Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

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HIPPOCAMPAL FUNCTION IS COMPROMISED IN AN ANIMAL MODEL OF MULTIPLE SCLEROSIS T. NOVKOVIC, a,b O. SHCHYGLO, a,b R. GOLD b,c AND D. MANAHAN-VAUGHAN a,b*

Key words: LTP, LTD, EAE, object recognition, spatial memory, inflammation.

a

Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, Germany b International Graduate School of Neuroscience, Ruhr University Bochum, Germany

INTRODUCTION Hippocampal function, both at the level of learning and synaptic plasticity, can be affected in neurodegenerative diseases, as well as in animal models of neurodegen erative diseases (Kim et al., 2001; Rowan et al., 2003; Costa et al., 2012; Balu et al., 2012; Denney et al., 2005; Usdin et al., 1999). Multiple sclerosis (MS) is a progressive inflammatory, demyelinating, and ultimately neurodegenerative disease of the central nervous system (CNS) that is characterized by the progressive accumulation of lesions in the brain and the spinal cord. Pathological features of MS comprise inflammation, demyelination, axonal loss and gliosis (Frohman et al., 2005). The most typical symptoms are visual, motor, sensory and autonomic dysfunctions, as well as a wide range of symptoms related to lesions located in the CNS (Compston and Coles, 2002). A hallmark of MS is also cognitive decline, which occurs even in the absence of physical impairments, and affects many aspects of a patient’s daily life and social status (Chiaravalloti and DeLuca, 2008). Patients with MS often experience longterm memory impairments (Gaudino et al., 2001; Drake et al., 2006), attention deficiency (Beatty et al., 1996), reduced information processing speed (Litvan et al., 1988) and impairment in executive functions (Denney et al., 2005). Inhibition of transcallosal impulse conduction has been postulated to underlie some of these changes (Schmierer et al., 2000). However, the underlying mechanisms remain largely unknown. In rodents, experimental autoimmune encephalomyelitis (EAE) is used to model disease progression in MS. EAE mirrors MS-like pathology and complexity (Gold et al., 2006; Constantinescu et al., 2011) and mimics most of the histopathological, clinical and immunological features of the disease (Fletcher et al., 2010). Moreover, the pathogenesis of EAE resembles that of MS (Baker and Jackson, 2007; Constantinescu et al., 2011). EAE that is induced by myelin oligodendrocyte glycoprotein 35–55 peptide (MOG35–55-induced EAE) is characterized by gliosis, demyelination, axonal and neuronal loss (Herrero-Herranz et al., 2008; Peruga et al., 2011). Given that clinical studies have reported cognitive decline that correlates with hippocampal atrophy in MS patients (Sicotte et al., 2008; Roosendaal et al., 2009), the present study explored whether neuronal circuit

c Neurological University Clinic, St. Josef Hospital, Medical Faculty, Ruhr University Bochum, Germany

Abstract—Multiple sclerosis (MS) is a progressive inflammatory autoimmune disease that is characterized by demyelination and axonal damage in the nervous system. One obvious consequence is a cumulative loss of muscle control. However, cognitive dysfunction affects roughly half of MS sufferers, sometimes already early in the disease course. Although long-term (remote) memory is typically unaffected, the ability to form new declarative memories becomes compromised. A major structure for the encoding of new declarative memories is the hippocampus. Encoding is believed to be mediated by synaptic plasticity in the form of long-term potentiation (LTP) and long-term depression (LTD) of synaptic strength. Here, in an animal model of MS we explored whether disease symptoms are accompanied by a loss of functional neuronal integrity, synaptic plasticity, or hippocampus-dependent learning ability. In mice that developed MOG35–55-induced experimental autoimmune encephalomyelitis (EAE), passive properties of CA1 pyramidal neurons were unaffected, although the ability to fire action potentials became reduced in the late phase of EAE. LTP remained normal in the early phase of MOG35–55induced EAE. However, in the late phase, LTP was impaired and LTP-related spatial memory was impaired. In contrast, LTD and hippocampus-dependent object recognition memory were unaffected. These data suggest that in an animal model of MS hippocampal function becomes compromised as the disease progresses. This article is part of a Special Issue entitled: Hippocampus. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

*Correspondence to: D. Manahan-Vaughan, Department of Neurophysiology, Medical Faculty, Ruhr University Bochum, Universita¨tsstraße 150, MA 4/150, 44780 Bochum, Germany. Tel: +49234-32-22042; fax: +49-234-32-14192. E-mail address: [email protected] (D. ManahanVaughan). Abbreviations: aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; CFA, complete Freund’s adjuvant; EAE, experimental autoimmune encephalomyelitis; fEPSP, field excitatory postsynaptic potential; I/O, input–output; LTD, long-term depression; LTP, long-term potentiation; MS, multiple sclerosis; OVA, ovalbumin; PP, paired-pulse; PTX, pertussis toxin; s.e.m., standard error of the mean; TBS, theta burst stimulation. http://dx.doi.org/10.1016/j.neuroscience.2015.03.008 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. 1

