GABAergic signaling and connectivity on Purkinje cells are impaired in experimental autoimmune encephalomyelitis

Neurobiology of Disease 46 (2012) 414–424

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GABAergic signaling and connectivity on Purkinje cells are impaired in experimental autoimmune encephalomyelitis Georgia Mandolesi a, 1, Giorgio Grasselli a, b, 1, Alessandra Musella a, b, Antonietta Gentile a, Gabriele Musumeci a, b, Helena Sepman a, Nabila Haji a, c, Diego Fresegna b, Giorgio Bernardi a, b, Diego Centonze a, b,⁎ a b c

Fondazione Santa Lucia/Centro Europeo per la Ricerca sul Cervello (CERC), 00143 Rome, Italy Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, 00133 Rome, Italy Department of Neuroscience and National Institute of Neuroscience-Italy, University of Turin, 10125 Turin, Italy

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Article history: Received 18 August 2011 Revised 30 January 2012 Accepted 4 February 2012 Available online 12 February 2012 Keywords: EAE Multiple sclerosis IPSC Cerebellar interneurons PC Parvalbumin IL1-beta VGAT

a b s t r a c t A significant proportion of multiple sclerosis (MS) patients have functionally relevant cerebellar deficits, which significantly contribute to disability. Although clinical and experimental studies have been conducted to understand the pathophysiology of cerebellar dysfunction in MS, no electrophysiological and morphological studies have investigated potential alterations of synaptic connections of cerebellar Purkinje cells (PC). For this reason we analyzed cerebellar PC GABAergic connectivity in mice with MOG(35–55)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We observed a strong reduction in the frequency of the spontaneous inhibitory post-synaptic currents (IPSCs) recorded from PCs during the symptomatic phase of the disease, and in presence of prominent microglia activation not only in the white matter (WM) but also in the molecular layer (ML). The massive GABAergic innervation on PCs from basket and stellate cells was reduced and associated to a decrease of the number of these inhibitory interneurons. On the contrary no significant loss of the PCs could be detected. Incubation of interleukin-1beta (IL-1β) was sufficient to mimic the electrophysiological alterations observed in EAE mice. We thus suggest that microglia and pro-inflammatory cytokines, together with a degeneration of basket and stellate cells and their synaptic terminals, contribute to impair GABAergic transmission on PCs during EAE. Our results support a growing body of evidence that GABAergic signaling is compromised in EAE and in MS, and show a selective susceptibility to neuronal and synaptic degeneration of cerebellar inhibitory interneurons. © 2012 Elsevier Inc. All rights reserved.

Introduction Common symptoms of multiple sclerosis (MS) such as gait ataxia, poor coordination of hands, and intention tremors, are usually the result of lesions in the cerebellum. Besides giving a significant contribution to disability, cerebellar deficits seem also relatively refractory to symptomatic therapy and progress even under disease-modifying agents (Waxman, 2005). The pathophysiology of the cerebellar symptoms in MS is complex and only partially understood (Giovannoni and Ebers, 2007). Cerebellar cortex is a major predilection site for demyelination, in particular in patients with primary and secondary progressive MS (Kutzelnigg et al., 2007). In MS patients and in EAE

⁎ Corresponding author at: Dipartimento di Neuroscienze, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. Fax: + 39 06 7259 6006. E-mail address: [email protected] (D. Centonze). 1 GM and GG contributed equally to this work. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2012.02.005

mice, cerebellar deficits have been associated with cerebellar atrophy caused by PCs death as well as degeneration of neurons in olivary nuclei (Kumar and Timperley, 1988; Chin et al., 2009; MacKenzieGraham et al., 2009). In addition, functional abnormalities in PCs, caused by aberrant expression of surface receptors or ion channels, have been reported both in MS patients and EAE mice. In particular, an atypical repertoire of sodium channels detected in PCs was related to an abnormal bursting activity of PCs (Black et al., 2000; Craner et al., 2003a, 2003b; Renganathan et al., 2003; Saab et al., 2004; Waxman, 2005). Recently, abnormal expression of metabotropic glutamate receptors (Fazio et al., 2008), cannabinoid CB1 receptors (Cabranes et al., 2006; Centonze et al., 2007), and glutamate transporters (MitosekSzewczyk et al., 2008) have been reported in cerebellum during EAE. Altogether these studies suggest that synaptic changes in the cerebellum could contribute to the pathophysiology of MS. A physiological hallmark of MS and of its animal model EAE is an unbalance between glutamatergic and GABAergic transmission accompanied by synaptic degeneration (Centonze et al., 2009, 2010;

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Ziehn et al., 2010; Rossi et al., 2011). These start early before symptoms onset and have been proposed to underlie gray matter dysfunction and also cognitive deficits (Mandolesi et al., 2010). Recently, it has been shown that a decrease in GABAergic signal gives a relevant contribution in the enhancement of neuronal excitability in striatum during EAE, likely representing a further cause of excitotoxic damage together with an increase of glutamatergic transmission (Centonze et al., 2009; Rossi et al., 2011). Moreover, a loss in GABAergic interneurons was observed early in the acute phase of EAE both in hippocampus (Ziehn et al., 2010) and striatum (Rossi et al., 2011), as well as in motor cortex of post-mortem MS patients (Clements et al., 2008). GABA is reduced in the cerebrospinal fluid of MS subjects (Qureshi and Baig, 1988). Furthermore, potentiation of GABA signaling significantly ameliorates EAE clinical course, through a mechanism likely involving a direct neuroprotective effect and an inhibitory action on antigen-presenting cells and the resulting inflammatory response (Bhat et al., 2010). To date, synaptic transmission and connectivity on cerebellar PCs during MS or EAE have never been investigated. In the present work we studied transmission, neuroinflammation and pathology of GABAergic inhibitory interneurons impinging on cerebellar PCs during EAE.

CsCl, 30 K-gluconate, 1.1 EGTA, 10 HEPES, 0.1 CaCl2, 4 Na-ATP, and 0.3 Na-GTP, adjusted to pH 7.3 with CsOH. MK-801 (30 μM) and CNQX (10 μM) were added to the external solution to block, NMDA and non-NMDA glutamate receptors, respectively. Patch pipette resistances were between 2 and 5.5 MΩ. Miniature IPSCs (mIPSCs) were recorded in the presence of the voltage-gated sodium channel blocker tetrodotoxin (TTX, 1 μM). Data were recorded and stored by using pCLAMP 10 (Molecular Devices, Sunnyvale, CA, USA), and analyzed offline on a personal computer by Mini Analysis 5.1 (Synaptosoft, Leonia, NJ, USA) software. The detection threshold of these 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 to 2 min), sampled every 2 or 3 min. Only cells that exhibited stable frequencies and amplitudes were taken into account. Drugs were applied by dissolving them to the desired final concentration in the bathing ACSF and were as follows: Bicuculline (10 μM, Sigma, St. Louis, MO, USA); CNQX (10 μM), MK-801 (30 μM), and TTX (1 μM) were from Tocris Cookson, Bristol, UK; IL-1β (30 ng/ml) was from R&D Systems, Minneapolis, MN, USA). Οne to six cells per animal were recorded. CFA data from pre-symptomatic and acute phase were pooled together since the mean values were not significant different.

