Laquinimod ameliorates excitotoxic damage by regulating glutamate re-uptake

Animals employed in this study were 7- to 8-week-old female mice, C57BL/6N, obtained from Charles-River (Italy) and CNR-EMMA Mouse Clinic facility (Monterotondo-Rome, Italy). Animals were randomly assigned to standard cages, with 4–5 animals per cage, and kept under standard housing conditions with a light/dark cycle of 12 h and free access to food and water. Minipump-implanted mice were housed in individual cages endowed with special bedding (TEK-FRESCH, Harlan) in order to avoid skin infections around the surgical scar.

Experiments were carried out in accordance with the Internal Institutional Review Committee, the European Directive 2010/63/EU and the European Recommendations 526/2007, and the Italian D.Lgs 26/2014. All efforts were made to minimize the number of animals used, as well as their suffering.

EAE was induced as previously described [25]. Mice were injected subcutaneously at the flanks with 200 μg of MOG35–55 emulsion to induce EAE by active immunization. The emulsion was prepared under sterile conditions using MOG_35–55 (85% purity; Espikem) in 300 μl of complete Freund’s adjuvant (CFA; Difco) containing Mycobacterium tuberculosis (8 mg/ml, strain H37Ra; Difco) and emulsified with phosphate buffer solution (PBS). All animals were injected with 500 ng of pertussis toxin (Sigma) intravenously on the day of immunization and 2 days later. Control animals received the same treatment as EAE mice without the immunogen MOG peptide, including complete CFA and pertussis toxin (referred to hereafter as “CFA”). Animals were daily scored for clinical symptoms of EAE according to the following scale: 0 = no clinical signs, 1 = flaccid tail, 2 = hindlimb weakness, 3 = hindlimb paresis, 4 = complete bilateral hindlimb paralysis, and 5 = death due to EAE; intermediate clinical signs were scored by adding 0.5. For each animal, the onset day was recorded as the day post-immunization (dpi) when it showed the first clinical manifestations.

Laquinimod (Teva Pharmaceutical Industries, Netanya, Israel) was dissolved in 0.9% NaCl. One week before immunization, mice were implanted with subcutaneous osmotic minipumps allowing continuous intracerebroventricular (icv) infusion of either vehicle (vhl) or laquinimod (1.25 mg/kg/day) for 4 weeks [25, 27].

Mice were killed by cervical dislocation, and cerebellar parasagittal slices (210 μm) were prepared from fresh tissue blocks of the brain using a vibratome. After 1 h of recovery time in a chamber containing oxygenated artificial cerebrospinal fluid (ACSF), single slices were transferred to a recording chamber and submerged in a continuously flowing ACSF at 2–3 ml/min gassed with 95% O₂–5% CO₂. The composition of the ACSF was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl₂, 1.2 NaH₂PO₄, 2.4 CaCl₂, 11 glucose, and 25 NaHCO₃. Purkinjie cells (PCs) could be easily identified using an Olympus BX50WI upright microscope with a ×40 water-immersion objective combined with an infrared filter. Whole-cell patch-clamp recordings were made with borosilicate glass pipettes (1.8 mm outer diameter; 2–5.5 MΩ) in voltage-clamp mode at the holding potential of − 70 mV. To detect spontaneous excitatory postsynaptic currents (sEPSCs), the recording pipettes were filled with internal solution containing the following (in mM): 125 K+-gluconate, 10 NaCl, 1.0 CaCl2, 2.0 MgCl2, 0.5 BAPTA, 10 HEPES, 0.3 GTP, 3.0 Mg-ATP, adjusted to pH 7.3 with KOH. Bicuculline (10 μM) was added to the external solution to block GABAA-mediated transmission. Laquinimod was added in the bath solution at final concentration of 30 μM for 2 h before recordings. Some experiments were performed in the presence of the GLT-1 inhibitor DHK (200 μM).

Spontaneous synaptic events were stored using P-CLAMP 10 (Molecular Devices) and analyzed offline on a personal computer with Mini Analysis Version 6.0.7 software (Synaptosoft). The detection threshold of spontaneous and miniature excitatory events was set at twice the baseline noise. Positive events were confirmed by visual inspection for each experiment. Analysis was performed on spontaneous synaptic events recorded during a fixed time epoch (1–2 min) sampled every 2 or 3 min. Only cells that exhibited stable frequencies and amplitudes were taken into account. For sEPSC kinetic analysis, events with peak amplitude between 5 and 40 pA were grouped, aligned by half-rise time, and normalized by peak amplitude. In each cell, all events between 5 and 40 pA were averaged to obtain rise times, decay times, and half widths.