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responsiveness and cognitive functions are affected following MOG35–55-induced EAE. Here, we examined synaptic plasticity in the CA1 stratum radiatum, hippocampus-dependent learning and memory, and the physiological properties of CA1 pyramidal neurons in the EAE model. We observed that impairments in hippocampal synaptic plasticity and hippocampus-dependent memory occur in EAE. Specifically, we observed impaired long-term potentiation (LTP) and spatial memory in the late, but not in the early phase of EAE. However, long-term depression (LTD) and hippocampus-dependent object recognition memory (Eichenbaum et al., 1999; Manns and Eichenbaum, 2009; Brown et al., 2012) were unaffected. Passive properties of CA1 pyramidal neurons were unaffected, although the ability to fire action potentials became reduced in the late phase of EAE, indicating that neuronal transmission becomes impaired. Taken together, our data suggest that as MS progresses hippocampal function becomes directly compromised. Given the presumed role of LTP in memory formation (Kemp and Manahan-Vaughan, 2007), the deficits that occur in hippocampal LTP may underlie memory-related cognitive deficits that occur in MS patients.

EXPERIMENTAL PROCEDURES The present study was carried out in accordance with the European Communities Council Directive of September 22nd 2010 (2010/63/EEC) for care of laboratory animals and after approval of the local government ethics committee (Bezirksamt, Arnsberg). All efforts were made to minimize the number of animals used. Active induction of EAE EAE is a CD4+ T cell-mediated autoimmune disease that generates a mouse model of MS (Miller et al., 2007), in which animals progressively exhibit motor disabilities and abnormalities that are similar to changes observed in MS patients. EAE is characterized by an early phase and a late phase (Fig. 1). Following seven days of acclimatization to the animal housing facility, 8–10-week-old female C57BL/6 mice were anaesthetized with Ketamin/Xylazin and subcutaneously immunized with 100 lg of myelin oligodendrocyte glycoprotein (MOG35–55) peptide (Institute for Medical Immunology, Charite, Berlin,

Germany) that is a target antigen that leads to autoimmune-mediated demyelination (Berger et al., 2003). MOG was emulsified with 100 lg of complete Freund’s adjuvant (CFA) containing Mycobacterium tuberculosis at a final concentration of 1 mg/ml. Additionally, mice received an intraperitoneal injection of 100 ng of pertussis toxin (PTX) (Sigma, St. Louis, MO, USA) on days 0 and 2 post immunization. CFA augments the effects of MOG and expedites the onset of symptoms (Freund, 1947; Gold et al., 2006). PTX is used to make the blood–brain barrier more permeable and thus increase the effectiveness of the immunization with MOG (Linthicum et al., 1982; Yong et al., 1993). One control group of mice was immunized with ovalbumin (OVA)/CFA emulsion (100 lg) together with PTX (200 ng), whereas a second control group was immunized with OVA/CFA emulsion only. This was done so that we could exclude that any possible effects detected in hippocampal function were not caused by changes in the blood–brain barrier (elicited by PTX) and/or the presence of the adjuvant, CFA, together with an antigen. The antigen, OVA is not a myelinassociated protein, thus served as a control for MOG. The mice were monitored for weight-loss and clinical symptoms of EAE on a daily basis. The immunization with MOG35–55 peptide results in an ascending paralysis usually starting after 10 days and reaching its maximum within one week. According to the symptoms, the progression of the disease was assessed using a disability scale ranging from 0 to 10 (Hartung et al., 1988). This was scored in the following way: 0 = normal, 1 = less lively, 2 = impaired righting/limp tail, 3 = absent righting, 4 = ataxic gait, abnormal position, 5 = mild paraparesis, 6 = severe paraparesis, 7 = tetraparesis, 9 = moribund, 10 = death. The early phase of EAE (between days 8 and 19 after immunization) is characterized by severe motor disabilities. Starting with the loss of tail tone, the first motor disabilities of EAE typically appear 8–12 days after immunization (Peruga et al., 2011; ZorzellaPezavento et al., 2013). The peak of clinical signs appears between days 14 and 19 after immunization, i.e. when clinical symptoms are maximally severe. In the current experiments, we assessed early EAE effects in mice 14 and 19 days after immunization. The maximal disability score (Hartung et al., 1988) we observed was three or four.

Fig. 1. Schematic illustration of the EAE progression. Two phases of the EAE course were distinguished – the early and the late phase. Mice that were in the early phase of EAE (between days 14 and 19 post immunization) as well as mice that were in the late phase of EAE (between days 40 and 45 post-immunization) were used for the experiments (indicated by dashed circle lines). Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