Material and methods

Western blot

EAE induction and clinical evaluation

Cerebellum was collected and frozen in liquid nitrogen and stored at −80 °C until use (n = 4 for each experimental group). Cerebellar protein extract was obtained by homogenizing the cerebellum in a buffer containing (in mM): 50 Tris (pH 7.5), 300 NaCl, 1.5 MgCl2, 1 CaCl2, 1 EGTA, and 1% Triton-X, 10% glycerol, 1% protease inhibitor cocktail (Sigma). Crude lysate was centrifuged at 16,000 ×g for 15 min at 4 °C, and the supernatant was collected. Protein concentration of the samples was quantified by Bradford colorimetric reaction. A quantity of 30 μg of cerebellar extract was denaturated at 98 °C for 5 min and loaded onto a sodium dodecyl-sulfate polyacrilamide gel [10% for vesicular GABA transporter (VGAT) and 15% for ionized calcium binding adaptor molecule 1(Iba1) and parvalbumin (PV)]. Gels were (wet) blotted onto a polyvinylidene fluoride (PVDF) membranes. These were blocked for 1 h at RT by 5% non-fat dry milk in 0.1% Tween20-PBS-(T-PBS). All following incubations were performed in T-PBS. Membranes were incubated with specific antibodies in 5% milk over-night at 4 °C (or for 15 min at RT for anti-beta-actin) and after washing they were incubated with secondary HRP-conjugated IgG (Millipore, AP308P, 1:5000) in 5% milk for 1 h at RT (or for 15 min at RT after incubation with anti-beta-actin). Primary antibodies were used as following: mouse anti-beta-actin (1:10000, Sigma-Aldrich, A5441), mouse anti-PV (1:1000 ab 10838 Immunological science), rabbit anti-VGAT (1:5000, Synaptic Systems, Germany, # 131002), mouse anti-Calbindin (Cb) (1:2000, Swant cod. 300), rabbit anti Iba1 (1:500 WAKO, cod. 019-19741). After washing immunodetection was performed by ECL-Plus reagent (Amersham) and the Storm 840 acquisition system (Amersham). Densitometric analysis of protein levels was performed by NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME, USA) were used for all the experiments. EAE was induced in 6–8 weeks animals as previously described (Centonze et al., 2009; Rossi et al., 2010, 2011). Mice were injected subcutaneously at the flanks with 200 μg of myelin oligodendrocyte glycoprotein p35–55 (MOG(35–55)) emulsion to induce EAE by active immunization. The emulsion was prepared under sterile conditions using MOG(35–55) (>85% purity, Espikem, Florence, Italy) in complete Freund's adjuvant (CFA, Difco), and Mycobacterium tuberculosis H37Ra (8 mg/ml; strain H37Ra, Difco, Lawrence, KS, USA) emulsified with phosphate buffered saline (PBS). The control emulsion was prepared the same way without MOG(35–55) for the control group (CFA group). All animals were injected with 500 ng pertussis toxin (Sigma, St. Louis, MO, USA) intravenously on the day of immunization and 2 days later according to standard protocols of EAE induction. Animals were scored daily for clinical symptoms of EAE, according to the following scale: 0, no clinical signs; 1, flaccid tail; 2, hind limb weakness; 3, hind limb paresis; 4, complete bilateral hind limb paralysis; 5, death due to EAE with intervals of 0.5. All efforts were made to minimize animal suffering and to reduce the number of mice used, in accordance with the European Communities Council Directive of 24 November, 1986 (86/609/EEC). Electrophysiology Mice were anesthetised with isoflurane and decapitated, the cerebellum was quickly removed and glued onto the stage of a chamber filled with ice-cold artificial cerebro-spinal fluid (ACSF; in mM: 125 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 20 Glucose) saturated with 95% O2 and 5% CO2. The cerebellar parasagittal slices were then cut with a vibroslicer and kept at 27 °C for 1 h in a chamber containing oxygenated ACSF. After this recovery time, individual slices will be transferred to the recording chamber of an upright microscope, and continuously perfused with oxygenated ACSF at room temperature (RT) during the course of the whole experiment. Whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 2–5 MΩ) at the holding potential (HP) of − 70 mV. To detect spontaneous GABAA-mediated IPSCs, intraelectrode solution with the following composition was used (in mM): 110

Immunohistochemistry and microscopy Immunohistochemistry, microscopy and image analyses were performed similarly to what previously described (Rossi et al., 2011). Briefly, mice at least from 2 to 3 different immunization experiments were sacrificed in the preclinical phase (7 days post immunization, dpi) or at the peak of the symptomatic phase (20 dpi). They were deeply anesthetized and intracardially perfused with ice-cold 4% paraformaldehyde. Brains were post-fixed for 2 h and equilibrated with 30% sucrose at least one overnight. Thirty micrometer-thick sagittal sections were permeabilized in PBS with Triton-X 0.25% (TxPBS). All following incubations were performed in Tx-PBS. Sections

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were pre-incubated with 10% normal donkey serum solution for 1 h at RT and incubated with the primary antibody overnight at + 4 °C, then, after washing, they were incubated with secondary antibodies for 2 h at RT and rinsed. Primary antibodies were used as following: rat anti CD3 (1:250, AbD Serotec, MCA1477.); rabbit anti-Cb (1:500, Swant); mouse anti-Cb (1:1000, Swant); rabbit anti-Iba1 (1:500, Wako, 019-19741); mouse anti-PV (1:250, Sigma-Aldrich, P3088), rabbit anti VGAT (1:500, SYSY, 131002). These were used in combination with the following secondary antibodies: Alexa-488 or Alexa-

647-conjugated donkey anti-mouse (1:200, Invitrogen); Cy3conjugated donkey anti-rabbit or anti-rat (1:200, Jackson). Images from immunolabeled samples were acquired with a Leica TCR SP5 confocal imaging system (Leica Microsystem, Germany). 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. The images had a pixel resolution of at least 1024 × 1024. For the acquisition of inhibitory interneurons a 40 × objective (zoom: 0.5×, z-step: 1.5 μm) was used to

Fig. 1. WM lesions and inflammation in the cerebellum of EAE mice.(A) Low magnification of a sagittal cerebellar section immunostained with Cb showing WM lesions in most of the cerebellum of EAE mice during the acute phase of the disease compared to control, CFA mice (D); (B–C) CD3 + cells are mainly localized in the WM lesions of EAE mice while they are absent in the cerebellum of CFA mice (E–F). (C) An high magnification of WM lesion shows Cb+PC axons bearing dystrophic alterations called torpedoes or thickened segments typical of axotomized PCs. (F) High magnification of WM of control mice showing normal Cb+ axons. (G) The number of PCs per μm of PC layer was not significant different between EAE and CFA groups. Scale bars 100 μm in A, B and D; 20 μm in C and F.

