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Neurobiology of Disease
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GABAergic signaling and connectivity on Purkinje cells are impaired in experimentalautoimmune 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 Fondazione Santa Lucia/Centro Europeo per la Ricerca sul Cervello (CERC), 00143 Rome, Italyb Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, 00133 Rome, Italyc Department of Neuroscience and National Institute of Neuroscience-Italy, University of Turin, 10125 Turin, Italy
⁎ Corresponding author at: Dipartimento di NeuroscVia Montpellier 1, 00133 Rome, Italy. Fax: +39 06 7259
E-mail address: [email protected] (D. Centonze1 GM and GG contributed equally to this work.
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 conductedto understand the pathophysiology of cerebellar dysfunction in MS, no electrophysiological and morpholog-ical 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 experi-mental autoimmune encephalomyelitis (EAE), a mouse model of MS. We observed a strong reduction inthe frequency of the spontaneous inhibitory post-synaptic currents (IPSCs) recorded from PCs during thesymptomatic phase of the disease, and in presence of prominent microglia activation not only in the whitematter (WM) but also in the molecular layer (ML). The massive GABAergic innervation on PCs from basketand 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 basketand stellate cells and their synaptic terminals, contribute to impair GABAergic transmission on PCs dur-ing EAE. Our results support a growing body of evidence that GABAergic signaling is compromised inEAE and in MS, and show a selective susceptibility to neuronal and synaptic degeneration of cerebellarinhibitory interneurons.
Common symptoms of multiple sclerosis (MS) such as gait ataxia,poor coordination of hands, and intention tremors, are usually the re-sult of lesions in the cerebellum. Besides giving a significant contribu-tion to disability, cerebellar deficits seem also relatively refractory tosymptomatic therapy and progress even under disease-modifyingagents (Waxman, 2005). The pathophysiology of the cerebellar symp-toms in MS is complex and only partially understood (Giovannoniand Ebers, 2007). Cerebellar cortex is a major predilection site for de-myelination, in particular in patients with primary and secondaryprogressive MS (Kutzelnigg et al., 2007). In MS patients and in EAE
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mice, cerebellar deficits have been associated with cerebellar atrophycaused by PCs death as well as degeneration of neurons in olivary nu-clei (Kumar and Timperley, 1988; Chin et al., 2009; MacKenzie-Graham 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 relatedto an abnormal bursting activity of PCs (Black et al., 2000; Craneret al., 2003a, 2003b; Renganathan et al., 2003; Saab et al., 2004;Waxman, 2005).
Recently, abnormal expression of metabotropic glutamate recep-tors (Fazio et al., 2008), cannabinoid CB1 receptors (Cabranes et al.,2006; Centonze et al., 2007), and glutamate transporters (Mitosek-Szewczyk et al., 2008) have been reported in cerebellum duringEAE. Altogether these studies suggest that synaptic changes in thecerebellum could contribute to the pathophysiology of MS.
A physiological hallmark of MS and of its animal model EAE is anunbalance between glutamatergic and GABAergic transmission ac-companied by synaptic degeneration (Centonze et al., 2009, 2010;
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Ziehn et al., 2010; Rossi et al., 2011). These start early before symp-toms onset and have been proposed to underlie gray matter dysfunc-tion and also cognitive deficits (Mandolesi et al., 2010). Recently, ithas been shown that a decrease in GABAergic signal gives a relevantcontribution in the enhancement of neuronal excitability in striatumduring EAE, likely representing a further cause of excitotoxic damagetogether with an increase of glutamatergic transmission (Centonze etal., 2009; Rossi et al., 2011). Moreover, a loss in GABAergic interneu-rons was observed early in the acute phase of EAE both in hippocam-pus (Ziehn et al., 2010) and striatum (Rossi et al., 2011), as well as inmotor cortex of post-mortem MS patients (Clements et al., 2008).GABA is reduced in the cerebrospinal fluid of MS subjects (Qureshiand Baig, 1988). Furthermore, potentiation of GABA signaling signifi-cantly ameliorates EAE clinical course, through amechanism likely in-volving a direct neuroprotective effect and an inhibitory action onantigen-presenting cells and the resulting inflammatory response(Bhat et al., 2010).
To date, synaptic transmission and connectivity on cerebellar PCsduring MS or EAE have never been investigated. In the present workwe studied transmission, neuroinflammation and pathology ofGABAergic inhibitory interneurons impinging on cerebellar PCs dur-ing EAE.