Cerebellar slices from 21 dpi EAE mice were incubated in oxygenated ACSF in the presence of vehicle (vhl) or laquinimod (30 μM) for 2 h. For each cerebellum, both experimental group (control and laquinimod) were included. Each slice was quickly dried and then snap frozen in dry ice. Four animals were used for protein extraction and western blot, and the same number of animals were employed for mRNA extraction and quantitative real-time PCR (qPCR).

Twenty-two dpi cerebella from EAE-vhl and EAE-laquinimod mice were isolated and snap frozen after sacrifice of the animals by cervical dislocation. Slices from ex vivo experiments and whole cerebella were next lysed in RIPA buffer supplemented with protease inhibitors. Protein quantification and western blot condition as in [25].

The following primary antibodies were used: mouse anti-β-actin (1:20,000; Sigma) for 1 h at RT; rabbit anti-GLAST/EAAT1 (1:5000; Abcam) 30 min at RT; mouse anti-glial fibrillary acidic protein (GFAP) (1:4000; Immunological Science) overnight at + 4 °C; guinea pig anti-GLT-1 (1:10,000; Millipore) 1 h at RT. Membranes were incubated with secondary HRP-conjugated IgG anti-rabbit (1:5000 for 30 min at RT), anti-mouse (1:4000 and 1:10,000 1 h at RT for GFAP and β-actin, respectively), and anti-guinea pig (1:10,000 1 h at RT) (all from Amersham GE Healthcare, formerly Amersham Biosciences). Immunodetection was performed by ECL reagent (Amersham GE Healthcare, formerly Amersham Biosciences), and membrane was exposed to film (Amersham GE Healthcare, formerly Amersham Biosciences). Densitometric analysis of protein levels was performed with ImageJ software (https://​imagej.​nih.​gov/​ij/​). WB results were presented as data normalized to control CFA values.

Twenty-two dpi cerebella from EAE-vhl and EAE-laquinimod mice were dissected in RNAse-free conditions. Total RNA was extracted according to the standard miRNeasy Micro kit protocol (Qiagen) from both cerebella (in vivo experiments) and cerebellar slices (ex vivo experiments). Next, 700–1650 ng of total RNA were reverse-transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), and 10–50 ng of complementary DNA (cDNA) were amplified in triplicate using the Applied Biosystem 7900HT Fast Real-Time PCR system. SensiMix SYBR Hi-Rox Kit (Bioline) was utilized for the quantification of messenger RNA (mRNAs) coding for ionized binding protein type-1 (IBA-1), glial fibrillary acidic protein (GFAP), GLAST, GLT-1, and BDNF by using the following primers:

For the mRNA quantification of Cd3, Il1b, and M1 (Cd86, MHC-II, CD16, and iNOS) and M2 (Arg1, Ym1, FIZZ1, and Tgfb1) markers, SensiMix II Probe Hi-Rox Kit (Bioline; Meridian Life Science) and the following TaqMan gene expression assays were used:

For both SYBR and TaqMan qPCR experiments, mRNA relative quantification was performed using the comparative cycle threshold (2^−ΔΔCt) method. β-actin was used as endogenous control. All data are expressed relative to EAE-vhl.

miR-142-3p expression was evaluated using miR-142-3p TaqMan miRNA assay (catalog ID 000464) and TaqMan miRNA Reverse Transcription Kit according to the manufacturer’s instructions (Applied Biosystems). Each reaction of amplification was performed in triplicates with SensiMix SYBR II Probe Hi-Rox Kit (Bioline, Meridian Life Science); data, normalized to U6B snRNA (catalog ID 001973) and control samples (EAE-vhl), are represented as 2^−ΔΔCt.

IF experiments were performed on slices taken from mice sacrificed at the peak of the EAE from at least two different immunization experiments, similarly to [25]. Animals were deeply anesthetized and perfused intracardially with ice-cold 4% paraformaldehyde (PFA).