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acquire z-stacks of images from the ML. From each z-stack, images were selected from the first and last 3 μm to reduce the possible variability deriving from the penetration of the anti-PV antibody. For the acquisition of VGAT + terminals on PC soma a 63× oil objective (numerical aperture: 1.4) was used (zoom: 1.5×, z-step: 0.5 μm) and the entire PC cell soma was included in the stack. For the acquisition of microglia, a 20 × objective was used (zoom 0.5×; z-step: 1 μm to acquire z-stacks of images from the ML or from the WM. Each stack was z-projected and exported in TIFF file format and adjusted for brightness and contrast as needed by NIH ImageJ software. Median filters were used to reduce noise on stacks and z-projections. Inhibitory interneurons were identified as parvalbumin+/calbindin(PV +/Cb –) cells and counted in the ML. For quantification of VGAT + synaptic contacts on PC soma we measured the percentage of Cb area that was in contact with VGAT; the overlapping signal was detected by a colocalization software (ImageJ). Microglial cells were

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identified as Iba1 + cells and counted in the ML or in the WM on different images. Their surface was measured on the basis of anti-Iba1 immunostaining. For a different anti-Iba1 immunostaining background, a higher threshold was needed in the ML compared to WM. All measurements were performed on at least 4 images acquired from at least 4 serial sections per animal, from at least 2 independent experiments. Statistical analysis For each type of experiment and time point, at least five mice in each group were employed, unless otherwise specified. Throughout the text “n” refers to the number of cells, unless otherwise specified. Data were presented as the mean ± S.E.M. The significance level was established at p b 0.05. Statistical analysis was performed using a paired or unpaired Student's t-test. Multiple comparisons were

Fig. 2. Activation of microglia in WM and ML of EAE cerebellum during the acute phase of the disease.(A) Western blot analysis of Iba1 expression in cerebella of EAE and CFA mice during the acute phase of the disease. Quantification of Iba1 expression demonstrates a strong up-regulation of Iba1 in EAE mice. Western blot data were normalized to actin and plotted as percentage of CFA mice. (B) Confocal images of Iba1 immunostaining clearly show morphological changes that characterize microglia cells following activation. C and D show Iba1 (red and gray) and Cb (green) staining in cerebellar sagittal sections derived from CFA and EAE mice respectively, during the acute phase of the disease. Both proliferation and hypertrophy of microglial cells were evident in the cerebellar cortex and in the WM. The measurements of the total surface covered by Iba1+ cells (F) (a parameter indicative of both proliferation and morphological changes of microglia cells), their density (E) and their mean cell area (G) indicate a strong microglial reaction in EAE mice both in the WM and the ML relative to CFA. * p b 0.05; ** p b 0.01; *** p b 0.001. Scale bar C–D: 100 μm.

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Table 1 Morphological analysis of microglia activation. Density (1/mm2)

Total surface (%)

Cell surface (μm2/cell)

Pre-symptomatic phase WM_CFA 159.75 ± 11.27 WM_EAE 199.80 ± 10.05 * WM_CFA 20.86 ± 1.80 WM_EAE 50.27 ± 6.63 ***

2.41 ± 0.002 3.6 ± 0.003 ** 0.33 ± 0.005 0.80 ± 0.001 ***

164.96 ± 21.74 188.94 ± 16.38 ns 161.62 ± 17.29 197.82 ± 25.16 ns

Acute phase WM_CFA WM_EAE WM_CFA WM_EAE

4.3 ± 0.4 14.6 ± 4.3 * 0.6 ± 0.1 2.8 ± 0.5 **

188 ± 8 394 ± 87 * 32 ± 2 90 ± 12 **

236 ± 19 374 ± 35 ** 202 ± 25 312 ± 24 *

All numbers are mean values and standard errors. T-test EAE vs CFA ⁎ p b 0.05, ⁎⁎p b 0.01, ⁎⁎⁎ p b 0.001, ns no significant.

analyzed by one-way ANOVA for independent and/or repeated measures followed by Tukey HSD. Results WM lesions and microglia activation in the cerebellum of EAE mice In EAE mice, brain infiltrating T lymphocytes, resident immune cells such as microglia and inflammatory cytokines have been found to be responsible of altered synaptic transmission in the striatum (Centonze et al., 2009; Rossi et al., 2011). Therefore, we first investigated the presence of infiltrating T lymphocytes and the degree of activation of the microglia/macrophage population by immunohistochemistry and western blot in the cerebellum of mice with EAE and compared the results with those in mice only treated with CFA

(control group). During the acute phase of EAE, we observed extensive lesions in the WM in most of the cerebellum labeled by Cb immunostaining (Fig. 1). CD3 + lymphocytes were present at the lesion sites (Figs. 1B–C) and Cb-positive PC axons had dystrophic alterations such as torpedoes or thickened segments (Figs. 1C), which represent a typical response to axotomy in this neuronal type (Dusart and Sotelo, 1994; Rossi et al., 1994; Rossi and Strata, 1995). In order to verify whether PCs undergo early degeneration in EAE during this phase of the disease, we quantified the number of PCs per length of PC layer (Fig. 1G). We did not find a significant difference between the groups (mean cells/100 μm ± SEM: CTR = 3.1 ± 0.15, n = 6 mice; EAE = 3.3 ± 0.15, n = 7 mice; t-test, p = 0.117). Accordingly, no significant difference of Cb expression was found between the groups by western blot (see below). Microglia cells and macrophages undergo proliferation and morphological changes following activation and overexpress Iba1 (Ponomarev et al., 2005). This was observed in striatum during EAE as a consequence of the autoimmune reaction and was proposed as source of the cytokines involved in the impairment of glutamatergic transmission (Centonze et al., 2009). Here we observed also in cerebellum a robust increase of Iba1 expression by western blot in EAE compared to control group (n = 3 per group; p b 0.01) during the acute phase of the disease (Fig. 2A), indicating a strong activation of microglia and macrophages. We then carried out a confocal quantitative analysis for the total surface covered by Iba1 + cells (a parameter indicative of both proliferation and morphological changes of microglia cells), their density and their mean cell area (parameters indicative respectively of proliferation and morphological change) in order to assess whether there was a differential microglia activation in the WM, the major site for the inflammatory reaction due to the presence of myelin, and in the ML, with less myelinated axons and where PC dendrites receive most of their inputs. We observed that both

Fig. 3. Activation of microglia in WM and ML of EAE cerebellum during the presymptomatic phase of the disease.(A–B) Confocal images of Iba1 (red and gray) and Cb (green) staining in the cerebellum of CFA and EAE mice during the presymptomatic phase of the disease. Measurements of the density (C) and of the total surface covered by Iba1 + cells (D) in the WM and ML indicated an early activation before the disease onset. On the other hand, by measuring their mean cell area (E), it seems that only a slight change in the morphology started during this phase. Accordingly, no significant differences were found between CFA and EAE. * p b 0.05; ** p b 0.01; *** p b 0.001. Scale bar: 100 μm.

