Abnormal activity of the Na/Ca exchanger enhances glutamate transmission in experimental autoimmune encephalomyelitis

Brain, Behavior, and Immunity 24 (2010) 1379–1385

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Abnormal activity of the Na/Ca exchanger enhances glutamate transmission in experimental autoimmune encephalomyelitis Silvia Rossi a,b, Valentina De Chiara a,b, Roberto Furlan c, Alessandra Musella a,b, Francesca Cavasinni a,b, Luca Muzio c, Giorgio Bernardi a,b, Gianvito Martino c, Diego Centonze a,b,* a b c

Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, 00133 Rome, Italy Fondazione Santa Lucia/Centro Europeo per la Ricerca sul Cervello (CERC), 00143 Rome, Italy Neuroimmunology Unit-DIBIT, INspE, Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy

a r t i c l e

i n f o

Article history: Received 23 March 2010 Received in revised form 11 July 2010 Accepted 14 July 2010 Available online 18 July 2010 Keywords: EPSC EAE Excitotoxicity Multiple sclerosis Neurodegeneraration Sodium channel Striatum

a b s t r a c t It is increasingly accepted that excessive glutamate release plays a key role in the pathophysiology of grey matter damage in multiple sclerosis (MS). The mechanisms causing abnormal glutamate transmission in this disorder are however largely unexplored. By means of electrophysiological recordings from single striatal neurons in slices, we found that the presymptomatic and acute phases of experimental autoimmune encephalomyelitis (EAE), a preclinical model of MS, are associated with enhanced synaptic release of glutamate. The reverse mode of action of axonal Na+/Ca++ exchanger, secondary to abnormal functioning of voltage-dependent Na+ channels, was identified as a major cause of this alteration. In fact, inhibition of the Na+/Ca++ exchanger with bepridil or with KB-R7943, which selectively blocks the reverse mode of the exchanger, reduced the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) recorded from striatal neurons in EAE mice but not in control animals. In the presence of tetrodotoxin (TTX), a blocker of voltage-dependent Na+ channels, the effect of bepridil was normalized in acute (25 days post-immunization) EAE mice, indicating that axonal accumulation of Na+ ions flowing through voltage-dependent Na+ channels plays a role in the abnormal activity of the Na+/Ca++ exchanger in EAE. Our data reveal an important role of Na+/Ca++ exchanger and of voltage-dependent Na+ channels in the pathological process of EAE, and provide a rationale for the use of neuroprotective strategies since the very early stages of MS. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a leading cause of neurological disability in young adults, and follows immune-mediated attack of the central myelin by auto-reactive T lymphocytes (Compston and Coles, 2008). Not only the white matter, but also the neuronal compartment of the central nervous system is affected in MS, and in fact brain atrophy, cortical thinning, reduction of neuronal-specific markers, iron deposition and other grey matter abnormalities have been found since the early phases of MS by means of conventional and advanced in vivo imaging techniques (Geurts and Barkhof, 2008; Chard and Miller, 2009; Fisniku et al., 2009; Audoin et al., 2010; Schmierer et al., 2010). Furthermore, cognitive deficits have also been described in a relevant percentage of MS patients already at the first episode of the disease (Calabrese et al., 2010;

* Corresponding author at: Clinica Neurologica, Dipartimento di Neuroscienze, Università Tor Vergata, Via Montpellier 1, 00133 Rome, Italy. Fax: +39 06 7259 6006. E-mail address: [email protected] (D. Centonze). 0889-1591/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbi.2010.07.241

Deloire et al., 2010; Zipoli et al., 2010), again supporting the idea that neuronal dysfunction occurs early in the disease course. Altered glutamate handling, resulting in excessive synaptic excitation, has been postulated to trigger neuronal damage in several acute and chronic neurological disorders also including MS, through a process termed excitotoxicity (Forder and Tymianski, 2009). Of note, both pre and postsynaptic mechanisms (i.e. increased glutamate release and increased sensitivity of postsynaptic receptors) might contribute to excitotoxic damage in MS and in its animal model, namely experimental autoimmune encephalomyelitis (EAE). Glutamate levels, in fact, increase in the cerebrospinal fluid (Stover et al., 1997; Sarchielli et al., 2003) and in the brains of MS patients (Srinivasan et al., 2005; Cianfoni et al., 2007), while AMPA, NMDA, and kainate glutamate receptors are up-regulated (Newcombe et al., 2008). Furthermore, expression of glutamate transporters are altered in MS (Vallejo-Illarramendi et al., 2006; Vercellino et al., 2007) and in EAE (Hardin-Pouzet et al., 1997; Ohgoh et al., 2002), and overactivation of glutamate receptors causes MS-like lesions (Matute et al., 2001). Conversely, both AMPA and NMDA glutamate receptor antagonists exert beneficial effects in EAE (Wallström et al., 1996; Bolton and Paul, 1997; Pitt et al.,