For ex vivo experiments, after incubation with laquinimod or vhl, cerebellar slices (200 μm) were fixed in 4% PFA and equilibrated with 30% sucrose before cutting 30 μm slices. The following primary antibodies were used overnight at 4 °C in Triton X-100 0.25%: rabbit anti-GFAP (1:500; Dako) and guinea-pig anti-GLT-1 (1:5000; Millipore). AlexaFluor-488-conjugated donkey anti-rabbit (1:200; Invitrogen) and Cy3-conjugated donkey anti-guinea pig (1:200; Jackson ImmunoResearch Laboratories) were used as secondary antibodies. Nuclei were stained with DAPI. All images were acquired using an LSM7 Zeiss confocal laser-scanner microscope (Zeiss) with a ×20 (zoom ×1 or ×2) objective. All images had a pixel resolution of 1024 × 1024. The confocal pinhole was kept at 1.0, the gain and the offset were lowered to prevent saturation in the brightest signals, and sequential scanning for each channel was performed. Images were exported in Tiff format and adjusted for brightness and contrast as needed using ImageJ software.

For each type of experiment, at least three mice per group were employed. Throughout the text, “n” refers to the number of animals, except for electrophysiology, where it means the number of cells, and ex vivo experiments of WB and qPCR. Data were presented as the mean ± SEM. The significance level was established at p < 0.05. Statistical analysis was performed using unpaired Student’s T test for comparisons between two groups and non-parametric Mann–Whitney test, where needed. Multiple comparisons were analyzed by one-way ANOVA for independent measures followed by Tukey’s HSD.

To address a direct neuroprotective effect of laquinimod in EAE mice, we delivered the drug icv for 4 weeks by means of osmotic minipumps, starting 1 week before EAE induction. This preventive treatment did not elicit significant changes in daily clinical score of the treated animals compared with the vhl group in the first, inflammatory phases of the disease (Fig. 1a, n = 5 EAE-vhl, n = 5 EAE-laquinimod 1.25 mg/kg/day; Mann–Whitney non-parametric test p > 0.05). Also, the day onset was unchanged between the groups (EAE-vhl 10.6 ± 1.29 days vs EAE-laquinimod 1.25 mg/kg/day 11.4 ± 1.21 day, unpaired T test p > 0.05). However, laquinimod resulted in significant amelioration of motor disability in the late stage of EAE, when neurodegenerative damage was more pronounced (Mann–Whitney non-parametric test p < 0.05 at 22 dpi).

Next, we used qPCR to assess the effect of laquinimod on the inflammatory reaction of the cerebellum in treated animals taken at 22 dpi, when neuroinflammation and glutamatergic alterations are exacerbated pathological processes in EAE. To this end, we analyzed several markers of inflammation, microgliosis, T cell infiltration, and astrogliosis [25, 28]. As shown in Fig. 1b, the Aif1 mRNA coding for IBA-1, marker of macrophage/microglia, was not different between EAE–vhl and EAE–laquinimod groups (fold change: EAE–vhl 1.18 ± 0.42 n = 3; EAE–laquinimod 1.19 ± 0.38 n = 4; unpaired T test p > 0.05). To better investigate the effect of laquinimod on microglia cells, we also analyzed the expression of M1- and M2-like microglia-specific markers by means of qPCR [29]. The following M1-like markers were investigated: CD86 mRNA coding for a costimulatory protein for antigen presentation functions of microglia; H2ab1 mRNA coding for MHC-II component; Fcgr3 mRNA coding for CD16 protein, required for antibody recognition; and Nos-2 mRNA coding for iNos, a protein involved in immune response. None of these markers were significantly affected by laquinimod treatment (Fig. 1c; CD86 fold change: EAE–vhl 1.093 ± 0.33, EAE–laquinimod 0.96 ± 0.37; H2ab fold change: EAE–vhl 1.346 ± 0.73, EAE–laquinimod 1.835 ± 0.95; Fcgr3 fold change: EAE–vhl 1.018 ± 0.14, EAE–laquinimod 1.151 ± 0.28; Nos-2 fold change: EAE–vhl 1.06 ± 0.23, EAE–laquinimod 0.797 ± 0.28; unpaired T test p > 0.05 for each marker, EAE–vhl n = 3, EAE–laquinimod n = 4). Furthermore, we checked the expression of four M2-like markers: Arg1 mRNA coding for a protein involved in matrix deposition; Chil3 mRNA coding for Ym1 protein, a macrophage enzyme proposed to prevent extracellular matrix degradation; Retnla mRNA coding for FIZZ1 protein, which promotes deposition of extracellular matrix; and Tgfb with a number of functions in immune system regulation. Interestingly, Retnla and Tgfb were significantly upregulated in EAE–laquinimod cerebellum, suggesting that the upregulation of M2-like-specific markers might contribute to promote central beneficial effects of the drug (Fig. 1d; Arg1 fold change: EAE–vhl 1.132 ± 0.38; EAE–laquinimod 0.875 ± 0.24, unpaired T test p > 0.05; Chil3 fold change: EAE-vhl 1.2 ± 0.46; EAE-laquinimod 1.151 ± 0.28, unpaired T test p > 0.05; Retnla fold change: EAE-vhl 1.026 ± 0.17; EAE-laquinimod 1.608 ± 0.11, unpaired T test p < 0.05; Tgfb fold change: EAE-vhl 1.006 ± 0.007; EAE-laquinimod 1.547 ± 0.12, unpaired T test p < 0.05; EAE-vhl n = 3, EAE-laquinimod n = 4).