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proliferation and hypertrophyc morphology (Fig. 2B) characterized not only the WM but also the ML in EAE (Figs. 2C–D). As shown in Figs. 2(E–F–G) all parameters were significant different relative to their control (Table 1). As an early consequence of the autoimmune reaction and neuroinflammation, activation of microglia was previously shown in the striatum of EAE mice also during the presymptomatic phase of the disease together with alterations in synaptic transmission (Centonze et al., 2009; Rossi et al., 2011). Consistently, while rare microglial cells were present in the ML in control animals, many more could be observed in EAE mice (Figs. 3A–B). We thus observed, even during this early phase, a significant alteration of the proliferative state both in WM and ML. Only a slight change in the morphology seemed to start during this phase (Figs. 3C–D–E and Table 1). GABA transmission is impaired in EAE, a role for IL-1β Cerebellar PCs exhibit sustained spontaneous inhibitory activity because of the massive GABAergic innervation from basket and stellate cells in the ML and from neighboring PCs via their collaterals

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(Eccles et al., 1967a,b). We performed whole-cell voltage-clamp recordings on cerebellar slices from EAE mice at the pre-symptomatic phase (7–9 dpi, n = 5 mice), in the acute phase (20–25 dpi; n = 8 mice) and from relative control animals (CFA and CTR, n = 8 and 3 mice respectively), to record sIPSC and mIPSC in PCs from vermal lobules. The drugs MK-801 and CNQX, antagonists respectively of NMDA and non-NMDA glutamate receptors, were used to isolate spontaneous GABAergic activity in EAE and CFA mice (Fig. 4A). The isolation of mIPSC was then obtained by adding TTX (1 μM) to the bathing fluid (Fig. 4B). Both spontaneous and miniature events could be entirely blocked following the application of bicuculline, a selective antagonist of GABAA receptors (not shown). We also recorded from healthy C57/ BL6 female mice, and found that sIPSCs (amplitude: 24.92 ± 3.6 pA, frequency: 4.47 ± 0.288 Hz; n = 7 for both parameters) were comparable to CFA mice (amplitude: 19.95 ± 1.61 pA, frequency 5.46 ± 1.06 Hz; n = 14, unpaired t test p = 0.19 and 0.25). We decided therefore to report only data from CFA mice as the appropriate control group.

Fig. 4. EAE alters GABAergic transmission in the cerebellum.(A and B) The electrophysiological traces are examples of sIPSCs (A) and mIPSC (B) recorded from PCs in control conditions (CFA), during the presymptomatic (7–9 dpi) and acute phase (20–23 dpi) of EAE. (E and F) The frequency of GABA-mediated sIPSCs (E) and mIPSC (F) recorded from PC neurons were normal in the pre-symptomatic phase of EAE, but were down-regulated in the acute phase. (C and D) sIPSC and mIPSC amplitude were unaffected in the presymptomatic and acute phases of EAE. (G–H) Synaptic effects of IL-1β in WT mice. Application of IL-1β in cerebellar slices from WT mice significantly reduced the frequency (G) but not the amplitude (H) of sIPSCs (p b 0.05). Therefore IL-1β mimics the effect of EAE on GABAergic transmission. ** p b 0.01.

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We found that in EAE mice the mean values of the sIPSC amplitude in the pre-symptomatic and acute phase were indistinguishable from those of control mice (EAE pre-symptomatic = 23.8 ± 2.9 pA; n = 8; EAE acute = 17.65 ± 2.81 pA, n = 12; CFA = 19.95 ± 1.61 pA, n = 14; one way ANOVA, p = 0.259) (Fig. 4C). Similar results were obtained for the mIPSC amplitude (EAE presymptomatic = 18.91 ± 2.68 pA; EAE acute= 15.15± 2.35 pA; CFA = 14.98± 2.64 pA; one way ANOVA, p = 0.67) (Fig. 4D). On the contrary, we observed a drastic decrease of the sIPSC frequency in EAE mice during the symptomatic phase compared both to the presymptomatic phase and to control (EAE acute= 1.36 ± 0.26 Hz, n = 12; EAE presymptomatic = 5.75± 1.39 Hz, n = 8; CFA = 5.46 ± 1.06 Hz, n = 14; one way ANOVA, p b 0.01) (Fig. 4E). We observed a significant reduction also for the mIPSC frequency (EAE acute = 0.97 ± 0.25 Hz; EAE presymptomatic = 3.18± 1.04 Hz; CFA = 3.47 ± 0.6 Hz; one way ANOVA, p b 0.01) (Fig. 4F). Increasing evidence shows that in EAE pro-inflammatory cytokines, likely released by infiltrated inflammatory cells and by resident

microglia, mediate tissue damage and neurological deficits by altering synaptic transmission, and thus promoting excitotoxic neuronal damage (Centonze et al., 2009; Rossi et al., 2010, 2011). Thus, in order to clarify how inflammation alters GABA synapses in EAE, we addressed the effect on cerebellar sIPSCs of IL-1β, one of the major pro-inflammatory cytokine involved in EAE-induced brain damage (Zhao and Schwartz, 1998), and recently shown to be involved in the alteration of GABAergic transmission in striatal neurons (Musumeci et al., 2011). To this aim we recorded sIPSCs from cerebellar slices of nonimmunized WT mice before and after incubation with 30 ng/ml IL1β a dose known to induce an electrophysiological effect in acute slices (Musumeci et al., 2011). After 5–10 min of incubation we observed a reduction in the frequency of sIPSCs (80.0 ± 7.2% of basal frequency, paired t-test p = 0.03; n = 4, Fig. 4G) but not in their amplitudes (99.6 ± 6.9%; paired t-test p = 0.6, Fig. 4H), thus mimicking the alterations of GABAergic synapses in the symptomatic EAE mice.

Fig. 5. Loss of GABAergic synaptic contacts on PC somata during the acute phase of EAE.(A) Western blot analysis of VGAT expression in cerebella of EAE and CFA mice during the acute phase of the disease. Quantification of VGAT expression shows no difference between CFA and EAE, suggesting that no massive synaptic degeneration of GABA inputs occurs in the cerebellum of EAE. Western blot data were normalized to actin and plotted as percentage of CFA mice. (B) VGAT immunostaining (red) in the ML of the cerebellar cortex labels the synaptic terminals of basket and stellate cells that impinge on PC dendrites and bodies (Cb green). (C–D) Confocal analysis of the overlapping signal (magenta) between VGAT (red) and Cb (green) to measure the percentage of Cb area in contact with VGAT. Quantification in D shows a reduction of this specific subset of synaptic terminals that can justify in part an impairment of GABA signaling. T-test * p b 0.05. Scale bars 10 μm.