Material and methods
EAE induction and clinical evaluation
Female C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME,USA) were used for all the experiments. EAE was induced in6–8 weeks animals as previously described (Centonze et al., 2009;Rossi et al., 2010, 2011). Mice were injected subcutaneously at theflanks with 200 μg of myelin oligodendrocyte glycoprotein p35–55(MOG(35–55)) emulsion to induce EAE by active immunization. Theemulsion 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 phosphatebuffered saline (PBS). The control emulsion was prepared the sameway without MOG(35–55) for the control group (CFA group). All ani-mals were injected with 500 ng pertussis toxin (Sigma, St. Louis,MO, USA) intravenously on the day of immunization and 2 dayslater according to standard protocols of EAE induction. Animalswere scored daily for clinical symptoms of EAE, according to the fol-lowing 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 reducethe number of mice used, in accordance with the European Commu-nities Council Directive of 24 November, 1986 (86/609/EEC).
Electrophysiology
Mice were anesthetised with isoflurane and decapitated, the cere-bellum was quickly removed and glued onto the stage of a chamberfilled with ice-cold artificial cerebro-spinal fluid (ACSF; in mM: 125NaCl, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, 26 NaHCO3, 20 Glucose)saturated with 95% O2 and 5% CO2. The cerebellar parasagittal sliceswere then cut with a vibroslicer and kept at 27 °C for 1 h in a chambercontaining oxygenated ACSF. After this recovery time, individualslices will be transferred to the recording chamber of an upright mi-croscope, and continuously perfused with oxygenated ACSF at roomtemperature (RT) during the course of the whole experiment.Whole-cell patch clamp recordings were made with borosilicateglass pipettes (1.8 mm o.d.; 2–5 MΩ) at the holding potential (HP)of −70 mV. To detect spontaneous GABAA-mediated IPSCs, intraelec-trode solution with the following composition was used (in mM): 110
CsCl, 30 K-gluconate, 1.1 EGTA, 10 HEPES, 0.1 CaCl2, 4 Na-ATP, and 0.3Na-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 andnon-NMDA glutamate receptors, respectively. Patch pipette resis-tances were between 2 and 5.5 MΩ. Miniature IPSCs (mIPSCs) wererecorded in the presence of the voltage-gated sodium channel blockertetrodotoxin (TTX, 1 μM). Data were recorded and stored by usingpCLAMP 10 (Molecular Devices, Sunnyvale, CA, USA), and analyzedoffline on a personal computer byMini Analysis 5.1 (Synaptosoft, Leo-nia, NJ, USA) software. The detection threshold of these events wasset at twice the baseline noise. Positive events were confirmed by vi-sual inspection for each experiment. Analysis was performed onspontaneous synaptic events recorded during a fixed time epoch (1to 2 min), sampled every 2 or 3 min. Only cells that exhibited stablefrequencies and amplitudes were taken into account.
Drugs were applied by dissolving them to the desired final concen-tration 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) wasfrom R&D Systems, Minneapolis, MN, USA). Οne to six cells per animalwere recorded. CFA data from pre-symptomatic and acute phase werepooled together since the mean values were not significant different.
Western blot
Cerebellum was collected and frozen in liquid nitrogen and storedat −80 °C until use (n=4 for each experimental group). Cerebellarprotein extract was obtained by homogenizing the cerebellum in abuffer containing (in mM): 50 Tris (pH 7.5), 300 NaCl, 1.5 MgCl2, 1CaCl2, 1 EGTA, and 1% Triton-X, 10% glycerol, 1% protease inhibitorcocktail (Sigma). Crude lysate was centrifuged at 16,000×g for15 min at 4 °C, and the supernatant was collected. Protein concentra-tion of the samples was quantified by Bradford colorimetric reaction.A quantity of 30 μg of cerebellar extract was denaturated at 98 °C for5 min and loaded onto a sodium dodecyl-sulfate polyacrilamide gel[10% for vesicular GABA transporter (VGAT) and 15% for ionized calci-um binding adaptor molecule 1(Iba1) and parvalbumin (PV)]. Gelswere (wet) blotted onto a polyvinylidene fluoride (PVDF) mem-branes. These were blocked for 1 h at RT by 5% non-fat dry milk in0.1% Tween20-PBS-(T-PBS). All following incubations were performedin 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) andafter washing they were incubated with secondary HRP-conjugatedIgG (Millipore, AP308P, 1:5000) in 5% milk for 1 h at RT (or for 15 minat RT after incubation with anti-beta-actin). Primary antibodies wereused as following: mouse anti-beta-actin (1:10000, Sigma-Aldrich,A5441), mouse anti-PV (1:1000 ab 10838 Immunological science), rab-bit anti-VGAT (1:5000, Synaptic Systems, Germany, # 131002), mouseanti-Calbindin (Cb) (1:2000, Swant cod. 300), rabbit anti Iba1 (1:500WAKO, cod. 019-19741). After washing immunodetection was per-formed by ECL-Plus reagent (Amersham) and the Storm 840 acquisitionsystem (Amersham). Densitometric analysis of protein levels was per-formed by NIH ImageJ software (http://rsb.info.nih.gov/ij/).