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2000; Smith et al., 2000) and in MS (Plaut, 1987), by limiting not only oligodendrocyte but also neuronal damage (Pitt et al., 2000; Smith et al., 2000; Centonze et al., 2009a). Finally, recent neurophysiological recordings from EAE mice reported changes of glutamate-mediated synaptic transmission suggestive of both presynaptic and postsynaptic alterations (Centonze et al., 2009a). Together, these data indicate that excessive release of glutamate, and not only abnormal sensitivity of glutamate receptors occur in MS and in EAE, but so far only the postsynaptic upregulation of AMPA receptor sensitivity has been detailed in studies aimed at characterizing synaptic functioning in EAE brain preparations (Centonze et al., 2009a). Thus, in the present work we wanted to focus our attention on the possible presynaptic alterations of glutamate transmission associated with myelin oligodendrocyte glycoprotein (MOG)-induced EAE, to provide a physiological correlate of increased glutamate release in this inflammatory disorder. To study excitatory synaptic transmission, we performed electrophysiological recordings from single neurons of the striatum, a brain area where the inflammatory process of EAE has already been shown to cause a profound perturbation of excitatory synaptic transmission (Centonze et al., 2009a,b).

2. Materials and methods Female mice with EAE (6–8 weeks at the time of immunization) were employed along with age- and sex-matched naïve, untreated mice, and with mice treated with Freund’s adjuvant without MOG. The latter two groups of mice were used as controls, and the data were pooled together. 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 November 24, 1986 (86/609/EEC). 2.1. EAE induction and clinical score As described (Centonze et al., 2009a; Rossi et al., 2009), EAE was induced in C57BL/6 mice by subcutaneous immunization with 300 ll of 200 lg MOG (35–55) (multiple peptide system) in incomplete Freund’s adjuvant containing 8 mg ml 1 Mycobacterium tuberculosis (strain H37Ra; Difco). Pertussis toxin (Sigma) (500 ng) was injected on the day of the immunization and again two days later. Body weight and clinical score (0 = healthy; 1 = limp tail; 2 = ataxia and/or paresis of hindlimbs; 3 = paralysis of hindlimbs and/or paresis of forelimbs; 4 = tetraparalysis; 5 = moribund or death) were recorded daily. 2.2. Electrophysiology For electrophysiological experiments, mice were killed by cervical dislocation under halothane anesthesia, and corticostriatal coronal slices (200–300 lm) were prepared from fresh tissue blocks of the brain with the use of a vibratome. A single slice was then transferred to a recording chamber and submerged in a continuously flowing artificial cerebrospinal fluid (ACSF) (34 °C, 2–3 ml/min) gassed with 95% O2–5% CO2. The composition of the control solution was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 Glucose, 25 NaHCO3. The striatum could be readily identified under low power magnification, whereas individual neurons were visualized in situ using a differential interference contrast (Nomarski) optical system. This employed an Olympus BX50WI (Japan) non-inverted microscope with x40 water immersion objective combined with an infra-red filter, a monochrome CCD camera (COHU 4912), and a PC compatible system for analysis of images and contrast