Regarding the expression of the T cell marker CD3, we did not observe any difference between the two experimental groups, indicating that the treatment did not affect T cell infiltration in the cerebellum (Fig. 1e; fold change: EAE-vhl 1.30 ± 0.52 n = 3, EAE-laquinimod 1.10 ± 0.46 n = 4; unpaired T test p > 0.05). Moreover, the mRNA of the glial marker, GFAP, was not modulated by laquinimod (Fig. 1f; fold change: EAE-vhl 1.02 ± 0.15 n = 3, EAE-laquinimod 0.67 ± 0.14 n = 4; unpaired T test p > 0.05). Taken together, these data indicate that the preventive and central laquinimod treatment did not induce a prominent effect on inflammation in the cerebellum of EAE mice.

Lastly, we investigated the expression of the Slc1a3 mRNA coding for the glutamate transporter GLAST. Laquinimod upregulated Slc1a3 mRNA (Fig. 1g; fold change: EAE-vhl 1.00 ± 0.05 n = 3, EAE-laquinimod 1.24 ± 0.04 n = 4; unpaired T test p < 0.05), suggesting a possible effect of laquinimod on glutamatergic transmission in EAE cerebellum.

Laquinimod is a small molecule able to passively cross the blood–brain barrier (BBB) that has been shown to rapidly reach the brain in both physiological and pathological conditions such as EAE [19]. To better investigate the direct effect of laquinimod on glutamatergic excitotoxicity, we adopted an ex vivo model of incubation of laquinimod on cerebellar slices taken from EAE mice (21–25 dpi). After 2 h of laquinimod bath application (30 μM), we recorded spontaneous glutamatergic currents (EPSCs) from Purkinje cells (PCs). As previously reported [25], under the EAE conditions, the duration of EPSCs—decay time and half width—were significantly increased compared to control slices (Fig. 2a–d; EAE n = 9, decay time 14.76 ± 1.29 ms, half width 11.36 ± 0.72 ms; CFA n = 7, decay time 8.6 ± 0.71 ms, half width 8.10 ± 0.58 ms; one-way ANOVA Tukey post hoc analysis: EAE vs CFA decay time p < 0.001, half width p < 0.01). Interestingly, laquinimod was able to significantly recover the kinetic alterations of glutamatergic transmission in EAE cerebellum (EAE-laquinimod n = 14, decay time 8.805 ± 0.67 ms, half width 8.81 ± 0.56 ms; one-way ANOVA Tukey post hoc analysis: decay time EAE vs EAE-laquinimod p < 0.001, half width p < 0.05) (Fig. 2a–d). Rise time was unchanged among groups (Fig. 2c, d; EAE 1.36 ± 0.08 ms, CFA 1.09 ± 0.08 ms, EAE-laquinimod 1.36 ± 0.08 ms; one-way ANOVA p > 0.05). Furthermore, laquinimod incubation on EAE slices did not affect the frequency and the amplitude of sEPSC (frequency: CFA 0.85 ± 0.12 Hz, EAE 0.83 ± 0.08 Hz, EAE-laquinimod 0.69 ± 0.10 Hz; amplitude: CFA 11.10 ± 0.57 pA, EAE 11.27 ± 0.42 pA, EAE-laquinimod 12.02 ± 1.29 pA; one-way ANOVA p > 0.05; Fig. 2e, f). Altogether, these results suggest that laquinimod exerts a beneficial effect against EAE-induced postsynaptic alteration.