Immunohistochemistry and microscopy
Immunohistochemistry, microscopy and image analyses wereperformed similarly to what previously described (Rossi et al.,2011). Briefly, mice at least from 2 to 3 different immunization exper-iments were sacrificed in the preclinical phase (7 days post immuni-zation, dpi) or at the peak of the symptomatic phase (20 dpi). Theywere deeply anesthetized and intracardially perfused with ice-cold4% paraformaldehyde. Brains were post-fixed for 2 h and equilibratedwith 30% sucrose at least one overnight. Thirty micrometer-thick sag-ittal sections were permeabilized in PBS with Triton-X 0.25% (Tx-PBS). All following incubations were performed in Tx-PBS. Sections
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were pre-incubated with 10% normal donkey serum solution for 1 hat RT and incubated with the primary antibody overnight at +4 °C,then, after washing, they were incubated with secondary antibodiesfor 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 combina-tion with the following secondary antibodies: Alexa-488 or Alexa-
Fig. 1. WM lesions and inflammation in the cerebellum of EAE mice.(A) Low magnificationthe cerebellum of EAE mice during the acute phase of the disease compared to control, CFAthey are absent in the cerebellum of CFA mice (E–F). (C) An high magnification of WM lesionments typical of axotomized PCs. (F) High magnification of WM of control mice showing nobetween EAE and CFA groups. Scale bars 100 μm in A, B and D; 20 μm in C and F.
Images from immunolabeled samples were acquired with a LeicaTCR SP5 confocal imaging system (Leica Microsystem, Germany).The confocal pinhole was kept at 1.0, the gain and the offset werelowered to prevent saturation in the brightest signals and sequentialscanning for each channel was performed. The images had a pixel res-olution of at least 1024×1024. For the acquisition of inhibitory inter-neurons a 40× objective (zoom: 0.5×, z-step: 1.5 μm) was used to
of a sagittal cerebellar section immunostained with Cb showing WM lesions in most ofmice (D); (B–C) CD3+ cells are mainly localized in the WM lesions of EAE mice whileshows Cb+PC axons bearing dystrophic alterations called torpedoes or thickened seg-
rmal Cb+ axons. (G) The number of PCs per μm of PC layer was not significant different
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acquire z-stacks of images from the ML. From each z-stack, imageswere selected from the first and last 3 μm to reduce the possible var-iability deriving from the penetration of the anti-PV antibody. For theacquisition of VGAT+ terminals on PC soma a 63× oil objective (nu-merical aperture: 1.4) was used (zoom: 1.5×, z-step: 0.5 μm) andthe entire PC cell soma was included in the stack. For the acquisitionof microglia, a 20× objective was used (zoom 0.5×; z-step: 1 μm toacquire z-stacks of images from the ML or from the WM. Each stackwas z-projected and exported in TIFF file format and adjusted forbrightness and contrast as needed by NIH ImageJ software. Medianfilters were used to reduce noise on stacks and z-projections. Inhibi-tory 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 Cbarea that was in contact with VGAT; the overlapping signal wasdetected by a colocalization software (ImageJ). Microglial cells were
Fig. 2. Activation of microglia in WM and ML of EAE cerebellum during the acute phase of thduring the acute phase of the disease. Quantification of Iba1 expression demonstrates a stroplotted as percentage of CFA mice. (B) Confocal images of Iba1 immunostaining clearly showshow Iba1 (red and gray) and Cb (green) staining in cerebellar sagittal sections derived fromand hypertrophy of microglial cells were evident in the cerebellar cortex and in the WM. Thof both proliferation and morphological changes of microglia cells), their density (E) and thand the ML relative to CFA. * pb0.05; ** pb0.01; *** pb0.001. Scale bar C–D: 100 μm.
identified as Iba1+ cells and counted in the ML or in the WM on dif-ferent images. Their surface was measured on the basis of anti-Iba1immunostaining. For a different anti-Iba1 immunostaining back-ground, a higher threshold was needed in the ML compared to WM.All measurements were performed on at least 4 imagesacquired from at least 4 serial sections per animal, from at least 2 in-dependent experiments.
Statistical analysis
For each type of experiment and time point, at least five mice ineach group were employed, unless otherwise specified. Throughoutthe text “n” refers to the number of cells, unless otherwise specified.Data were presented as the mean±S.E.M. The significance level wasestablished at pb0.05. Statistical analysis was performed using apaired or unpaired Student's t-test. Multiple comparisons were
e disease.(A) Western blot analysis of Iba1 expression in cerebella of EAE and CFA miceng up-regulation of Iba1 in EAE mice. Western blot data were normalized to actin andmorphological changes that characterize microglia cells following activation. C and D
CFA and EAEmice respectively, during the acute phase of the disease. Both proliferatione measurements of the total surface covered by Iba1+ cells (F) (a parameter indicativeeir mean cell area (G) indicate a strong microglial reaction in EAE mice both in the WM
All numbers are mean values and standard errors. T-test EAE vs CFA ⁎ pb0.05,⁎⁎pb0.01, ⁎⁎⁎ pb0.001, ns no significant.