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Twenty days after immunization, the late phase of EAE begins. It is characterized by the recovery of motor disabilities, although recovery remains incomplete. After day 25 motor abilities do not change (Peruga et al., 2011; Zorzella-Pezavento et al., 2013). In the current experiments, we assessed late EAE effects in mice 40 and 45 days after immunization. In vitro electrophysiology Mice were anaesthetized by inhalation of the anesthetic isoflurane, and then decapitated. Brains were dissected in cold (1–4 °C), oxygenated artificial cerebrospinal fluid (aCSF) (in mM: NaCl, 124; KCl, 4.9; KH2PO4, 1.2; MgSO4, 1.3; CaCl2, 2.5; NaHCO3, 25.6 and d-glucose, 10; pH: 7.4). Hippocampal slices were used for both field excitatory postsynaptic potential (fEPSP) recordings, and for patch-clamp analyses. Immediately after preparation, slices (400 lm) that were used for fEPSP recording were placed on a nylon net in separate 2 ml submerged chambers to enable recording from four slices simultaneously (Synchroslice system, Lohmann Research Equipment, Castrop-Rauxel, Germany). Slices were continuously perfused (at a constant flow rate of 2 ml/min) with an oxygenated aCSF solution at 30 °C. Slices were allowed to recover from cutting for 2 h. A bipolar stimulation electrode (Fredrick Haer, Bowdowinham, ME, USA) was positioned in the stratum radiatum of the CA1 region. A metal recording electrode (platinum/tungsten core, impedance: 0.5 MX; Thomas Recording, Germany) was paced in the apical dendrites of the CA1 region (Dunwiddie et al., 1978; Frey et al., 1988). Test-pulse stimuli, at low frequency (0.025 Hz) and 0.2-ms duration, were applied and evoked fEPSPs were recorded with a sample rate of 10,000 Hz. Five responses were averaged for each time-point. As an estimate of the overall synaptic strength, input–output (I/O) curves were determined to detect the maximum fEPSP (stimulation range of 60–480 lA), and a stimulation strength that evoked fEPSP that comprised 50% of the I/O maximum was used for test-pulse stimulation to generate baseline responses. Following baseline recordings for 30 min, and without changing the stimulus duration/intensity, LTP was induced via theta burst stimulation (TBS), given as three trains, 10 s apart. Each train consisted of 10 bursts of four pulses each, at 100 Hz, delivered at 200-ms intervals. In order to induce LTD, paired-pulse (PP) stimulation (50-ms PP-interstimulus interval) at 1 Hz was applied for 20 min. For patch-clamp recordings, the slices were transferred to a recording chamber, following incubation at room temperature (23 °C) for 45 min. The chamber was located in the fixed-stage of an upright OLYMPUS BX51WI microscope, and was continuously perfused with oxygenated artificial aCSF. Recording electrodes were pulled from borosilicate glass pipettes (1.5 mm external diameter). Glass electrodes with a resistance of 6–10 MX were filed with the intracellular solution (in mM: 97.5 potassium gluconate, 32.5 KCl, 5 EGTA, 10 Hepes, 1 MgCl2, 4 Na2ATP, adjusted to pH 7.2 with KOH). Recordings were performed from visually determined pyramidal neurons in the CA1 soma region.

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In some cases, cells were labeled by means of biocytin diffusion. Intrinsic membrane properties were recorded in the whole-cell current-clamp mode. Electrical signals were acquired with a HEKA EPC10 amplifier using the PATCHMASTER acquisition software (HEKA). Data were subjected to low-pass filtering at 2.9 kHz and digitized at 10 kHz. FITMASTER analysis software was used for off-line analysis. Input resistance was determined from the slope of the linear fit of the relationship between the change in membrane potential (DV) and the intensity of the injected current (between 120 pA and +60 pA). The time constant was determined from an exponential fit of the averaged voltage decay. The minimum current needed to induce an action potential was determined as a threshold current. The action potential amplitude was measured as the voltage difference between the threshold and the peak. Firing properties were examined by applying current steps of D50 pA hyperpolarizing and depolarizing square pulses (1-s duration) through the patch-clamp electrode (in the range of 200 pA to 350 pA). The adaptation ratio was calculated by dividing the initial instantaneous frequency of the first interspike interval by the final instantaneous frequency of the last interspike interval. The reciprocal of the interspike interval defined the instantaneous firing frequency. In the graphs, the data of the electrophysiology experiments are represented as ‘n’ that signifies the number of hippocampal slices and ‘N’ that signifies the number of mice. Object recognition protocol The object recognition paradigm used is one that triggers information processing by the hippocampus (Eichenbaum et al., 1999; Manns and Eichenbaum, 2009) and results in the induction of LTD in the mouse hippocampus (Goh and Manahan-Vaughan, 2013a). Experiments were conducted as described previously (Goh and ManahanVaughan, 2013a). Before the behavioral experiments were started, the mice were placed in the test chamber for the purpose of habituation, during which time the mice were allowed to explore an empty arena for four consecutive days (5 min each day). Following habituation to the test chamber, two novel objects (i.e. A and B) were presented (day 1: training phase). One familiar and one novel object (i.e. A and C) were presented 24 h after the training phase (day 2: first testing phase). One week later (day 7: second testing phase), the familiar object and a novel object (i.e. A and D) were presented to test for object recognition memory (Fig. 4A). The presentation of objects lasted for 5 min, during which time the animals were left to explore the objects freely, before being removed from the recording chamber. The familiar and novel objects, as well as their relative positions, were randomly assigned for each animal, but the positions were then kept constant for all subsequent tests. The objects and the recording chambers were cleaned thoroughly between task trials to ensure the absence of olfactory cues. The test objects were selected before the study began on the basis of their equivalent preference by a separate animal group. This was done to exclude that differences in exploration times