enhancement (WinVision 2000, Delta Sistem, Italy). Recording pipettes were advanced towards individual striatal cells in the slice under positive pressure and, on contact, tight GX seals were made by applying negative pressure. The membrane patch was then ruptured by suction and membrane current and potential monitored using an Axopatch 1D patch clamp amplifier (Axon Instruments, Foster City, CA, USA). Whole-cell access resistances measured in voltage clamp were in the range of 5–20 MX. To study evoked (eEPSCs), spontaneous (sEPSCs), and miniature glutamate-mediated excitatory postsynaptic currents (mEPSCs), whole-cell patch clamp recordings were made with borosilicate glass pipettes (1.8 mm o.d.; 2–4 MX) in voltage-clamp mode and at the holding potential (HP) of –80 mV. Recording pipettes were filled with internal solution of the following composition (mM): K+-gluconate (125), NaCl (10), CaCl2, (1.0), MgCl2 (2.0), 1,2-bis (2aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA; 0.5), N(2-hydroxyethyl)-piperazine-N-s-ethanesulfonic acid (HEPES; 19), guanosine triphosphate (GTP; 0.3), Mg-adenosine triphosphate (Mg-ATP; 1.0), adjusted to pH 7.3 with KOH. Offline analysis was performed on spontaneous and miniature synaptic events recorded during fixed time epochs (1–2 min), sampled every 2–3 min (5–12 samplings) (Centonze et al., 2009a; Rossi et al., 2009). Only data from putative medium spiny projection neurons were included in the present study. These neuronal subtypes represent over 95% of the entire population of striatal neurons, and were identified for their morphological and electrophysiological properties. In fact, while putative cholinergic interneurons were easily recognized in striatal slices because of their large somata (40– 60 lm diameter compared to 25–35 lm for projection neurons), low resting membrane potential ( 50 to 60 mV compared to 75 to 85 mV for projection neurons) and typical membrane responses to both depolarizing and hyperpolarizing current steps (Kawaguchi et al., 1995), we identified medium spiny projection neurons immediately after rupture of the GX seal, by evaluating their firing response to the injecting of depolarizing current (typically tonic, with little or no adaptation). Fast-spiking GABAergic interneurons were recognized for their typical firing activity (Kawaguchi et al., 1995), and discarded. For sEPSC kinetic analysis, events with peak amplitude between 10 and 50 pA were grouped, aligned by half-rise time, and normalized by peak amplitude. In each cell, all events between 10 and 50 pA were averaged to obtain rise times, decay times, and half widths (Centonze et al., 2009a; Rossi et al., 2009). eEPSCs were elicited at 0.1 Hz frequency by using bipolar electrodes located in the white matter between the cortex and the striatum, to activate corticostriatal glutamatergic fibers. PCLAMP 9 (Axon Instruments) was used to store the data. In distinct neurons, eEPSCs of similar amplitude were obtained with variable intensities of stimulation, mainly depending on the distance between the stimulating and recording sites. eEPSCs normally ranged between 50 and 400 pA. No gross difference in the intensity of stimulation required to obtain a given synaptic event was observed across the experimental groups. For the experiments on paired pulse ratio (PPR), pulse interval between two consecutive eEPSCs was 40 ms. PPR of eEPSCs (eEPSC2/ eEPSC1) is commonly calculated to investigate the preferential (pre versus postsynaptic) site of action of a drug at glutamate synapses. Accordingly, compounds that inhibit or promote glutamate release enhance or reduce the ratio respectively, while compounds acting postsynaptically do not affect this parameter (Nicoll and Malenka, 1999; Thomson, 2000). 2.3. Statistical analysis One to six cells per animal were recorded. For each type of experiment and time-point, at least six control mice and EAE mice

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were employed. 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 < 0.05. Statistical analysis was performed using a paired or unpaired Student’s t-test or Wilcoxon’s test for comparisons between two groups. Multiple comparisons were analyzed by one-way ANOVA for independent and/or repeated measures followed by Tukey HSD. To determine whether two cumulative distributions of spontaneous synaptic activity were significantly different, the Kolmogorov–Smirnov (K–S test) was used. 2.4. Drugs Drugs were applied by dissolving them to the desired final concentration in the bathing ACSF. Drugs were (in lM): CNQX (10), KB-R7943 (10), MK-801 (30), tetrodotoxin (TTX, 1) (from Tocris Cookson, Bristol, UK). Bepridil (100), Bicuculline (10) (from Sigma-RBI, St. Louis, USA).