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analyzed by one-way ANOVA for independent and/or repeated mea-sures followed by Tukey HSD.
Results
WM lesions and microglia activation in the cerebellum of EAE mice
In EAE mice, brain infiltrating T lymphocytes, resident immunecells such as microglia and inflammatory cytokines have beenfound to be responsible of altered synaptic transmission in the stria-tum (Centonze et al., 2009; Rossi et al., 2011). Therefore, we first in-vestigated the presence of infiltrating T lymphocytes and the degreeof activation of the microglia/macrophage population by immunohis-tochemistry and western blot in the cerebellum of mice with EAE andcompared the results with those in mice only treated with CFA
Fig. 3. Activation of microglia in WM and ML of EAE cerebellum during the presymptomaticing in the cerebellum of CFA and EAE mice during the presymptomatic phase of the disease.the WM and ML indicated an early activation before the disease onset. On the other hand, bogy started during this phase. Accordingly, no significant differences were found between C
(control group). During the acute phase of EAE, we observed exten-sive lesions in theWM in most of the cerebellum labeled by Cb immu-nostaining (Fig. 1). CD3+ lymphocytes were present at the lesionsites (Figs. 1B–C) and Cb-positive PC axons had dystrophic alterationssuch as torpedoes or thickened segments (Figs. 1C), which representa typical response to axotomy in this neuronal type (Dusart andSotelo, 1994; Rossi et al., 1994; Rossi and Strata, 1995). In order toverify whether PCs undergo early degeneration in EAE during thisphase of the disease, we quantified the number of PCs per length ofPC layer (Fig. 1G). We did not find a significant difference betweenthe 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 sig-nificant difference of Cb expression was found between the groupsby western blot (see below).
Microglia cells and macrophages undergo proliferation and mor-phological changes following activation and overexpress Iba1(Ponomarev et al., 2005). This was observed in striatum during EAEas a consequence of the autoimmune reaction and was proposed assource of the cytokines involved in the impairment of glutamatergictransmission (Centonze et al., 2009). Here we observed also in cere-bellum a robust increase of Iba1 expression by western blot in EAEcompared to control group (n=3 per group; pb0.01) during theacute phase of the disease (Fig. 2A), indicating a strong activation ofmicroglia and macrophages. We then carried out a confocal quantita-tive analysis for the total surface covered by Iba1+ cells (a parameterindicative of both proliferation and morphological changes of micro-glia cells), their density and their mean cell area (parameters indica-tive respectively of proliferation and morphological change) in orderto assess whether there was a differential microglia activation in theWM, the major site for the inflammatory reaction due to the presenceof myelin, and in the ML, with less myelinated axons and where PCdendrites receive most of their inputs. We observed that both
phase of the disease.(A–B) Confocal images of Iba1 (red and gray) and Cb (green) stain-Measurements of the density (C) and of the total surface covered by Iba1+ cells (D) iny measuring their mean cell area (E), it seems that only a slight change in the morphol-FA and EAE. * pb0.05; ** pb0.01; *** pb0.001. Scale bar: 100 μm.
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proliferation and hypertrophyc morphology (Fig. 2B) characterizednot only the WM but also the ML in EAE (Figs. 2C–D). As shown inFigs. 2(E–F–G) all parameters were significant different relative totheir control (Table 1).
As an early consequence of the autoimmune reaction and neuroin-flammation, activation of microglia was previously shown in the stri-atum of EAE mice also during the presymptomatic phase of thedisease together with alterations in synaptic transmission (Centonzeet al., 2009; Rossi et al., 2011). Consistently, while rare microglialcells were present in the ML in control animals, many more couldbe observed in EAE mice (Figs. 3A–B). We thus observed, even duringthis early phase, a significant alteration of the proliferative state bothin WM and ML. Only a slight change in the morphology seemed tostart during this phase (Figs. 3C–D–E and Table 1).