We speculated whether laquinimod beneficial effect on EAE synaptic alterations could be due to the interference with the excitotoxic mechanism involving the IL-1β-miR-142-3p-GLAST regulatory axis, recently described in [26]. Indeed, GLAST dysfunction in EAE cerebellum depends on a downregulation of the protein which is caused by an IL-1β-dependent upregulation of miR-142-3p, which targets the Slc1a3 mRNA coding for GLAST [25, 26]. Therefore, we first used qPCR to quantify the expression level of IL-1β mRNA, miR-142-3p, and Slc1a3 mRNA in EAE cerebellar slices incubated with 30 μM laquinimod compared with EAE-vhl slices. As shown in Fig. 3a, b, neither IL-1β transcript nor miR142–3p was corrected by laquinimod acute treatment (IL-1β fold change: EAE 1.07 ± 0.15, EAE-laquinimod 1.03 ± 0.21; miR-142-3p fold change: EAE 1.07 ± 0.15, EAE-laquinimod 1.03 ± 0.21; n = 7 per group; unpaired T test p > 0.05 for both comparisons), while we found a significant increase of Slc1a3 mRNA compared to vehicle slices (Fig. 3c; GLAST fold change: EAE-vhl 1.01 ± 0.063, EAE-laquinimod 1.57 ± 0.15; n = 7 per group; unpaired T test p < 0.01). As observed in the icv laquinimod experiments, Slc1a3 mRNA GLAST upregulation was not accompanied by significant changes in GFAP expression (Fig. 3d; GFAP fold change: EAE-vhl 1.01 ± 0.06, EAE-laquinimod 1.03 ± 0.16; n = 7 per group; unpaired T test p > 0.05).

To better characterize the involvement of GLAST in the laquinimod anti-excitotoxic effect, we used WB analysis to quantify GLAST and GFAP protein levels. Consistent with the qPCR quantification, GFAP protein content was unchanged between the two groups (Fig. 3e–g; fold change to EAE-vhl: EAE-vhl 1 ± 0.08 n = 13, EAE-laquinimod 1.24 ± 0.18, n = 14; unpaired T test, p > 0.05), suggesting that laquinimod did not affect astrogliosis in EAE cerebellar slices. Conversely, we could not detect significant differences in GLAST content between the two experimental groups (Fig. 3e, f; fold change to EAE-vhl: EAE-vhl 1 ± 0.09 n = 13, EAE-laquinimod 1.204 ± 0.152, n = 14; unpaired T test, p > 0.05). These data suggest that, although laquinimod induced an upregulation of the Slc1a3 mRNA, GLAST protein synthesis was impaired likely due the presence of high levels of miR-142-3p in EAE cerebellum.

Altogether, these results indicate that laquinimod recovers glutamatergic transmission through a mechanism that is not dependent on GLAST upregulation.

The above results prompted us to investigate another possible mechanism of laquinimod-mediated reduced glutamate excitotoxicity. Although glutamate removal at tripartite synapse in the molecular layer of cerebellum is mediated mainly by GLAST, Bergmann glial (BG) cells express another glutamate transporter GLT-1, which is functionally less relevant and less expressed than GLAST in BG in healthy conditions [30]. We hypothesized that, under laquinimod administration, GLT-1 could compensate for GLAST dysfunction, thereby contributing to maintenance of proper glutamate homeostasis. To address this hypothesis, we first investigated GLT-1 expression in EAE condition and then the possible involvement of GLT-1 in laquinimod-mediated synaptic recovery.

We assessed GLT-1 expression by performing both immunofluorescence experiments and western blot analysis in EAE cerebella taken at 22 dpi, in comparison to control CFA. As shown in Fig. 4a, cerebellar sagittal sections were immunostained for GFAP (green) and GLT-1 (red) to perform confocal imaging. As previously observed, GFAP staining was strikingly increased in EAE slices. Regarding GLT-1 protein, it localized in BG cells and astrocytes in the molecular layer (ml) and granular layer (gl) of the cerebellar cortex as reported by others [31]. In EAE cerebellar slices, GLT-1 staining seemed a bit more intense than CFA (Fig. 4a, a’). To better characterize GLT-1 expression, we assessed Slc1a2 mRNA coding for GLT-1 and protein content in 22 dpi EAE cerebellar lysates compared with experimental control CFA. We did not detect significant differences between the two groups in either Slc1a2 mRNA (Fig. 4a, b; fold change: CFA 1.01 ± 0.06, n = 7, EAE 1.06 ± 0.14, n = 8; unpaired T test p > 0.05) or GLT-1 protein (Fig. 4c, c’; fold change to CFA values: CFA 1 ± 0.17, n = 10, EAE 1.44 ± 0.23, n = 9; unpaired T test p > 0.05), despite a small but non-significant increase of the protein observed in EAE conditions.