3. Results 3.1. Effects of EAE induction on motor performances and intrinsic membrane properties of striatal neurons According to previous findings (Centonze et al., 2007, 2009a; Rossi et al., 2009), EAE induction produced overt motor deficits starting about 12–15 dpi. The severity of these clinical manifestations was maximal at 20–25 dpi (median value at 25 dpi: 3.0), and was less pronounced at 40 and 50 dpi (median value at 50 dpi: 2.0) (n = 14 mice). No motor disturbances were seen in control, CFA-treated mice (n = 10 mice, p < 0.05 at each time-points starting from 12 dpi) (Fig. 1). Whole-cell patch clamp recordings were made from striatal spiny neurons identified by morphological and electrophysiological criteria. RMP of the recorded neurons (n = at lest 30 for each group) did not significantly differ in EAE and control mice in the presymptomatic (7 dpi; EAE: 83 ± 6 mV, control: 82 ± 4 mV; p > 0.05), acute (25 dpi; EAE: 81 ± 4 mV, control: 83 ± 4 mV; p > 0.05), and chronic phases of the disease (50 dpi) (EAE: 83 ± 4 mV, control: 81 ± 3 mV; p > 0.05). In voltage-clamp mode, control and EAE mice showed similar current–voltage relationships at each time-point explored (7, 25 and 50 days after immunization or CFA administration). These membrane properties were indistinguishable from those previously reported for striatal medium spiny neurons (Wilson and Kawaguchi, 1996; Centonze et al., 2007, 2009a) (not shown).

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3.2. Spontaneous glutamate transmission in EAE mice To address excitatory transmission in EAE, we recorded spontaneous synaptic activity from striatal neurons of EAE mice. In all the electrophysiological recordings, bicuculline (10 lM) was added to the perfusing solution to block GABAA-mediated transmission for the entire duration of the experiment. Conversely, glutamate AMPA and NMDA receptors were blocked with CNQX (antagonist of AMPA receptors) plus MK-801 (NMDA receptor antagonist) at the end of each experiment, to further confirm that the recorded synaptic currents were entirely mediated by glutamate receptors. According to a previous report (Centonze et al., 2009a), frequency of glutamate-mediated sEPSCs was increased 7 dpi, before the appearance of clinical deficits (EAE: n = 22; control: n = 21; p < 0.05) (Figs. 1 and 2A, C and D). For each time-point considered in this study, the analysis in the two groups of control neurons (naïve, untreated mice and mice treated with Freund’s adjuvant without MOG) gave comparable results also in terms of frequency of sEPSC (frequency at 25 days p.i.: 2.4 ± 0.6 Hz for naïve mice; 2.3 ± 0.8 Hz for Freund’s adjuvant treated mice, n = at least 10 for each group and time-point, p > 0.1), so that the data were pooled together for these and following analysis. Twenty-five dpi, mice exhibited severe neurological disturbances (Fig.1), and sEPSC frequency was still abnormally elevated (EAE: n = 26; control: n = 16; p < 0.05) (Fig. 2A, C and D). Normal sEPSC frequency was conversely observed in the chronic phase of the disease (50 days p.i.) (EAE: n = 19; control: n = 15; p > 0.1) (Fig. 2A, C and D), when the animals experienced a partial rescue of their motor symptoms (Fig.1). In the presymptomatic, acute, and chronic phases of EAE, sEPSC amplitude was normal (Fig. 2B and E). 3.3. Role of Na+/Ca++ exchanger in the abnormal glutamate transmission in EAE mice We hypothesized a presynaptic mechanism at the basis of increased frequency of sEPSCs, since abnormal functioning of axonal Na+/Ca++ exchanger has been proposed to occur in MS and in EAE (Craner et al., 2004). To study the activity of Na+/Ca++ exchanger in EAE and in control mice, we first incubated corticostriatal slices from the two experimental groups with bepridil, a blocker of this ion exchanger. In control slices, bepridil increased the frequency of sEPSCs (n = 8) and reduced PPR of eEPSCs (n = 7) (Fig. 3A). In the acute phase of EAE, but not in the chronic phase, this pharmacological agent produced opposite effects, since it decreased to close to normal values sEPSC frequency (25 day p.i.: 2.8 ± 0.3 Hz, p > 0.05 respect to control, n = 12) (Fig. 3A), and increased PPR of eEPSCs (n = 6) (Fig. 3B and C). Also in the presymptomatic phase of EAE bepridil failed to increase sEPSC frequency (7 day p.i.: n = 16, p > 0.1 respect to pre-drug values) (Fig. 3A), indicating an early dysfunction. In both experimental groups, bepridil failed to affect the postsynaptic properties of sEPSCs (amplitude, rise time, decay time, and half width) (n = at least 10 and p > 0.05 for each electrophysiological parameter, time-point, and experimental group) (not shown). Notably, basal PPR was reduced in presymptomatic and acute EAE compared to control mice (p < 0.05 for both time-points) (Fig. 3B), a finding which is in agreement with the idea that presynaptic glutamatergic terminals are more prone to release glutamate in response to synaptic activation in EAE than in controls (Nicoll and Malenka, 1999; Thomson, 2000). 3.4. Reverse mode of the Na+/Ca++ exchanger in EAE