GABA transmission is impaired in EAE, a role for IL-1βCerebellar PCs exhibit sustained spontaneous inhibitory activity
because of the massive GABAergic innervation from basket and stel-late cells in the ML and from neighboring PCs via their collaterals
Fig. 4. EAE alters GABAergic transmission in the cerebellum.(A and B) The electrophysiologditions (CFA), during the presymptomatic (7–9 dpi) and acute phase (20–23 dpi) of EAE. (neurons were normal in the pre-symptomatic phase of EAE, but were down-regulated insymptomatic and acute phases of EAE. (G–H) Synaptic effects of IL-1β in WT mice. Appli(G) but not the amplitude (H) of sIPSCs (pb0.05). Therefore IL-1β mimics the effect of EAE
(Eccles et al., 1967a,b). We performed whole-cell voltage-clamp re-cordings on cerebellar slices from EAE mice at the pre-symptomaticphase (7–9 dpi, n=5 mice), in the acute phase (20–25 dpi; n=8mice) and from relative control animals (CFA and CTR, n=8 and 3mice respectively), to record sIPSC and mIPSC in PCs from vermal lob-ules. The drugs MK-801 and CNQX, antagonists respectively of NMDAand non-NMDA glutamate receptors, were used to isolate spontane-ous GABAergic activity in EAE and CFA mice (Fig. 4A). The isolationof mIPSC was then obtained by adding TTX (1 μM) to the bathingfluid (Fig. 4B).
Both spontaneous and miniature events could be entirely blockedfollowing the application of bicuculline, a selective antagonist ofGABAA 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 compa-rable 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 there-fore to report only data from CFA mice as the appropriate controlgroup.
ical traces are examples of sIPSCs (A) and mIPSC (B) recorded from PCs in control con-E and F) The frequency of GABA-mediated sIPSCs (E) and mIPSC (F) recorded from PCthe acute phase. (C and D) sIPSC and mIPSC amplitude were unaffected in the pre-
cation of IL-1β in cerebellar slices from WT mice significantly reduced the frequencyon GABAergic transmission. ** pb0.01.
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We found that in EAE mice the mean values of the sIPSC amplitudein the pre-symptomatic and acute phase were indistinguishable fromthose 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 obtainedfor 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 decreaseof the sIPSC frequency in EAEmice during the symptomatic phase com-pared both to the presymptomatic phase and to control (EAEacute=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, pb0.01)(Fig. 4E). We observed a significant reduction also for the mIPSC fre-quency (EAE acute=0.97±0.25 Hz; EAE presymptomatic=3.18±1.04 Hz; CFA=3.47±0.6 Hz; one way ANOVA, pb0.01) (Fig. 4F).
Increasing evidence shows that in EAE pro-inflammatory cytokines,likely released by infiltrated inflammatory cells and by resident
Fig. 5. Loss of GABAergic synaptic contacts on PC somata during the acute phase of EAE.(A)acute phase of the disease. Quantification of VGAT expression shows no difference between Cthe cerebellum of EAE. Western blot data were normalized to actin and plotted as percentagthe synaptic terminals of basket and stellate cells that impinge on PC dendrites and bodies ((red) and Cb (green) to measure the percentage of Cb area in contact with VGAT. Quantificatpart an impairment of GABA signaling. T-test * pb0.05. Scale bars 10 μm.
microglia, mediate tissue damage and neurological deficits by alteringsynaptic transmission, and thus promoting excitotoxic neuronal damage(Centonze et al., 2009; Rossi et al., 2010, 2011). Thus, in order to clarifyhow inflammation alters GABA synapses in EAE, we addressed the effecton cerebellar sIPSCs of IL-1β, one of themajor pro-inflammatory cytokineinvolved in EAE-induced brain damage (Zhao and Schwartz, 1998), andrecently shown to be involved in the alteration of GABAergic transmis-sion in striatal neurons (Musumeci et al., 2011).
To this aim we recorded sIPSCs from cerebellar slices of non-immunized WT mice before and after incubation with 30 ng/ml IL-1β a dose known to induce an electrophysiological effect in acuteslices (Musumeci et al., 2011). After 5–10 min of incubation we ob-served a reduction in the frequency of sIPSCs (80.0±7.2% of basal fre-quency, paired t-test p=0.03; n=4, Fig. 4G) but not in theiramplitudes (99.6±6.9%; paired t-test p=0.6, Fig. 4H), thus mimick-ing the alterations of GABAergic synapses in the symptomatic EAEmice.
Western blot analysis of VGAT expression in cerebella of EAE and CFA mice during theFA and EAE, suggesting that no massive synaptic degeneration of GABA inputs occurs ine of CFA mice. (B) VGAT immunostaining (red) in the ML of the cerebellar cortex labelsCb green). (C–D) Confocal analysis of the overlapping signal (magenta) between VGATion in D shows a reduction of this specific subset of synaptic terminals that can justify in
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Altogether these results demonstrate that GABAergic transmissionin the cerebellum of EAE mice is compromised during the acute phaseof the disease, and suggest that IL-1β could be one of the proinflam-matory cytokines involved in such inhibition.