We then analyzed the acute effect of laquinimod on GLT-1 expression and protein level in EAE cerebellar slices. Although Slc1a2 mRNA was unchanged between EAE-vhl and EAE-laquinimod slices (Fig. 5a; fold change: EAE-vhl 1.01 ± 0.05 n = 7, EAE-laquinimod 1.06 ± 0.06 n = 7, unpaired T test p > 0.05), a significant increase of the protein in the presence of laquinimod was observed (Fig. 5b, b’; fold change to EAE-vehicle: EAE-vhl 1 ± 0.18 n = 13, EAE-laquinimod 1.88 ± 0.34 n = 14; unpaired T test, p < 0.05). Based on these results, we used IF to investigate the effect of laquinimod on GLT-1 distribution in the cerebellar cortex (Fig. 5c). Astroglia activation was unaffected by laquinimod treatment, as shown by the comparable GFAP (green) staining in EAE and EAE-laquinimod sections. On the contrary, the overall intensity staining for GLT-1 was slightly increased in EAE slices treated with laquinimod (Fig. 5c, c’), corroborating WB findings.

These results point to GLT-1 as a putative target of laquinimod anti-excitotoxic action in the EAE cerebellum. Since laquinimod is reported to induce the expression of the neurotrophin, brain-derived neurotrophic factor (BDNF) in the brain of EAE mice [8] and BDNF positively regulates GluT expression [32]; we wondered whether BDNF could be involved in laquinimod-induced GLT-1 induction. We used qPCR to check the expression of BDNF in cerebellar slices exposed to laquinimod relative to vehicle slices, finding no differences between the two groups (Fig. 5d; fold change: EAE-vhl 1.06 ± 0.15 n = 7; EAE-laquinimod 1.04 ± 0.13 n = 7; unpaired T test p > 0.05) and suggesting that BDNF does not mediate the effect of laquinimod on GLT-1.

To assess the contribution of GLT-1 to the functional recovery of glutamatergic transmission observed in EAE slices in the presence of laquinimod, we recorded sEPSC from PCs of EAE slices co-incubated with both laquinimod and the GLT-1 antagonist DHK. Under this experimental condition, we observed that the beneficial effect of laquinimod on sEPSC kinetic properties was largely prevented by the concomitant incubation with the GLT-1 antagonist DHK. As shown in Fig. 6a, decay time of sEPSC of EAE slices incubated with laquinimod was significantly reduced compared to both EAE-vhl and EAE-laquinimod -DHK (EAE-vhl n = 11, EAE-laquinimod n = 14, EAE-laquinimod -DHK n = 14; decay time: EAE-vhl 12.52 ± 0.617 ms, EAE-laquinimod 8.796 ± 0.670 ms, EAE-laquinimod -DHK 11.25 ± 0.899 ms; one-way ANOVA, Tukey post hoc analysis: EAE-vhl vs EAE-laquinimod p < 0.01). The same effect was observed when analyzing the half-width parameter of sEPSCs (Fig. 6b; half width: EAE-vhl 10.97 ± 0.40 ms; EAE-laquinimod 8.809 ± 0.55 ms; EAE-laquinimod -DHK 9.33 ± 0.78 ms; one-way ANOVA Tukey post hoc analysis: EAE-vhl vs EAE-laquinimod half width p < 0.05). Again, rise time values were unchanged among groups (Fig. 6c; rise time: EAE-vhl 1.416 ± 0.062 ms; EAE-laquinimod 1.362 ± 0.077 ms; EAE-laquinimod-DHK 1.57 ± 0.107 ms).

Taken together, these results indicate that laquinimod acute treatment ameliorates glutamatergic transmission in EAE cerebellum by inducing GLT-1 expression and improving its function.