Fig. 1. Clinical effects of EAE induction in mice. A. The graph shows that the severity of EAE-induced clinical disturbances is maximal at 20–25 dpi, and less pronounced in the chronic phases of the disease. Control mice (naïve or only receiving CFA) did not show any motor deficit. (Data are presented as mean ± SEM; ANOVA for repeated measures).

A possible interpretation of these results is that bepridil favors presynaptic Ca++ accumulation and transmitter release by blocking Na+/Ca++ exchanger during its normal mode of operation (extrusion

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Fig. 2. EAE alters glutamatergic transmission. (A) The frequency of glutamatergic sEPSCs recorded from striatal neurons was up-regulated in the presymptomatic (7 days p.i.) and in the acute phase of EAE (25 days p.i.), while it was normal in the chronic stages of the disease (50 days p.i.). (unpaired Student’s t-test to compare mean values of EAE and control group at different time-points, ANOVA for independent measures to compare the effect of time on sEPSC frequency). (B) sEPSC amplitude was conversely unaffected in presymptomatic, acute, and chronic stages of EAE. (Unpaired Student’s t-test to compare mean values of EAE and control group at different time-points, ANOVA for independent measures to compare the effect of time on sEPSC amplitude). (C) The electrophysiological traces are examples of sEPSCs (downward deflections) recorded from striatal neurons in control conditions and 7, 25, and 50 days p.i. with MOG. (D and E) Cumulative distributions of sEPSC inter-event interval (D) and amplitude (E) recorded in control condition and in the presymptomatic, acute and chronic stages of EAE. (Kolmogorov–Smirnov test). Asterisks mean p < 0.05.

of Ca++ ions and uptake of Na+ ions) in control mice, while causing opposite effects in EAE mice, since in these mice the exchanger works in the reverse mode. Accordingly, the selective blockade of the reverse mode of Na+/Ca++ exchanger with KB-R7943 did not produce measurable effects in control mice, but mimicked the effects of bepridil on sEPSC frequency (Fig. 4A) and PPR (Fig. 4B and C) in EAE (n = at least 6 for each group and time-point).

experimental group; p > 0.1 comparing values 5 and 10 min after TTX application, and 5 and 10 min after TTX plus bepridil application) (Fig. 5), indicating that in the acute phase of EAE the reverse mode of functioning of Na+/Ca++ exchanger was normalized by preventing Na+ influx into the axons. Notably, TTX failed to significantly alter per se sEPSC frequency in control and EAE mice, indicating that the majority of these spontaneous currents in striatal slices are action-potential independent events (Fig. 5).

3.5. Role of voltage-dependent Na+ channels in the abnormal glutamate transmission in EAE mice

4. Discussion

Increased Nav1.6 sodium channel expression and the resulting intra-axonal accumulation of Na+ ions through voltage-dependent Na+ channels have been proposed as the main determinant of the reverse mode of action of Na+/Ca++ exchanger in MS and EAE (Craner et al., 2004). Our data are in tune with this idea, since pharmacological inhibition of this ion exchanger caused remarkably different effects in EAE and in controls. To study the role of Na+ influx through voltage-gated Na+ channels in the mode of action of Na+/Ca++ exchanger, we recorded mEPSCs (in the presence of TTX), a selective blocker of voltage-gated Na+ channels. Application of bepridil caused similar increases of mEPSC frequency in control neurons, as well as in EAE (25 dpi) mice (n = at least 10 for each

MS has long been viewed as a primarily inflammatory and demyelinating disorder, but neuronal degeneration and axonal loss are common and abundant, affecting both overt inflammatory lesions and normal-appearing white matter (Filippi and Rocca, 2005). Glutamate excitotoxicity has been proposed as a major determinant of neurodegeneration in MS and in EAE. Activated leukocytes and microglia are the most accredited source of glutamate during inflammation of the CNS (Piani et al., 1991; Werner et al., 2001), while synaptic release has received so far little or no attention. By means of conventional whole-cell patch-clamp recordings from single neurons, we recently addressed glutamate-mediated