Degeneration of inhibitory interneurons in the ML of EAE mice
To evaluate if the observed alteration of GABA transmissioncould derive from lower number of GABAergic synapses in EAEmice, we studied the VGAT and PV, respectively markers of GABAreleasing terminals and GABAergic neurons, by means of westernblot and immunohistochemistry. No significant difference in VGATexpression levels could be observed by western blot (n=4 pergroup, t-test p=0.15), indicating that no massive synaptic degener-ation of GABA releasing terminals occurs in the cerebellum of EAEmice (Fig. 5A). However, many diverse VGAT+ inhibitory synapsesare present in all the layers of cerebellar cortex and in deep cere-bellar nuclei: those made by Golgi cells onto mossy fibers in thegranular layer, those made by PCs on the body of other PCs bytheir collaterals and in the deep cerebellar nuclei, and those im-pinging on PC soma and dendrites originating from the inhibitoryinterneurons present in the ML (stellate and basket cells). To assesswhether a more specific impairment of GABAergic innervation ontoPCs could account for the decreased GABAergic transmission, welooked at GABAergic innervation on PC somata. This consists mainlyon basket cells and collateral PC synapses. By measuring the densityof VGAT+ synaptic contacts on the PC body through immunofluo-rescent staining (Fig. 5B), we found a reduction of GABA releasingterminals 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
Fig. 6. Neurodegeneration of PV+ interneurons in the ML of EAE cerebellum.(A–B) Westernphase of the disease. (B) Quantification of PV and Cb normalized to actin content (left anddifferences between EAE and CFA mice. (C) Confocal images of PV (green) and Cb immunostduring the acute phase of disease. Interneurons were identified in the ML as the PV+/Cb− ceand expressed as density (1/mm2), was significantly reduced in EAE mice to 83% of control
phase (CFA n=18, EAE n=21; n=3 mice each group t-testp=0.56) (Figs. 5C–D). These results indicate that, despite an overallpreservation of VGAT expression throughout the cerebellum,GABAergic innervation on PC somata undergoes neurodegeneration,during the acute phase of EAE. This could contribute to the decreaseof GABAergic transmission.
A reduction of inhibitory interneurons has been shown to occurduring EAE both in striatum and hippocampus (Rossi et al., 2011;Ziehn et al., 2010). We then asked whether the number of stellateand basket cells, which are PV+ GABAergic interneurons, was also af-fected in the cerebellum during EAE thus contributing to reduceGABAergic innervation of PCs. No dramatic overall alterations of PVexpression were detected in EAE mice by western blot if normalizedon β-actin levels (t-test, p=0.31; Fig. 6A–B). Expression of PV wasalso calculated in relation to Cb content to verify if any alteration ofPV levels could be correlated to PC population. Similarly, no signifi-cant differences could be observed in PV expression normalizing iton Cb levels (t-test, p=0.90; Fig. 6B).
Since PCs, preserved at this disease phase, contribute relevantly tothe overall cerebellar levels of PV, we analyzed the density of only in-hibitory interneurons by immunohistochemistry. We focused ouranalysis on the ML, where both basket cells and stellate cells reside(Fig. 6C). We found a significant decrease in EAE mice to 81% oftheir density compared to control (CFA=320±9 neurons/mm2;EAE=260±11 neurons/mm2; n=6 animals per group; pb0.01)(Fig. 6D).
These data thus show that the impairment of GABAergic transmis-sion on PC during the acute phase of EAE is, at least partially, due to areduction in GABAergic innervation and a reduction of GABAergic in-terneurons themselves.
blot analysis of PV and Cb expression in cerebella of EAE and CFA mice during the acutecenter) and quantification of PV normalized to Cb content (right) show undetectableaining (red) in cerebellar sagittal sections derived from CFA and EAE mice respectively,lls. (D) The number of interneurons, counted in the ML on confocal microscopy imagesin the acute phase of the disease. T-test ** pb0.01. Scale bar 100 μm.