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Fig. 2. LTP, but not LTD, is impaired in the late phase of experimental autoimmune encephalomyelitis. (A) Both control groups of mice, OVA and OVA + PTX, and MOG + PTX mice exhibit stable basal synaptic transmission at Schaffer collateral – CA1 synapses for duration of 180 min. (B) In response to TBS application, LTP was recorded for 3 h. LTPs of control OVA and control OVA + PTX were comparable to LTP of MOG + PTX mice. (C) Following TBS, both vehicle controls OVA and OVA + PTX showed significantly higher LTP in comparison to MOG-treated mice. LTP was equivalent between OVA and OVA + PTX controls. (D) Paired-pulse (PP) stimulation (50-ms PP inter-stimulus interval) at 1 Hz for 20 min exhibited LTD at the SC–CA1 synapses in both MOG-treated and OVA + PTX control mice. With regard to the induction and maintenance phase, evoked LTD did not differ between MOG-treated and OVA + PTX control mice. Inset: The line-break indicates a change in time scale. Representative fEPSP traces (at the indicated time points) are shown within the graph. The vertical scale-bar corresponds to 1.5 mV and the horizontal scale-bar corresponds to 10 ms.

could arise from general behavioral differences in object preferences. The objects were distinctly different from one another and heavy, so that they could not be displaced by the mice. Several copies of each object were available. Spatial memory test (cookie finding test) Before testing abilities to remember a previously learned location, the mice were trained in standard cages (dimensions: 42.5 cm length, 26.6 cm width and 18.5 cm height) marked with the letter X as a visual cue, as described previously (Prochnow et al., 2012a,b). Seven training trials were performed within four subsequent consecutive days. The time delay between two training trials, when they were performed on the same day, was 8 h. A cookie fragment (trial 1 & 2: 500 mg, trial 3: 100 mg, trial 4 & 5: 50 mg) was used as a treat in training trials 1–5, whereas in training trials 6 and 7 (day 4) the cookie was replaced with mouse chow. Overnight prior to day 4, the animals received no food. The food was always hidden at the same spot in the woodchip bedding. The treat/food size was systematically reduced with the progression of the training trials in order to reduce olfactory learning cues. The mice were allowed to explore the cage for 10 min during each trial. The treat finding time was defined as the time until the mice held the treat

(Prochnow et al., 2012a,b). The last training trial (trial 7) on day 4 was followed by the testing trial, in which no treat was present. The mice trajectories were recorded and analyzed with EthoVision XT9. The mice’s spatial memory was evaluated by measuring the distance between the mouse and the place where the cookie was hidden during the training trials. Behavioral learning assessments were conducted only with the late-phase EAE group, as the motor impairments during the early phase of EAE (disability score of 3–4) precluded an accurate behavioral comparison with control animals. In the late phase EAE group, the disability score was typically 0–1 (scaling according to Hartung et al., 1988), with a limp tail sometimes occurring (score of two), but exploration times and velocity were not statistically significant from values obtained in control mice (see Results section and Figs. 4B, C, D & 5C). The brain slices used for electrophysiological analysis were obtained from the same animals that were tested behaviorally. Data analysis All experiments were carried out in experimenter-blind conditions. To analyze LTP and LTD responses between the EAE-and two control groups, a 3-way analysis of variance (ANOVA) with repeated measures

Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

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Fig. 3. Firing properties of CA1 pyramidal neurons are altered over the course of experimental autoimmune encephalomyelitis. (A) Application of depolarizing currents (from 100 to 350 pA) revealed no differences in spike frequency patterns. The firing rates of CA1 pyramidal cells were comparable between all groups in the early phase of EAE. (B) Application of depolarizing currents (from 100 to 350 pA) revealed a difference in spike frequency patterns in the late phase of EAE. The firing rate observed in MOG + PTX mice was significantly lower in comparison to both controls, OVA and OVA + PTX. (C) Current clamp recordings of repetitive firing evoked by current application (300-pA current, 1-s duration) to hippocampal cells from mice that were in the late phase of EAE.

was applied for the LTP experiment, and a 2-way ANOVA with repeated measures was applied for the LTD experiment, following confirmation that the data were normally distributed. Each point shown in the summary graphs represents the mean value ± the standard error of the mean (s.e.m.). Since the amplitude of fEPSP at these synapses correlates strongly with changes of the slope, mean slope values are exclusively presented, while amplitude data are not shown. Data obtained in the patch clamp studies of the EAEand two control groups were also assessed using a 3-way ANOVA. When appropriate, a post hoc Fisher’s test was used to determine which comparisons generated significant differences. Data are displayed as mean values ± s.e.m. Object recognition memory was analyzed (experimenter-blind) using the within-object area scoring system that was defined as sniffing of the object (with nose contact or head directed to the object) within a 2 cm radius of the object (Bevins and Besheer, 2006). Standing, sitting or leaning on the object was not scored as object exploration. Object recognition data were expressed as a percentage of the total exploration time for each object per experiment (Clarke et al., 2010; Goh and Manahan-Vaughan, 2013a). The results across animals were expressed in terms of mean ± s.e.m. Discrimination ratios were determined, and were calculated as the time spent exploring novel, or familiar, objects divided by the total exploration time. Data were presented as average discrimination ratio ± s.e.m. The data, which followed a normal distribution, were then statistically assessed using Student’s t-test.

Data obtained in the ‘cookie finding test’ were presented as mean value ± s.e.m. Statistical data analyses were performed using a 2-way ANOVA with repeated measures to compare learning performance over the seven training trials. Student’s t-test was used to evaluate memory between two groups in the test trial, and to compare the average velocities. For all statistical results, the significance level was set to p < 0.05.