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Altogether these results demonstrate that GABAergic transmission in the cerebellum of EAE mice is compromised during the acute phase of the disease, and suggest that IL-1β could be one of the proinflammatory cytokines involved in such inhibition. Degeneration of inhibitory interneurons in the ML of EAE mice To evaluate if the observed alteration of GABA transmission could derive from lower number of GABAergic synapses in EAE mice, we studied the VGAT and PV, respectively markers of GABA releasing terminals and GABAergic neurons, by means of western blot and immunohistochemistry. No significant difference in VGAT expression levels could be observed by western blot (n = 4 per group, t-test p = 0.15), indicating that no massive synaptic degeneration of GABA releasing terminals occurs in the cerebellum of EAE mice (Fig. 5A). However, many diverse VGAT + inhibitory synapses are present in all the layers of cerebellar cortex and in deep cerebellar nuclei: those made by Golgi cells onto mossy fibers in the granular layer, those made by PCs on the body of other PCs by their collaterals and in the deep cerebellar nuclei, and those impinging on PC soma and dendrites originating from the inhibitory interneurons present in the ML (stellate and basket cells). To assess whether a more specific impairment of GABAergic innervation onto PCs could account for the decreased GABAergic transmission, we looked at GABAergic innervation on PC somata. This consists mainly on basket cells and collateral PC synapses. By measuring the density of VGAT + synaptic contacts on the PC body through immunofluorescent staining (Fig. 5B), we found a reduction of GABA releasing terminals on PCs during EAE acute phase (CFA n = 36, EAE n = 48; n = 5 mice each group; t test p = 0.03) but not in presymptomatic

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phase (CFA n = 18, EAE n = 21; n = 3 mice each group t-test p = 0.56) (Figs. 5C–D). These results indicate that, despite an overall preservation of VGAT expression throughout the cerebellum, GABAergic innervation on PC somata undergoes neurodegeneration, during the acute phase of EAE. This could contribute to the decrease of GABAergic transmission. A reduction of inhibitory interneurons has been shown to occur during EAE both in striatum and hippocampus (Rossi et al., 2011; Ziehn et al., 2010). We then asked whether the number of stellate and basket cells, which are PV + GABAergic interneurons, was also affected in the cerebellum during EAE thus contributing to reduce GABAergic innervation of PCs. No dramatic overall alterations of PV expression were detected in EAE mice by western blot if normalized on β-actin levels (t-test, p = 0.31; Fig. 6A–B). Expression of PV was also calculated in relation to Cb content to verify if any alteration of PV levels could be correlated to PC population. Similarly, no significant differences could be observed in PV expression normalizing it on Cb levels (t-test, p = 0.90; Fig. 6B). Since PCs, preserved at this disease phase, contribute relevantly to the overall cerebellar levels of PV, we analyzed the density of only inhibitory interneurons by immunohistochemistry. We focused our analysis on the ML, where both basket cells and stellate cells reside (Fig. 6C). We found a significant decrease in EAE mice to 81% of their density compared to control (CFA = 320 ± 9 neurons/mm 2; EAE = 260 ± 11 neurons/mm 2; n = 6 animals per group; p b 0.01) (Fig. 6D). These data thus show that the impairment of GABAergic transmission on PC during the acute phase of EAE is, at least partially, due to a reduction in GABAergic innervation and a reduction of GABAergic interneurons themselves.

Fig. 6. Neurodegeneration of PV+ interneurons in the ML of EAE cerebellum.(A–B) Western blot analysis of PV and Cb expression in cerebella of EAE and CFA mice during the acute phase of the disease. (B) Quantification of PV and Cb normalized to actin content (left and center) and quantification of PV normalized to Cb content (right) show undetectable differences between EAE and CFA mice. (C) Confocal images of PV (green) and Cb immunostaining (red) in cerebellar sagittal sections derived from CFA and EAE mice respectively, during the acute phase of disease. Interneurons were identified in the ML as the PV+/Cb− cells. (D) The number of interneurons, counted in the ML on confocal microscopy images and expressed as density (1/mm2), was significantly reduced in EAE mice to 83% of control in the acute phase of the disease. T-test ** p b 0.01. Scale bar 100 μm.

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Discussion In the present work we have shown that in EAE mice PCs receive a reduced inhibitory tone during the symptomatic phase of the disease. The reduction of GABAergic sIPSC frequency was accompanied by synaptic degenerative processes and loss of stellate and basket cells. Such populations of inhibitory neurons belong to the PV + interneurons, a class of neurons particularly susceptible to degeneration in MS and EAE (Dutta et al., 2006; Clements et al., 2008; Rossi et al., 2011). Our findings suggest that an aberrant GABAergic synaptic transmission in the cerebellar cortex may contribute to cerebellar deficits in EAE and likely in MS patients. Previous studies have demonstrated impaired GABA transmission in MS and in EAE (Gottesfeld et al., 1976; Qureshi and Baig, 1988; Clements et al., 2008; Wang et al., 2008; Bhat et al., 2010; Ziehn et al., 2010). Recently, we provided evidence of irreversible alterations of GABA transmission in the striatum of EAE mice and in network of neurons (Rossi et al., 2011). Such changes together with increased glutamatergic transmission in the acute phase of the disease (Centonze et al., 2009; Rossi et al., 2010) seem to represent early events triggering secondary excitotoxicity and inflammatory neurodegeneration. Microglia and proinflammatory cytokines are emerging as fundamental players of such synaptic modifications. In fact, the same electrophysiological changes observed in EAE were mimicked by direct incubation of corticostriatal slices with activated microglial cells, as well as with cytokines produced by microglia (Centonze et al., 2010). Based on this consideration, here we first explored microglia activation in the cerebellum of EAE mice. As reported by other groups in different EAE models, we observed extensive WM lesions and prominent inflammatory cellular staining in all cerebellar cortex (MacKenzie-Graham et al., 2009; Zheng and Bizzozero, 2010). Due to the complexity of microglia activation we investigated two parameters, proliferation and hypertrophic state, during the course of EAE. Early in the disease, the proliferative state was more pronounced than morphological changes. During the acute phase, microglial cells proliferated even more and became hypertrophic. Interestingly, a strong activation was evident also in the ML where there are rare microglial cells and where PC dendrites receive most of the synaptic contacts. By characterizing microglia activation during the disease course and in different neuronal compartments, we aimed at defining morphological hallmarks of EAE in association with the electrophysiological findings. In previous studies we reported early microglia activation in the striatum in association with up-regulation of glutamate-mediated transmission (Centonze et al., 2009) but not with GABAergic downregulation (Rossi et al., 2011). In the present work, we showed that GABA spontaneous release was normal in the presymptomatic phase while a remarkable reduction of the sIPSC frequency was evident during the acute phase. Similar results were observed in EAE striatum (Rossi et al., 2011). Altogether our observations suggest that in EAE the course of GABAergic transmission impairment occur similarly in striatum and cerebellum both in pre symptomatic and acute phase. Interestingly, it seems that the mechanisms at the basis of GABA hypofunctioning are similar in the two brain structures, involving the degeneration of PV + interneurons and potentially the proinflammatory cytokine IL-1β. However, exogenous IL-1β seems to interfere with synaptic transmission at different levels: presynaptically in the cerebellum and postysinaptically in the striatum. In fact, identical application of IL1β (time and exposure) in striatal slices from non-immunized WT mice significantly reduced the amplitude without altering the frequencies of sIPSCs (Musumeci et al., 2011) while in cerebellum the effect was opposite. Recently, we have shown that IL-1β-mediated inhibition of the GABAergic transmission in striatum is enhanced by transient receptor potential vanilloid 1 channels (TRPV1) (Musumeci et al., 2011). Interestingly, a growing body of evidence shows that cytokines regulate neuronal excitability, synaptic

