}) values. Effects of AMA on L-cystine-induced astroglial releases of L-glutamate and D-serine were two-way analysis of variance with Tukey’s post hoc test (BellCurve for Excel). Concentration-dependent effects of AMA on astroglial Sxc activity and glutathione synthesis of primary cultured astrocytes were analyzed by logistic regression analysis (BellCurve for Excel). Interaction between glutamate receptor antagonists (AMA, MK801 and PER) and CO on intra-astroglial glutathione level was analyzed by two-way analysis of variance with Tukey’s post hoc test (BellCurve for Excel).

All drugs used in the current experiments were from Tocris Bioscience. Drugs used in the study and their concentrations are as follows: dihydrokainic acid (DHK), a competitive and selective GLT-1 blocker (300 µM; low DHK experiments used 1, 2, or 5 µM as indicated); _DL-threo-β-benzloxsapartic acid (_DL-TBOA), a competitive and nonselective EAAT blocker (100 µM); DNQX disodium salt, an AMPA/kainate receptor antagonist (20 µM); D-APV, a selective NMDA receptor antagonist (50 µM); MSOP, a selective group III metabotropic glutamate receptor antagonist (100 µM); and MTEP hydrochloride, a selective mGluR5 antagonist (100 µM). In DHK, TBOA, or MSOP/MTEP experiments, slices were incubated for 5–10 min before imaging or electrophysiology was conducted.

Mouse cerebellar slices were prepared as previously described^{64 (link)}. The mice were deeply anesthetized via isoflurane inhalation and then decapitated. Sagittal slices (250-μm thick) of the cerebellar vermis were obtained using a vibrating microtome (catalog number VT1200S, Leica) in an ice-cold extracellular solution containing (in mM) 252 sucrose, 3.35 KCl, 21 NaHCO₃, 0.6 NaH₂PO₄, 9.9 glucose, 0.5 CaCl₂, and 10 MgCl₂ and gassed with a mixture of 95% O₂ and 5% CO₂ (pH 7.4). The slices were maintained at 30 ºC for 30 min in a holding chamber, where they were submerged in ACSF containing (in mM) 138.6 NaCl, 3.35 KCl, 21 NaHCO₃, 0.6 NaH₂PO₄, 9.9 glucose, 2 CaCl₂, and 1 MgCl₂ (bubbled with 95% O₂ and 5% CO₂ to maintain the pH at 7.4). Thereafter, the slices were maintained at room temperature. Individual slices were transferred to a recording chamber attached to the stage of a microscope (catalog number BX51WI, Olympus) and superfused with oxygenated ACSF. Recordings were performed from PCs and MLIs located exclusively in lobules IV–VII to limit the variability associated with the specialization of different regions of the cerebellar cortex. Spike activity in PCs and MLIs was observed using loose cell-attached voltage-clamp recordings, which allowed long recordings without changing cytoplasmic content^{65 (link)}. Glass electrodes (2–3 MΩ) used for cell-attached recordings were filled with ACSF and gently placed in contact with PCs and interneurons located in the molecular layer. Slight suction was applied, and the holding potential was set to 0 mV. Here, we did not identify each interneuron as either a basket or a stellate cell according to the criteria for their morphology and physiology^{18 (link)}, thus, we generically referred to the cells as MLIs. To inhibit synaptic transmission onto MLIs and PCs, we bath-applied the synaptic blockers, 100 μM PTX, 5 μM NBQX, and/or 15 μM APV.
IPSCs of PCs were examined using whole-cell voltage-clamp recordings with patch pipettes (2–3 MΩ). A non-selective ionotropic glutamate receptor antagonist kynurenic acid (1 mM) was added to the ACSF throughout the IPSC recordings. At the first experiment of each data set, we examined whether IPSCs were completely inhibited by bicuculline (10 μM) or PTX (100 μM). To isolate spontaneous IPSCs as outward current responses, patch pipettes were filled with an intracellular solution containing (in mM) 120 K-gluconate, 9 KCl, 10 KOH, 4 NaCl, 10 Na-HEPES, 17.5 sucrose, 10 phosphocreatine, 0.6 QX-314, 3 Mg-ATP, and 0.4 Na-GTP (pH 7.4), and the holding potential was set at − 35 mV. To detect miniature IPSCs (mIPSCs) as larger inward current responses, we used a CsCl-based internal solution (in mM) 140 CsCl, 0.1 CaCl₂ 1 K-EGTA, 10 Na-HEPES, 10 phosphocreatine, 0.6 QX-314, 3 Mg-ATP, and 0.4 Na-GTP (pH 7.4), and the holding potential was set at − 60 mV in the presence of tetrodotoxin (TTX, 0.5 μM). Stimulation-evoked IPSCs (eIPSCs) were recorded using a cesium methanesulfonate-based internal solution (in mM) 140 CsCH₃SO₃, 5 CsCl, 0.1 CaCl₂ 1 K-EGTA, 10 Na-HEPES, 10 phosphocreatine, 0.6 QX-314, 3 Mg-ATP, and 0.4 Na-GTP (pH 7.4), and the holding potential was set at 10 mV. Focal stimulation (20–50 V, 0.1 ms) was applied using a glass microelectrode containing ACSF (1–2 MΩ) placed within the ML of the cerebellar slices. PF-EPSCs were recorded using the cesium methanesulfonate-based internal solution and the holding potential was − 60 mV. Focal stimulation (20–50 V, 0.05–0.1 ms) was applied via a glass microelectrode containing ACSF (1–2 MΩ) placed within the ML of the cerebellar slices. Paired-pulse stimulation was delivered at an interval of 7.5 s in the presence of PTX (100 μM). We did not correct the junction potential.
Series and input resistances were monitored continuously online with 2-mV hyperpolarizing voltage steps at an interval of 7.5 or 30 s. Series resistance (10–18 MΩ) was compensated by 60–70%, and the experiments were discarded if the value changed by ~ 20%. Experiments were performed at room temperature (24–26 °C). TTX was obtained from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan), JMV3002 (Cayman Chemical, Ann Arbor, MI), 2-APB (Abcam Biochemicals, Cambridge, UK), CGP55845, QX-314 and XE991 (Tocris Bioscience, Bristol, UK), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The membrane currents were recorded using an amplifier MultiClamp 700B (Molecular Devices, Sunnyvale, CA) and pCLAMP 10.3 software (Molecular Devices), digitized, and stored on a computer disk for offline analysis. All signals were filtered at 2–4 kHz and sampled at 5–20 kHz, and synaptic events were analyzed with a threshold of 10 pA. The frequencies of synaptic events are shown as the number of synaptic events (for 30 s) divided by the time duration. Spike firing and synaptic events were analyzed using the Mini Analysis Program 6.0 (Synaptosoft, Decatur, GA), Clampfit 10.3 software (Molecular Devices), and KyPlot software (version 6.0; KyensLab, Tokyo, Japan).