RESULTS Basal synaptic transmission is unaffected by the early phase of EAE First of all, we examined if basal synaptic transmission that is elicited by test-pulse stimulation, was altered by EAE, as this could influence the induction thresholds for LTP (Fig. 2A). Fourteen or 19 days after initiation of MOG35– 55-induced EAE, we compared EAE-mice (n = 11, N = 5, whereby ‘n’ signifies the number of hippocampal slices, and ‘N’ signifies the number of mice) with two control groups: one had received OVA (n = 9, N = 6) and one had received OVA in the presence of PTX (n = 11, N = 6). No differences were seen in basal synaptic transmission that was followed for 3.5 h between all three groups of mice (ANOVA: F(2,28) = 0.008, p = 0.99), 14–19 days after immunisation (Fig. 2A). LTP is not impaired in the early phase of EAE We then assessed if LTP is affected in the early phase of EAE. Fourteen or 19 days after treatment/immunisation,

Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

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Fig. 4. Intact object recognition memory in the late phase of experimental autoimmune encephalomyelitis. (A) During the training phase (day 1), the mice of all four groups showed similar interest in both objects (object A and object B). (B) On day 2, control (OVA + PTX) and MOG-treated (MOG + PTX) mice showed a significantly higher interest in the novel object (object C), reflected by a longer exploration time of the novel object in comparison to the familiar object (object A) (p < 0.05). Values represent mean ± s.e.m. (C) Seven days after the training phase, both groups of mice showed a greater preference for the novel object (object D) than for the familiar object (object A). (D, E) The discrimination ratios on day 2 and day 7 revealed no effect of EAE on memory performance. Data are presented as average discrimination ratio ± s.e.m. ⁄p < 0.05, ⁄⁄p < 0.01, ⁄⁄⁄ p < 0.001.

theta-burst stimulation (TBS) was applied to hippocampal slices from OVA-treated (n = 9, N = 6), OVA/PTXtreated (n = 9, N = 7), or EAE-mice (n = 9, N = 8). In all three animal groups, LTP was elicited that persisted for at least 3 h (Fig. 2B) and exhibited no difference in magnitude or profile when compared across the three groups (ANOVA: F(2,24) = 0.21, p = 0.8) (Fig. 2B). LTP, but not LTD, is impaired in the late phase of EAE We then went on to assess possible changes in synaptic plasticity that occurs in the late phase of EAE, 40– 45 days after treatment. Here, in hippocampal slices from OVA-treated (n = 12, N = 10) and OVA/PTX-treated animals (n = 19, N = 12), LTP was elicited by TBS that lasted for at least 3 h (Fig. 2C). In slices from EAEanimals (n = 19, N = 10), the magnitude of induction of LTP was significantly less than in both control groups (ttest: OVA vs. EAE p = 0.02; OVA/PTX vs. EAE p = 0.03, t = 5 min), and a significant impairment of LTP was evident throughout the entire recording period after

TBS (ANOVA: F(2,45) = 3.7, p = 0.03) (Fig. 2C). More specifically, EAE-animals exhibited a lower magnitude of LTP than OVA (post hoc Fisher’s test, p = 0.03) or OVA/ PTX animals (post hoc Fisher’s test, p = 0.01). Having seen that LTP is significantly impaired in the late phase of EAE, we examined whether LTD is also affected. Low-frequency stimulation comprising PPs (50 ms PP-interstimulus interval) applied at a frequency of 1 Hz for 20 min, elicited LTD in slices from OVA/PTXanimals (n = 7, N = 6), which lasted for at least 3 h (Fig. 2D). The effects were significant compared to OVA/PTX-animals (n = 11, N = 6) that received testpulse stimulation only (ANOVA: F(1,16) = 23.7, p = 0.0001). Similarly, slices from EAE-animals (n = 6, N = 6) expressed LTD following low-frequency stimulation (Fig. 2D) that was significant compared to EAE-animals that received test-pulse stimulation only (n = 11, N = 5) (ANOVA: F(1,15) = 17.1, p = 0.008). The LTD elicited in the control and EAE-group was equivalent (ANOVA: F(1,11) = 0.05, p = 0.8) (Fig. 2D).

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current (OVA: 111.2 ± 7.4, OVA + PTX: 113.5 ± 9.9, MOG + PTX: 104.1 ± 5.8; F(2,26) = 0.37, p = 0.7) did not differ between EAE-mice (n = 11, N = 9) and mice immunized with OVA (n = 8, N = 7) or OVA + PTX (n = 10, N = 6) (Table 1). Furthermore, the action potential amplitude (OVA: 104.8 ± 1.1, OVA + PTX: 105.5 ± 0.9, MOG + PTX: 103.4 ± 1.9; F(2,26) = 0.23, p = 0.8), and the adaptation ratio (OVA: 1.88 ± 0.35, OVA + PTX: 1.85 ± 0.3, MOG + PTX: 1.97 ± 0.27; F(2,26) = 0.04, p = 0.9) were equivalent between groups (Table 1). The relationship between the firing rate of CA1 pyramidal cells and the membrane depolarization level was also assessed by examined action potential responses elicited by current step injections (D50 pA, from 100 to +350 pA). In the early phase of EAE, no differences were apparent in the different animal groups (ANOVA: F(2,26) = 0.08, p = 0.9) (Fig. 3A). Passive properties of pyramidal neurons are unchanged but active properties are altered in the late phase of EAE