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Fig. 3. Involvement of Na+/Ca++ exchanger in the alteration of glutamate transmission in EAE. (A) Pharmacological inhibition of Na+/Ca++ exchanger by bepridil increased sEPSC frequency in control animals and during the chronic stage of EAE. This compound failed to alter sEPSC frequency in presymptomatic EAE mice and caused opposite effects in acute EAE mice, by reducing sEPSC frequency. (Paired Student’s t-test to compare pre and post-drug values). (B) Bepridil reduced PPR in control slices but enhanced this physiological parameter in acute EAE mice. Note that basal PPR was reduced in neurons from EAE mice (7 and 25 dpi) compared to control neurons. (Wilcoxon’s test). (C) The electrophysiological traces here shown are examples of PPR recorded before and during bepridil application. Asterisks mean p < 0.05.

Fig. 4. Effects of pharmacological inhibition of the reverse mode of Na+/Ca++ exchanger in EAE. (A) KB-R7943, a blocker of the reverse mode of the Na+/Ca++ exchanger, did not affect sEPSC frequency in control mice and in the presymptomatic and chronic stages of EAE. This compound, however, mimicked the effects of bepridil in acute EAE. (Paired Student’s t-test to compare pre and post-drug values). (B) KB-R7943 did not affect PPR in control slices but enhanced this physiological parameter in acute EAE mice. Note that basal PPR was reduced in neurons from EAE mice (7 and 25 dpi) compared to control neurons. (Wilcoxon’s test). (C) The electrophysiological traces here shown are examples of PPR recorded before and during KB-R7943 application. Asterisks mean p < 0.05.

synaptic currents in corticostriatal brain slices from EAE mice. We found that the duration of glutamate-mediated sEPSCs and mEPSCs was increased in striatal cells in both the presymptomatic and clinical phase of the disease, as expected for enhanced sensitivity of postsynaptic receptors in these neurons. Glutamate NMDA receptors were not involved in this alteration, which was entirely med-

iated by AMPA receptors. These results suggested us that postsynaptic AMPA receptors are sensitized to endogenous glutamate during EAE, possibly causing synaptic degeneration since the very early phases of the disease (Centonze et al., 2009a,b). The present neurophysiological investigation extends to the presynaptic site of glutamate synapses the study of excitatory

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Fig. 5. Role of voltage-dependent Na+ channels in the mode of action of Na+/Ca++ exchanger in EAE. In the presence of TTX, to prevent Na+ influx through voltageactivated Na+ channels, bepridil increased sEPSC frequency not only in control mice but also in the acute phases of EAE. (Paired Student’s t-test to compare pre and post-drug mean values).

transmission in EAE. The alterations of glutamate transmission found in EAE mice, causing increased sEPSC frequency and reduced PPR or eEPSCs, involved at least two distinct determinants of transmitter release from presynaptic terminals: Na+/Ca++ exchanger and voltage-dependent Na+ channels. We have in fact shown that a substantial contribution to increased glutamate release in EAE mice was provided by the reverse mode of action of Na+/Ca++ exchanger. The abnormal functioning of this exchanger likely followed Na+ accumulation through altered voltage-dependent Na+ channels, since TTX was able to prevent the paradoxical effects of bepridil (that blocks both the forward and reverse mode of the Na+/Ca++ exchanger) on sEPSC frequency recorded from EAE mice. However, the finding that KB-R7943 only altered glutamate transmission in acute (25 dpi) EAE mice, while causing no changes in presymptomatic animals, indicate that reverse Na+/Ca++ exchanger is likely a major determinant of increased glutamate transmission during acute EAE, but not in presymptomatic EAE. Thus, other studies are necessary to further explore the mechanisms at the basis of abnormal glutamate transmission in the very early phases, as well as in the acute and chronic stages of EAE. In fact, the major role of voltage-dependent Na+ channels in the abnormal activity of Na+/Ca++ exchanger is challenged by the finding that TTX did not reduce per se sEPSC frequency in acute EAE mice, as expected based on the notion that TTX correct Na+/Ca++ exchanger activity in these mice. Again, these discrepant results need clarification in future studies. Despite these limitations, our current data show alterations of synaptic transmission in the striatum of EAE mice since the presymptomatic phase of the disease, providing support to the emerging concept that the neuronal compartment of the CNS suffers even before overt white matter damage in MS due to abnormal glutamate transmission. Along with AMPA receptors, metabotropic glutamate (mGlu) receptors are also likely to contribute to glutamate transmission changes in EAE and in MS brains. For example, a significant reduction in the expression of mGlu 1 receptors has been reported to occur concomitantly with increased mGlu 5 receptors in the cerebellum of mice with EAE and of patients with MS (Fazio et al., 2008), while both receptors increase in the context of MS lesions (Geurts et al., 2003). Also mGlu 2, 3, 4 and 8 have been found to increase in active MS lesions, especially in reactive astrocytic and microglial cells (Geurts et al., 2003, 2005). Importantly, the identification of CNS-resident astroglia and microglia as important components of the innate immunity was indeed crucial to understand the role of inflammation in neuronal damage. It is now clear, in fact, that both astroglia and microglia are of pivotal importance in the control of neuronal survival, because they release a number of potentially neurotoxic factors and regulate glutamate homeosta-