422 G. Mandolesi et al. / Neurobiology of Disease 46 (2012) 414–424
Discussion
In the present work we have shown that in EAE mice PCs receive areduced inhibitory tone during the symptomatic phase of the disease.The reduction of GABAergic sIPSC frequency was accompanied bysynaptic degenerative processes and loss of stellate and basket cells.Such populations of inhibitory neurons belong to the PV+ interneu-rons, a class of neurons particularly susceptible to degeneration inMS and EAE (Dutta et al., 2006; Clements et al., 2008; Rossi et al.,2011). Our findings suggest that an aberrant GABAergic synaptictransmission in the cerebellar cortex may contribute to cerebellardeficits in EAE and likely in MS patients. Previous studies have dem-onstrated impaired GABA transmission in MS and in EAE (Gottesfeldet al., 1976; Qureshi and Baig, 1988; Clements et al., 2008; Wang etal., 2008; Bhat et al., 2010; Ziehn et al., 2010). Recently, we providedevidence of irreversible alterations of GABA transmission in the stria-tum of EAE mice and in network of neurons (Rossi et al., 2011). Suchchanges together with increased glutamatergic transmission in theacute phase of the disease (Centonze et al., 2009; Rossi et al., 2010)seem to represent early events triggering secondary excitotoxicityand inflammatory neurodegeneration. Microglia and pro-inflammatory cytokines are emerging as fundamental players ofsuch synaptic modifications. In fact, the same electrophysiologicalchanges observed in EAE were mimicked by direct incubation of cor-ticostriatal slices with activated microglial cells, as well as with cyto-kines produced by microglia (Centonze et al., 2010). Based on thisconsideration, here we first explored microglia activation in the cere-bellum of EAE mice. As reported by other groups in different EAEmodels, we observed extensive WM lesions and prominent inflam-matory cellular staining in all cerebellar cortex (MacKenzie-Grahamet al., 2009; Zheng and Bizzozero, 2010). Due to the complexity ofmicroglia activation we investigated two parameters, proliferationand hypertrophic state, during the course of EAE. Early in the disease,the proliferative state was more pronounced than morphologicalchanges. During the acute phase, microglial cells proliferated evenmore and became hypertrophic. Interestingly, a strong activationwas evident also in the ML where there are rare microglial cells andwhere PC dendrites receive most of the synaptic contacts. By charac-terizing microglia activation during the disease course and in differ-ent neuronal compartments, we aimed at defining morphologicalhallmarks of EAE in association with the electrophysiological findings.In previous studies we reported early microglia activation in thestriatum in association with up-regulation of glutamate-mediatedtransmission (Centonze et al., 2009) but not with GABAergic downre-gulation (Rossi et al., 2011). In the present work, we showed thatGABA spontaneous release was normal in the presymptomaticphase while a remarkable reduction of the sIPSC frequency was evi-dent during the acute phase. Similar results were observed in EAEstriatum (Rossi et al., 2011). Altogether our observations suggestthat in EAE the course of GABAergic transmission impairment occursimilarly in striatum and cerebellum both in pre symptomatic andacute phase. Interestingly, it seems that the mechanisms at the basisof GABA hypofunctioning are similar in the two brain structures, in-volving the degeneration of PV+ interneurons and potentially theproinflammatory cytokine IL-1β.
However, exogenous IL-1β seems to interfere with synaptic trans-mission at different levels: presynaptically in the cerebellum andpostysinaptically in the striatum. In fact, identical application of IL-1β (time and exposure) in striatal slices from non-immunized WTmice significantly reduced the amplitude without altering the fre-quencies of sIPSCs (Musumeci et al., 2011) while in cerebellum the ef-fect was opposite. Recently, we have shown that IL-1β-mediatedinhibition of the GABAergic transmission in striatum is enhancedby transient receptor potential vanilloid 1 channels (TRPV1)(Musumeci et al., 2011). Interestingly, a growing body of evidenceshows that cytokines regulate neuronal excitability, synaptic
plasticity and injury by interacting specifically with receptors andion channels (Schäfers and Sorkin, 2008). It has been shown that IL-1β can affect TRPV1 receptors (Piper et al., 1999). At the presynapticlevel, IL-1β can induce inhibition of voltage-dependent calcium chan-nels and the resulting Ca2+-influx may impact on its ability to reduceneurotransmitter release (Murray et al., 1997; Rada et al., 1991). Re-garding its interaction with GABAA receptors, IL-1β can exert dual ef-fects. Exogenous IL-1β was shown to reduce synaptically mediatedGABAergic inhibition in dentate gyrus and CA3 pyramidal neurons(Zeise et al., 1997) while it induces opposite effects in CA1 pyramidalneurons (Bellinger et al., 1993). In hypothalamus neurons it increasesthe presynaptic release of GABA (Tabarean et al., 2006). In our exper-imental conditions we observed a rapid cytokine action (within mi-nutes) suggesting that it may act through a mechanism based atleast partly on the posttranslational modifications of proteins in-volved in the regulation of GABA release events, such as pre-synaptic ion channels. With regard to the specific molecular pathwaythat mediates acute cytokines effect, literature provides evidence forthe recruitment of different kinases (e.g. p38, PI3K) (Schäfers andSorkin, 2008). On the other hand, prolonged exposure of cytokinesmay also affect ion channels function (Furukawa and Mattson,1998; Liu et al., 2006) requiring altered gene expression rather thanposttranslational modifications of channel proteins.