plasticity and injury by interacting specifically with receptors and ion channels (Schäfers and Sorkin, 2008). It has been shown that IL1β can affect TRPV1 receptors (Piper et al., 1999). At the presynaptic level, IL-1β can induce inhibition of voltage-dependent calcium channels and the resulting Ca2+-influx may impact on its ability to reduce neurotransmitter release (Murray et al., 1997; Rada et al., 1991). Regarding its interaction with GABAA receptors, IL-1β can exert dual effects. Exogenous IL-1β was shown to reduce synaptically mediated GABAergic inhibition in dentate gyrus and CA3 pyramidal neurons (Zeise et al., 1997) while it induces opposite effects in CA1 pyramidal neurons (Bellinger et al., 1993). In hypothalamus neurons it increases the presynaptic release of GABA (Tabarean et al., 2006). In our experimental conditions we observed a rapid cytokine action (within minutes) suggesting that it may act through a mechanism based at least partly on the posttranslational modifications of proteins involved in the regulation of GABA release events, such as presynaptic ion channels. With regard to the specific molecular pathway that mediates acute cytokines effect, literature provides evidence for the recruitment of different kinases (e.g. p38, PI3K) (Schäfers and Sorkin, 2008). On the other hand, prolonged exposure of cytokines may also affect ion channels function (Furukawa and Mattson, 1998; Liu et al., 2006) requiring altered gene expression rather than posttranslational modifications of channel proteins. Together with our current and previous observations (Centonze et al., 2009; Rossi et al., 2010; Musumeci et al., 2011), these data show that exogenous application of cytokines on acute brain slices can modulate synaptic transmission. In addition, since we observed that their application in normal slices replicates the electrophysiological effects observed in EAE slices, we do suggest a role of proinflammatory cytokines as mediators of EAE synaptic alterations. In the ML pro-inflammatory cytokines are likely released from activated microglia and potentially also from Bergmann glia, which tightly wraps somata, dendrites, and dendritic spines of PCs and their excitatory and inhibitory synapses (Palay and Chan-Palay, 1974; Brambilla et al., 2005, 2009). Although Bergmann glia also plays essential role in synaptic homeostasis by expressing GABA and glutamate transporters and controlling GABA and glutamate clearance from synapses (Chaudhry et al., 1995; Conti et al., 1999; Tao et al., 2011; Wang et al., 2011), its involvement in the EAE-induced alterations of GABAergic transmission is unlikely, because the reduction of sIPSC and mIPSC currents here reported reflects mainly a presynaptic mechanism. Besides an inhibitory effect of IL-1β on GABAergic signaling, we observed synaptic degeneration and reduction in the number of the PV + interneurons basket and stellate cells. Interestingly, microglia could be instrumental in the synaptic stripping process, as observed in a model of facial nerve transection (Kreutzberg, 1993). Regarding neuronal cell death, selective loss of PV + interneurons was also reported in striatum (Rossi et al., 2011) and hippocampus of EAE mice, in association with significant defects of hippocampus-related memory abilities (Ziehn et al., 2010). In addition, a reduced extension of PV + neurites in the normal appearing gray matter and in the motor cortex of MS patients was reported (Clements et al., 2008; Dutta et al., 2006). In MS patients, defective GABAergic transmission within the motor cortex has also been hypothesized on the basis of neurophysiological findings with paired-pulse transcranial magnetic stimulation (Caramia et al., 2004). The reasons of the selective susceptibility of PV + inhibitory interneurons to degenerate in MS and in EAE have to be further investigated. We observed that PCs, which express both calcium-binding proteins PV and Cb, survive during the acute phase of the disease. However, swollen PC axons and axon retraction bulbs, called torpedos, were observed in the lesion site in the presence of infiltrating lymphocytes. Such virtually absent PC degeneration is consistent with their known low susceptibility to cell death induced by axotomy (Carulli et al., 2004). In fact, axotomised PCs survive after axotomy

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for a very long time, and only rare neurons close to the lesion site display degenerative changes (Buffo et al., 1997; Dusart and Sotelo, 1994). However, we cannot exclude the possibility that a PC loss occurs later on, consistently with data from some EAE models and MS patients showing a moderate reduction of PCs (Kutzelnigg et al., 2007; MacKenzie-Graham et al., 2009; Giuliani et al., 2011). Recent findings have shown PC degeneration only at later stages of the disease in a model of EAE mice in which the disease was exacerbated by a booster immunization. In particular, a reduction in the PC number (20%) was correlated to a decrease in ML volume (7.3%) (MacKenzie-Graham et al., 2009). Therefore, PC degeneration may occur at later stages induced by other insults typical of MS such as acquired channelopathies, altered activity of sodium channel exchanger, glutamate mediated ecitotoxicity, intraneuronal calcium accumulation and inhibition of mitochondrial respiratory chain. In addition, our observation suggests that, beside PC degeneration, loss of PV + interneurons may contribute to generate an atrophy of the ML as the disease progresses. In conclusion, the alterations of the GABAergic system observed in the EAE cerebellum and recurrent in other brain regions seem to represent clear hallmarks of the EAE model and play a crucial role in EAE pathology. In addition, our findings highlight important aspect for understanding cerebellar neuropathology in MS and EAE. Although further studies are needed to determine whether GABAergic transmission modulation could be successful in MS therapy, pharmacological compounds able to interfere with the chain of events that brings to GABAergic impairment are likely to exert neuroprotective effects in MS patients. Acknowledgments We wish to thank Massimo Tolu and Vladimiro Batocchi for helpful technical assistance. We want also to thank Prof. Piergiorgio Strata for his support. This investigation was supported by the Italian National Ministero della Salute to DC, by Fondazione TERCAS to DC, and by Fondazione Italiana Sclerosi Multipla (FISM) to DC, by a grant from the European Community (AXREGEN: Axonal regeneration, plasticity & stem cells— Grant agreement 21 4003) founding PhD fellowship of NH. References Bellinger, F.P., Madamba, S., Siggins, G.R., 1993. Interleukin 1 beta inhibits synaptic strength and long-term potentiation in the rat CA1 hippocampus. Brain Res. 628, 227–234. Bhat, R., Axtell, R., Mitra, A., Miranda, M., Lock, C., Tsien, R.W., Steinman, L., 2010. Inhibitory role for GABA in autoimmune inflammation. Proc. Natl. Acad. Sci. U. S. A. 107, 2580–2585. Black, J.A., Dib-Hajj, S., Baker, D., Newcombe, J., Cuzner, M.L., Waxman, S.G., 2000. Sensory neuron-specific sodium channel SNS is abnormally expressed in the brains of mice with experimental allergic encephalomyelitis and humans with multiple sclerosis. Proc. Natl. Acad. Sci. U. S. A. 97, 11598–11602. Brambilla, R., Bracchi-Ricard, V., Hu, W.H., Frydel, B., Bramwell, A., Karmally, S., Green, E.J., Bethea, J.R., 2005. Inhibition of astroglial nuclear factor kappaB reduces inflammation and improves functional recovery after spinal cord injury. J. Exp. Med. 202 (1), 145–156. Brambilla, R., Persaud, T., Hu, X., Karmally, S., Shestopalov, V.I., Dvoriantchikova, G., Ivanov, D., Nathanson, L., Barnum, S.R., Bethea, J.R., 2009. Transgenic inhibition of astroglial NF-kappa B improves functional outcome in experimental autoimmune encephalomyelitis by suppressing chronic central nervous system inflammation. J. Immunol. 182 (5), 2628–2640. Buffo, A., Holtmaat, A.J., Savio, T., Verbeek, J.S., Oberdick, J., Oestreicher, A.B., Gispen, W.H., Verhaagen, J., Rossi, F., Strata, P., 1997. Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants. J. Neurosci. 17, 8778–8791. Cabranes, A., Pryce, G., Baker, D., Fernández-Ruiz, J., 2006. Changes in CB1 receptors in motor-related brain structures of chronic relapsing experimental allergic encephalomyelitis mice. Brain Res. 1107, 199–205. Caramia, M.D., Palmieri, M.G., Desiato, M.T., Boffa, L., Galizia, P., Rossini, P.M., Centonze, D., Bernardi, G., 2004. Brain excitability changes in the relapsing and remitting phases of multiple sclerosis: a study with transcranial magnetic stimulation. Clin. Neurophysiol. 115, 956–965.