Fig. 5. Impaired spatial memory in the late phase of experimental autoimmune encephalomyelitis. (A) Two training trials per day were performed (8 h delay between). In trial 1, 2 and 3 a large cookie was used (500 mg, 500 mg and 100 mg respectively). In trial 4 and 5 a small cookie was used (50 mg), whereas in 6 and 7 a cookie was replaced by a weak-odorous chow (50 mg). In each training trial, time to find the treat did not differ between OVA + PTX and MOG + PTX mice. Values represent mean ± s.e.m. (B) After the training trial, the testing trial was performed when no treat was hidden. The graph represents the mean distance of the mice to the location where the treat was hidden during the training trials for the first 60 s. MOG + PTX mice spend less time searching at the position where the treat was previously hidden. Values represent mean ± s.e.m. (C) The velocity of OVA + PTX and MOG + PTX mice was similar during the testing trial. Values represent mean ± s.e.m.

Passive and active properties of pyramidal neurons are not altered in the early phase of EAE Alterations in membrane properties could change the thresholds for the induction of synaptic plasticity and contribute to deficits in LTP observed in our study. Thus we assessed intrinsic characteristics and firing properties of CA1 pyramidal neurons in the early and late phases of EAE. In the early phase of EAE, passive cellular properties of CA1 pyramidal cells were equivalent in EAE, OVA and OVA/PTX mice 14–19 days after treatment (Table 1). Here, intrinsic properties such as the resting membrane potential (OVA: 65.75 ± 1.1; OVA + PTX: 66.6 6 ± 0.9; MOG + PTX: 66.36 ± 0.9; F(2,26) = 0.149, p = 0.9), input resistance (OVA: 140.9 ± 6.5, OVA + PTX: 152.4 ± 8.7, MOG + PTX: 144.8 ± 6.2; F(2,26) = 0.54, p = 0.6), membrane time constant (OVA: 9.9 ± 0.8, OVA + PTX: 11.02 ± 1.3, MOG + PTX: 9.4 ± 1.0; F(2,26) = 0.52, p = 0.6) and the threshold

In the late phase of EAE no significant differences in passive membrane properties were evident (Table 1). Thus, in EAE-mice (n = 10, N = 7), and mice treated with OVA (n = 9, N = 7) or OVA + PTX (n = 11, N = 7), the resting membrane potential (OVA: 64.6 7 ± 1.0, OVA + PTX: 64.27 ± 0.9, MOG + P TX: 65.05 ± 0.8, F(2,27) = 0.18, p = 0.8), input resistance (OVA: 145. 5 ± 10.4, OVA + PTX: 124.3 ± 10.6, MOG + PTX: 134.7 ± 17.7; F(2,27) = 0.7, p = 0.5), membrane time constant (OVA: 9.6 ± 1.4, OVA + PTX: 10.7 ± 0.6, MOG + PTX: 10.1 ± 0.7; F(2,27) = 0.33, p = 0.72) and the threshold current (OVA: 108.9 ± 8.6, OVA + PTX: 120 ± 7.4, MOG + P TX: 124 ± 11.3; F(2,27) = 0.6, p = 0.5), did not differ between EAE-mice (n = 10, N = 7) and mice treated with OVA (n = 9, N = 7) or OVA + PTX (n = 17, N = 7) (Table 1). Although the action potential amplitude (OVA: 103.5 ± 0.7, OVA + PTX: 103.4 ± 0.9, MOG + PTX: 102.6 ± 1.6; F(2,27) = 0.08, p = 0.9), and the adaptation ratio (OVA: 2 ± 0.18, OVA + PTX: 1.69 ± 0.16, MOG + PTX: 2.12 ± 0.25; F(2,27) = 1.2, p = 0.3) – were equivalent, changes in firing properties were evident. We observed that the firing rate-to-current injection response was shifted to the right in slices from EAEanimals in the late-phase (ANOVA: F(2,27) = 3.5, p = 0.04), compared to slices from OVA-animals, (post hoc Fisher’s test, p = 0.03) and OVA + PTX-animals (post hoc Fisher’s test, p = 0.03) (Fig. 3B). Furthermore, the firing frequency of action potentials elicited with a 300-pA current step, was significantly lower in EAE-animals in the late-phase, compared to both control groups, OVA (t-test, p = 0.006) and OVA/ PTX (t-test, p = 0.03) (Fig. 3C). This indicates that although the minimum current required to trigger an action potential was comparable across all test groups, a higher current was required to elicit equivalent firing. The firing rate elicited with the 300-pA current step in EAE animals was slower than in controls, and

Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

1.97 ± 0.27 1.85 ± 0.3 1.88 ± 0.35 Adaptation ratio

104.1 ± 5.8 113.5 ± 9.9 Current threshold, pA

111.2 ± 7.4

103.4 ± 1.9 104.5 ± 0.9 104.8 ± 1.1 Amplitude of action potential, mV

9.4 ± 1.0 11.02 ± 1.3 Membrane time constant, ms

9.9 ± 0.8

144.8 ± 6.2 152.4 ± 8.7 140.9 ± 6.5 Input resistance, MX

The table describes the mean values obtained for resting membrane potential, input resistance, membrane time constant, action potential amplitude, current threshold, and adaptation ratio for CA1 pyramidal cells from the hippocampi of animals that were treated with myelin oligodendrocyte glycoprotein (MOG35–55) peptide together with pertussis toxin (PTX), or control animals that were treated with the vehicles, Ovalbumin (OVA), or OVA together with PTX.