To visually identify LC NE neurons during patch clamp electrophysiology, we injected adult (12 weeks) male TH-Cre rats with a virus that expresses enhanced green fluorescent protein (EGFP) in the presence of Cre (AAV pCAG-FLEX-EGFP-WPRE; titer: 2 × 10¹², Addgene). Approximately 8 to 12 weeks after injection, rats were anesthetized with isoflurane and brains were removed. The brainstem was placed in an ice-cold bath of artificial cerebral spinal fluid. Horizontal sections of the brainstem containing the LC were sectioned at 250 mm on a vibrating microtome (Leica VT1200S). Slices were cut with a sapphire knife (Delaware Diamond Knives, Wilmington, DE) and secured using a fine polyethylene mesh in a perfusion chamber with continuous perfusion of artificial cerebrospinal fluid (aCSF) bubbled with 95% O₂ to 5% CO₂ at 32°C.
We recorded in aCSF with the following ionic conditions (in millimolar): 125 NaCl, 3 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 2 CaCl₂, and 10 dextrose, bubbled with 95% O₂ to 5% CO2 and the internal (in millimolar): 6 NaCl, 4 NaOH, 130 K-gluconate, 11 EGTA, 1 CaCl₂, 1 MgCl₂, 10 Hepes, 2 Na₂ATP, and 0.2 Na₂GTP. On average, recorded NE neurons had access resistance of 14 ± 2 megohms and an average holding current of −71 ± 15 pA, consistent with reported resting membrane potentials between −50 and −60 mV (120 (link)).
Neurons were studied under voltage-clamp conditions with a MultiClamp 700A amplifier (Molecular Devices, Union City, CA) and held at V_H = −60 mV in whole-cell patch configuration. Only recordings with a series resistance of <20 megohms were used for experiments to ensure good access and maintenance of the voltage clamp. Signals were filtered with a 1 kHz Bessel filter and sampled at 20 kHz using Axon pClamp10 software (Molecular Devices). Voltage-clamp recordings of TH⁺ LC neurons were performed under conditions to isolate glutamate-mediated EPSCs. Our internal and external bath conditions produced an ECl− = V_H = −60 mV, thus minimizing the influence of GABAergic signaling. Analysis of EPSC waveforms was consistent with AMPA receptor–mediated glutamatergic events and confirmed in a subgroup of neurons with bath perfusion of the AMPA-type glutamate receptor antagonist NBQX (10 mM). Following a baseline period in standard aCSF, Ex-4 was bath applied for 5 to 10 min at a flow rate of 2 ml/min and recording chamber volume of approximately 1.5 ml.