423

Carulli, D., Buffo, A., Strata, P., 2004. Reparative mechanisms in the cerebellar cortex. Prog. Neurobiol. 72, 373–398. Centonze, D., Bari, M., Rossi, S., Prosperetti, C., Furlan, R., Fezza, F., De Chiara, V., Battistini, L., Bernardi, G., Bernardini, S., Martino, G., Maccarrone, M., 2007. The endocannabinoid system is dysregulated in multiple sclerosis and in experimental autoimmune encephalomyelitis. Brain 130, 2543–2553. Centonze, D., Muzio, L., Rossi, S., Cavasinni, F., De Chiara, V., Bergami, A., Musella, A., D'Amelio, M., Cavallucci, V., Martorana, A., Bergamaschi, A., Cencioni, M.T., Diamantini, A., Butti, E., Comi, G., Bernardi, G., Cecconi, F., Battistini, L., Furlan, R., Martino, G., 2009. Inflammation triggers synaptic alteration and degeneration in experimental autoimmune encephalomyelitis. J. Neurosci. 29, 3442–3452. Centonze, D., Muzio, L., Rossi, S., Furlan, R., Bernardi, G., Martino, G., 2010. The link between inflammation, synaptic transmission and neurodegeneration in multiple sclerosis. Cell Death Differ. 17, 1083–1091. Chaudhry, F.A., Lehre, K.P., van Lookeren Campagne, M., Ottersen, O.P., Danbolt, N.C., Storm-Mathisen, J., 1995. Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultra structural immunocytochemistry. Neuron 15, 711–720. Chin, C.L., Pai, M., Bousquet, P.F., Schwartz, A.J., O'Connor, E.M., Nelson, C.M., Hradil, V.P., Cox, B.F., McRae, B.L., Fox, G.B., 2009. Distinct spatiotemporal pattern of CNS lesions revealed by USPIO-enhanced MRI in MOG-induced EAE rats implicates the involvement of spino-olivocerebellar pathways. J. Neuroimmunol. 211, 49–55. Clements, R.J., McDonough, J., Freeman, E.J., 2008. Distribution of parvalbumin and calretinin immunoreactive interneurons in motor cortex from multiple sclerosis postmortem tissue. Exp. Brain Res. 187, 459–465. Conti, F., Zuccarello, L.V., Barbaresi, P., Minelli, A., Brecha, N.C., Melone, M., 1999. Neuronal, glial and epithelial localization of γ-aminobutyric acid transporter 2, a highaffinity γ-aminobutyric acid plasma membrane transporter, in the cerebral cortex and neighboring structures. J. Comp. Neurol. 409, 482–494. Craner, M.J., Lo, A.C., Black, J.A., Baker, D., Newcombe, J., Cuzner, M.L., Waxman, S.G., 2003a. Annexin II/p11 is up-regulated in Purkinje cells in EAE and MS. NeuroReport 14, 555–558. Craner, M.J., Kataoka, Y., Lo, A.C., Black, J.A., Baker, D., Waxman, S.G., 2003b. Temporal course of upregulation of Nav1.8 in Purkine neurons parallels the progression of clinical deficit in EAE. J. Neuropathol. Exp. Neurol. 62, 968–976. Dusart, I., Sotelo, C., 1994. Lack of Purkinje cell loss in adult rat cerebellum following protracted axotomy: degenerative changes and regenerative attempts of the severed axons. J. Comp. Neurol. 347, 211–232. Dutta, R., McDonough, J., Yin, X., Peterson, J., Chang, A., Torres, T., Gudz, T., Macklin, W.B., Lewis, D.A., Fox, R.J., Rudick, R., Mirnics, K., Trapp, B.D., 2006. Mitochondrial dysfunction as a cause of axonal degeneration in multiple sclerosis patients. Ann. Neurol. 59, 478–489. Eccles, J.C., Sasaki, K., Strata, P., 1967a. A comparison of the inhibitory actions of Golgi cells and of basket cells. Exp. Brain Res. 3, 81–94. Eccles, J.C., Ito, M., Szentágothai, J., 1967b. The Cerebellum as a Neuronal Machine. Springer-Verlag Press, Berlin. Fazio, F., Notartomaso, S., Aronica, E., Storto, M., Battaglia, G., Vieira, E., Gatti, S., Bruno, V., Biagioni, F., Gradini, R., Nicoletti, F., Di Marco, R., 2008. Switch in the expression of mGlu1 and mGlu5 metabotropic glutamate receptors in the cerebellum of mice developing experimental autoimmune encephalomyelitis and in autoptic cerebellar samples from patients with multiple sclerosis. Neuropharmacology 55, 491–499. Furukawa, K., Mattson, M.P., 1998. The transcription factor NF-kappaB mediates increases in calcium currents and decreases in NMDA- and AMPA/kainateinduced currents induced by tumor necrosis factor-alpha in hippocampal neurons. J. Neurochem. 70, 1876–1886. Giovannoni, G., Ebers, G., 2007. Multiple sclerosis: the environment and causation. Curr. Opin. Neurol. 20, 261–268. Giuliani, F., Catz, I., Johnson, E., Resch, L., Warren, K., 2011. Loss of purkinje cells is associated with demyelination in multiple sclerosis. Can. J. Neurol. Sci. 38, 529–531. Gottesfeld, Z., Teitelbaum, D., Webb, C., Arnon, R., 1976. Changes in the GABA system in experimental allergic encephalomyelitis-induced paralysis. J. Neurochem. 27, 695–699. Kreutzberg, G.W., 1993. Dynamic changes in motoneurons during regeneration. Restor. Neurol. Neurosci. 5, 59–60. Kumar, D., Timperley, W.R., 1988. The clinical, pathological and genetic aspects of sporadic late onset cerebellar ataxia: observations on a series of ten patients. Acta Neurol. Scand. 77, 181–186. Kutzelnigg, A., Faber-Rod, J.C., Bauer, J., Lucchinetti, C.F., Sorensen, P.S., Laursen, H., Stadelmann, C., Brück, W., Rauschka, H., Schmidbauer, M., Lassmann, H., 2007. Widespread demyelination in the cerebellar cortex in multiple sclerosis. Brain Pathol. 17, 38–44. Liu, L., Yang, T.M., Liedtke, W., Simon, S.A., 2006. Chronic IL-1 beta signaling potentiates voltage-dependent sodium currents in trigeminal nociceptive neurons. J. Neurophysiol. 95, 1478–1490. MacKenzie-Graham, A., Tiwari-Woodruff, S.K., Sharma, G., Aguilar, C., Vo, K.T., Strickland, L.V., Morales, L., Fubara, B., Martin, M., Jacobs, R.E., Johnson, G.A., Toga, A.W., Voskuhl, R.R., 2009. Purkinje cell loss in experimental autoimmune encephalomyelitis. NeuroImage 48, 637–651. Mandolesi, G., Grasselli, G., Musumeci, G., Centonze, D., 2010. Cognitive deficits in experimental autoimmune encephalomyelitis: neuroinflammation and synaptic degeneration. Neurol. Sci. 31, S255–S259. Mitosek-Szewczyk, K., Sulkowski, G., Stelmasiak, Z., Struzyńska, L., 2008. Expression of glutamate transporters GLT-1 and GLAST in different regions of rat brain during the course of experimental autoimmune encephalomyelitis. Neuroscience 155, 45–52.