2.12 ± 0.25 1.69 ± 0.16 2.00 ± 0.18

124 ± 11.3 120 ± 7.4 108.9 ± 8.6

102.6 ± 1.6 103.4 ± 0.9 103.5 ± 0.7

10.1 ± 0.7 10.7 ± 0.6 9.6 ± 1.4

134.7 ± 12.7 124.3 ± 10.6 145.5 ± 10.4

65.05 ± 0.8 64.27 ± 0.9 64.67 ± 1.0

F(2,26) = 0.149 p = 0.9 F(2,26) = 0.54 p = 0.6 F(2,26) = 0.52 p = 0.6 F(2,26) = 0.23 p = 0.8 F(2,26) = 0.37 p = 0.7 F(2,26) = 0.04 p = 0.9 66.36 ± 0.9 66.66 ± 0.9 65.75 ± 1.1 Resting membrane potential, mV

MOG + PTX OVA + PTX Late phase

OVA OVA + PTX

MOG + PTX

ANOVA F= p= Early phase

OVA

Membrane property

Table 1. Comparison of membrane properties of hippocampal neurons during the early and late phases of EAE compared to controls

F(2,27) = 0.18 p = 0.8 F(2,27) = 0.7 p = 0.5 F(2,27) = 0.33 p = 0.72 F(2,27) = 0.08 p = 0.9 F(2,27) = 0.6 p = 0.5 F(2,27) = 1.2 p = 0.3

T. Novkovic et al. / Neuroscience xxx (2015) xxx–xxx

ANOVA F= p=

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equivalent to that elicited with a 300-pA current step in control slices. Thus, in the late-phase of EAE, the action potential firing rates are significantly impaired. Intact object recognition memory in the late phase of EAE Having found that LTP is impaired in the late phase of EAE, we explored whether hippocampus-dependent memory is also affected. We first assessed object recognition, using a protocol that requires hippocampal information processing (Manns and Eichenbaum, 2009) and that is tightly associated with hippocampal LTD (Goh and Manahan-Vaughan, 2013a,b,c). Not surprisingly, given that we saw no effects on LTD in late-phase EAE, object recognition memory was intact in EAE-treated animals (Fig. 4). The object recognition test consisted of the training phase (day 1), in which two novel objects were presented and two testing phases: on day 2, the familiar object A was compared with a novel object C, and on day 7, the familiar object A was compared with a novel object D (Fig. 4A). We compared learning behavior in EAE-mice (N = 10) with mice treated with OVA/PTX (N = 10). During the training phase, both mice groups explored the novel objects equally, and showed no object preference (Fig. 4B). In contrast, when one familiar (A) and one novel object (C) were presented 24 h later (day 2), the mice of both groups were able to distinguish between the familiar and the novel objects (t-test, OVA/ PTX: p = 0.01, EAE: p = 0.02) (Fig. 4C). Similarly, seven days after the initial exposure to object A and object B, both mice groups exhibited a significant preference for the novel object D, compared to the familiar object A, indicating that they could differentiate between the new and old objects (t-test, OVA/PTX: p = 0.002, EAE: p = 0.02) (Fig. 4D). Discrimination ratios were also determined to verify that the animals recognized the new from the old objects. Both OVA/PTX (N = 10) and EAE mice (N = 10) were equally successful in distinguishing between novel and familiar objects (t-test, p = 0.5) on day 1 (24 h after the training phase) (Fig. 4E). Seven days after the initial object exposure, the discrimination ratios confirmed that both treatment groups could distinguish between the familiar and novel object (t-test, p = 0.2) (Fig. 4F). Discrimination ratios determined that no difference in learning behavior was evident between groups. Impaired spatial memory in the late phase of EAE LTP is tightly associated with specific aspects of spatial memory (Kemp and Manahan-Vaughan, 2007). Thus, given the finding that LTP was impaired in the late EAE phase, we assessed spatial memory in mice 40–45 days after treatment. Mice have difficulties performing in paradigms that were originally designed for rats, such as the water maze or radial arm maze, as being prey, they are reluctant to venture out into empty spaces. Furthermore, motor deficits that might arise in EAE could confound the interpretation of behavioral data derived from tasks

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that involve substantial running or swimming. For this reason, we implemented a spatial memory test that can be conducted in a familiar environment (a test cage, that is equivalent to the home cage), and capitalizes on the intrinsic motivation of mice to forage for food and learn its location (Prochnow et al., 2012a,b). Over a 4-day period, mice were trained to remember the location of a small piece of food that was hidden deep below the bedding in a familiar cage environment. During the seven training trials OVA/PTX (N = 6) and EAE mice (N = 6) were equally successful in finding the treat (ANOVA: F(1,10) = 1.37, p = 0.3). Moreover, learning performance over the 7-trial period was improved in both groups (ANOVA: F(6,30) = 11.8, p = 0.0001) (Fig. 5A). In the test trial, the treat was removed and the animals’ memory for the treat location was assessed. Here, we observed that the OVA/PTX mice were significantly better in remembering the treat location in comparison to the EAE-mice (t-test, p = 0.03, N = 6) (Fig. 5B). Additionally, the velocity of the mice was tested, as MOG-treated mice were in the late phase of EAE. No difference in velocity was detected between the animal groups (t-test, p = 0.5, N = 5) (Fig. 5C), signifying that the difference in motor ability did not underlie the performance deficits seen.