Together with our current and previous observations (Centonze etal., 2009; Rossi et al., 2010; Musumeci et al., 2011), these data showthat exogenous application of cytokines on acute brain slices canmodulate synaptic transmission. In addition, since we observed thattheir application in normal slices replicates the electrophysiologicaleffects observed in EAE slices, we do suggest a role of pro-inflammatory cytokines as mediators of EAE synaptic alterations. Inthe ML pro-inflammatory cytokines are likely released from activatedmicroglia and potentially also from Bergmann glia, which tightlywraps somata, dendrites, and dendritic spines of PCs and their excit-atory and inhibitory synapses (Palay and Chan-Palay, 1974; Brambillaet al., 2005, 2009). Although Bergmann glia also plays essential role insynaptic homeostasis by expressing GABA and glutamate transportersand controlling GABA and glutamate clearance from synapses(Chaudhry et al., 1995; Conti et al., 1999; Tao et al., 2011; Wang etal., 2011), its involvement in the EAE-induced alterations of GABAer-gic transmission is unlikely, because the reduction of sIPSC and mIPSCcurrents here reported reflects mainly a presynaptic mechanism.
Besides an inhibitory effect of IL-1β on GABAergic signaling, weobserved synaptic degeneration and reduction in the number of thePV+ interneurons basket and stellate cells. Interestingly, microgliacould be instrumental in the synaptic stripping process, as observedin a model of facial nerve transection (Kreutzberg, 1993). Regardingneuronal cell death, selective loss of PV+ interneurons was alsoreported in striatum (Rossi et al., 2011) and hippocampus of EAEmice, in association with significant defects of hippocampus-relatedmemory abilities (Ziehn et al., 2010). In addition, a reduced extensionof PV+ neurites in the normal appearing gray matter and in themotor cortex of MS patients was reported (Clements et al., 2008;Dutta et al., 2006).
In MS patients, defective GABAergic transmission within themotor cortex has also been hypothesized on the basis of neurophysi-ological findings with paired-pulse transcranial magnetic stimulation(Caramia et al., 2004). The reasons of the selective susceptibility ofPV+ inhibitory interneurons to degenerate in MS and in EAE haveto be further investigated.
We observed that PCs, which express both calcium-binding pro-teins PV and Cb, survive during the acute phase of the disease. How-ever, swollen PC axons and axon retraction bulbs, called torpedos,were observed in the lesion site in the presence of infiltrating lym-phocytes. Such virtually absent PC degeneration is consistent withtheir known low susceptibility to cell death induced by axotomy(Carulli et al., 2004). In fact, axotomised PCs survive after axotomy
423G. Mandolesi et al. / Neurobiology of Disease 46 (2012) 414–424
for a very long time, and only rare neurons close to the lesion site dis-play degenerative changes (Buffo et al., 1997; Dusart and Sotelo,1994). However, we cannot exclude the possibility that a PC loss oc-curs later on, consistently with data from some EAE models and MSpatients showing a moderate reduction of PCs (Kutzelnigg et al.,2007; MacKenzie-Graham et al., 2009; Giuliani et al., 2011). Recentfindings have shown PC degeneration only at later stages of the dis-ease in a model of EAE mice in which the disease was exacerbatedby a booster immunization. In particular, a reduction in the PC num-ber (20%) was correlated to a decrease in ML volume (7.3%)(MacKenzie-Graham et al., 2009). Therefore, PC degeneration mayoccur at later stages induced by other insults typical of MS such asacquired channelopathies, altered activity of sodium channel ex-changer, glutamate mediated ecitotoxicity, intraneuronal calcium ac-cumulation and inhibition of mitochondrial respiratory chain. Inaddition, our observation suggests that, beside PC degeneration, lossof PV+ interneurons may contribute to generate an atrophy of theML as the disease progresses.
In conclusion, the alterations of the GABAergic system observed inthe EAE cerebellum and recurrent in other brain regions seem to rep-resent clear hallmarks of the EAE model and play a crucial role in EAEpathology. In addition, our findings highlight important aspect for un-derstanding cerebellar neuropathology in MS and EAE. Althoughfurther studies are needed to determine whether GABAergic trans-mission modulation could be successful in MS therapy, pharmacolog-ical compounds able to interfere with the chain of events that bringsto GABAergic impairment are likely to exert neuroprotective effects inMS patients.
Acknowledgments
We wish to thank Massimo Tolu and Vladimiro Batocchi for help-ful technical assistance. We want also to thank Prof. Piergiorgio Stratafor his support. This investigation was supported by the Italian Na-tional Ministero della Salute to DC, by Fondazione TERCAS to DC,and by Fondazione Italiana Sclerosi Multipla (FISM) to DC, by agrant from the European Community (AXREGEN: Axonal regenera-tion, plasticity & stem cells— Grant agreement 21 4003) foundingPhD fellowship of NH.
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