sis (Muzio et al., 2007; Centonze et al., 2009b; Shijie et al., 2009; Hamilton and Attwell, 2010; Paixão and Klein, 2010). Further studies are however necessary to clarify how mGlu receptors and glial cells intervene in the complex pre- and postsynaptic alterations of synaptic transmission seen in EAE. Acquired channelopathy with preferential expression of the Nav1.6 Na+ channels over the Nav1.2 Na+ channels has been proposed to favor persistent Na+ currents and Na+ overload in axons of MS patients and EAE mice, leading to abnormal Na+/Ca++ activity and Ca++ accumulation (Craner et al., 2004; Waxman, 2006). It has been suggested that the co-localization of Nav1.6 with Na+/Ca++ exchanger may be a mechanism underlying axonal injury. In agreement with these findings, inhibition of the exchanger protects axons during inflammation (Kapoor et al., 2003), and sodium channel blockers, such as phenytoin and lamotrigine, prevents axonal degeneration and significantly improve clinical scores in EAE (Lo et al., 2003; Bechtold et al., 2006; Black et al., 2006). Furthermore, the tissue sodium concentration levels in areas of normal-appearing white matter have been found, by sodium magnetic resonance imaging, to be significantly higher in the brain of MS patients, suggesting changes in cellular and metabolic integrity (Inglese et al., 2010). In the present physiological study, we have confirmed that the activity of Na+/Ca++ exchanger is under the control of Na+ ions entering the axons through voltage-dependent Na+ channels, and we have demonstrated that this interaction is important for transmitter release, possibly through the control of Ca++ ion concentrations in the presynaptic boutons. Facilitated synaptic excitation is likely able to prime excitotoxic damage by increasing neuronal vulnerability to MS-specific mitochondrial dysfunction (Vyshkina et al., 2005), or to the neurotoxic effects of inflammation (Xiong and McNamara, 2002; Centonze et al., 2009a,b) Finally, glutamate transmission hyperfunctioning, which is typical of acute phase of the disease, might also play a role in the generation of symptoms during clinical relapses. Accordingly, pharmacological inhibition of glutamate receptors decreases the clinical expression of relapse in MS patients (Plaut, 1987) and in EAE (Pitt et al., 2000; Smith et al., 2000; Centonze et al., 2009a,b) while the underlying immune response is unaffected by this treatment (Bolton and Paul, 1997; Pitt et al., 2000; Smith et al., 2000). In conclusion, here we have demonstrated that abnormal glutamate transmission occurs in EAE mice, raising the possibility that similar synaptic alterations may take place in MS, influencing gray matter functioning and survival since the very early stages of the disease (Pirko et al., 2007; Compston and Coles, 2008). The identification of the specific molecular determinants involved in dysregulated glutamate transmission in EAE might be important for the development of neuroprotective strategies in MS with agents targeting glutamate receptors, the Na+/Ca++ exchanger or voltage-dependent Na+ channels.

Acknowledgments This investigation was supported by the Italian National Ministero dell’Università e della Ricerca to DC; by the Italian National Ministero della Salute to DC, by Fondazione Italiana Sclerosi Multipla (FISM) to DC, RF, LM and GM, and by BMW to GM.

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