424

G. Mandolesi et al. / Neurobiology of Disease 46 (2012) 414–424

Murray, C.A., McGahon, B., McBennett, S., Lynch, M.A., 1997. Interleukin-1 beta inhibits glutamate release in hippocampus of young, but not aged, rats. Neurobiol. Aging 18, 343–348. Musumeci, G., Grasselli, G., Rossi, S., De Chiara, V., Musella, A., Motta, C., Studer, V., Bernardi, G., Haji, N., Sepman, H., Fresegna, D., Maccarrone, M., Mandolesi, G., Centonze, D., 2011. Transient receptor potential vanilloid 1 channels modulate the synaptic effects of TNF-alpha and of IL-1beta in experimental autoimmune encephalomyelitis. Neurobiol. Dis. 43, 669–677. Palay, S.L., Chan-Palay, V., 1974. Cerebellar Cortex: Cytology and Organisation. Springer Press, Berlin. Piper, A.S., Yeats, J.C., Bevan, S., Docherty, R.J., 1999. A study of the voltage dependence of capsaicin-activated membrane currents in rat sensory neurones before and after acute desensitisation. J. Physiol. 518, 721–733. Ponomarev, E.D., Shriver, L.P., Maresz, K., Dittel, B.N., 2005. Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J. Neurosci. Res. 81, 374–389. Qureshi, G.A., Baig, M.S., 1988. Quantitation of free amino acids in biological samples by high-performance liquid chromatography. Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis. J. Chromatogr. 459, 237–244. Rada, P., Mark, G.P., Vitek, M.P., Mangano, R.M., Blume, A.J., Beer, B., Hoebel, B.G., 1991. Interleukin-1 beta decreases acetylcholine measured by microdialysis in the hippocampus of freely moving rats. Brain Res. 550, 287–290. Renganathan, M., Gelderblom, M., Black, J.A., Waxman, S.G., 2003. Expression of Nav1.8 sodium channels perturbs the firing patterns of cerebellar Purkinje cells. Brain Res. 959, 235–243. Rossi, F., Strata, P., 1995. Reciprocal trophic interactions in the adult climbing fibrePurkinje cell system. Prog. Neurobiol. 47, 341–369. Rossi, F., Borsello, T., Strata, P., 1994. Embryonic Purkinje cells grafted on the surface of the adult uninjured rat cerebellum migrate in the host parenchyma and induce sprouting of intact climbing fibres. Eur. J. Neurosci. 6, 121–136. Rossi, S., De Chiara, V., Furlan, R., Musella, A., Cavasinni, F., Muzio, L., Bernardi, G., Martino, G., Centonze, D., 2010. Abnormal activity of the Na/Ca exchanger enhances glutamate transmission in experimental autoimmune encephalomyelitis. Brain Behav. Immun. 24, 1379–1385. Rossi, S., Muzio, L., De Chiara, V., Grasselli, G., Musella, A., Musumeci, G., Mandolesi, G., De Ceglia, R., Maida, S., Biffi, E., Pedrocchi, A., Menegon, A., Bernardi, G.,

Furlan, R., Martino, G., Centonze, D., 2011. Impaired striatal GABA transmission in experimental autoimmune encephalomyelitis. Brain Behav. Immun. 25, 947–956. Saab, C.Y., Craner, M.J., Kataoka, Y., Waxman, S.G., 2004. Abnormal Purkinje cell activity in vivo in experimental allergic encephalomyelitis. Exp. Brain Res. 158, 1–8. Schäfers, M., Sorkin, L., 2008. Effect of cytokines on neuronal excitability. Neurosci. Lett. 437, 188–193. Tabarean, I.V., Korn, H., Bartfai, T., 2006. Interleukin-1beta induces hyperpolarization and modulates synaptic inhibition in preoptic and anterior hypothalamic neurons. Neuroscience 141, 1685–1695. Tao, J., Wu, H., Lin, Q., Wei, W., Lu, X.H., Cantle, J.P., Ao, Y., Olsen, R.W., Yang, X.W., Mody, I., Sofroniew, M.V., Sun, Y.E., 2011. Deletion of astroglial dicer causes noncell-autonomous neuronal dysfunction and degeneration. J. Neurosci. 31, 8306–8319. Wang, Y., Feng, D., Liu, G., Luo, Q., Xu, Y., Lin, S., Fei, J., Xu, L., 2008. Gamma-aminobutyric acid transporter 1 negatively regulates T cell-mediated immune responses and ameliorates autoimmune inflammation in the CNS. J. Immunol. 181, 8226–8236. Wang, X., Imura, T., Sofroniew, M.V., 2011. Fushiki S Loss of adenomatous polyposis coli in Bergmann glia disrupts their unique architecture and leads to cell nonautonomous neurodegeneration of cerebellar Purkinje neurons. Glia 59, 857–868. Waxman, S.G., 2005. Cerebellar dysfunction in multiple sclerosis: evidence for an acquired channelopathy. Prog. Brain Res. 148, 353–365. Zeise, M.L., Espinoza, J., Morales, P., Nalli, A., 1997. Interleukin-1beta does not increase synaptic inhibition in hippocampal CA3 pyramidal and dentate gyrus granule cells of the rat in vitro. Brain Res. 768, 341–344. Zhao, B., Schwartz, J.P., 1998. Involvement of cytokines in normal CNS development and neurological diseases: recent progress and perspectives. J. Neurosci. Res. 52, 7–16. Zheng, J., Bizzozero, O.A., 2010. Accumulation of protein carbonyls within cerebellar astrocytes in murine experimental autoimmune encephalomyelitis. J. Neurosci. Res. 88, 3376–3385. Ziehn, M.O., Avedisian, A.A., Tiwari-Woodruff, S., Voskuhl, R.R., 2010. Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE. Lab. Invest. 90, 774–786.