DISCUSSION The data of this study describe that changes in hippocampal synaptic plasticity and hippocampusdependent memory occur in an animal model of MS that involves the induction of EAE (Gold et al., 2006; Constantinescu et al., 2011) using MOG35–55. The EAE status elicited by MOG35–55 in this mouse strain mimics many histopathological, clinical and immunological features of MS (Fletcher et al., 2010). Our data show that although hippocampal function is normal in the early phases of EAE, selective changes develop in late-phase EAE. Thus, LTP and spatial memory become impaired, whereas LTD and contextual object recognition memory (Goh and Manahan-Vaughan, 2013a,b) are unaffected. Passive membrane properties of hippocampal neurons are largely unaffected, but action potentials fire at a lower frequency and have a higher threshold in EAE animals in the late phase compared to controls. These data suggest that deficits in specific aspects of cognition in MS may derive from altered hippocampal synaptic plasticity that underlies hippocampus-dependent memory impairments. MS is characterized by motor, sensory and cognitive decline, which may prevail independent of each other. Furthermore, learning and memory, information processing speed, attention, and visual-spatial abilities are affected in 40–70% of MS patients (Rao et al., 1993; Bobholz and Rao, 2003; DeLuca et al., 2004; Benedict et al., 2006; Amato et al., 2010). Neuroinflammation, mediated by microglia activation (McGeer and McGeer, 2003) and directed against myelin, contributes to the pathogenesis of MS. However, not only white matter, but also gray matter atrophy and degeneration/rarification of synaptic buttons occur in cortical and subcortical regions (Bo¨ et al., 2006; Dutta and Trapp, 2007). Degeneration of gray matter appears in the early

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stages of MS and progresses with the disease’s progression, whereby it correlates with cognitive decline (Amato et al., 2004; Magnano et al., 2006). Since the hippocampus is vulnerable to metabolic challenges that characterize neurodegenerative processes (O’Banion et al., 1994; Morrison et al., 1998), disruption of its function is likely to contribute to cognitive impairment associated with MS (Geurts et al., 2007; Sicotte et al., 2008). Moreover, postmortem studies revealed neuronal demyelination in the hippocampus of MS patients, which is associated with a decreased expression of proteins involved in synaptic functions (Geurts et al., 2007; Dutta et al., 2011). Our study is the first to describe mechanistic changes in hippocampal function, i.e. which arise in an animal model of MS in conjunction with deficits in spatial memory, and which could underlie cognitive impairments that are attributed to the hippocampus in MS. Our data revealed no alteration in hippocampal LTP during the early phase of EAE. Likewise, using the same method of EAE induction, Prochnow et al. (2013) observed no apparent effect on LTP in the early phase of EAE. Interestingly, Di Filippo et al. (2013) revealed impaired hippocampal LTP in early EAE that is associated with a decreased expression of GluN2B subunits of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Lack of change of LTP in our study, may derive from differences in inducing EAE in both studies, or from the fact that, in comparison with the Di Filippo study, our mice developed relatively mild symptoms of early EAE (see Peruga et al., 2011). Thus, more severe forms of EAE might already affect synaptic plasticity at the peak of the early phase of EAE. In the late phase of EAE, when axonal loss, gliosis and tissue degeneration become pronounced (Lu¨hder et al., 2013), LTP expression became disrupted in our animals. Using mice with the same genetic background and the same EAE mouse model, Peruga et al. (2011) reported a reduction of hippocampal CA1 neurons in late-phase EAE that was associated with increased hippocampal tissue tumor necrosis factor alpha levels and neuronal loss. Thus, we assume that not only neuroinflammation but also neurodegeneration accompanied the progression of EAE in our study, with the latter becoming more pronounced during the late phase due to the accumulation of demyelinated lesions (Back et al., 2005). Although studies with regard to EAE-effects on striatal function have indicated that neuronal hyperexcitability occurs during EAE (Bermel et al., 2003; Centonze et al., 2009; Rossi et al., 2012a,b), we found no evidence of this in the hippocampus. Rather, we observed no changes in passive membrane properties, but observed that neuronal firing frequencies were decreased in the hippocampus during the late phases of EAE. This suggests that although the resting membrane potential, for example, was unchanged, other factors that contribute to the successful and serial triggering of an action potential were impaired in EAE. This may relate to neuroinflammatory processes that are triggered by EAE and affect hippocampal function. In line with this, Tong et al. (2012) reported that the incubation of hippocampal slices with IL-ß1, a proinflammatory cytokine involved in the progression of

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EAE (Chitnis and Khoury, 2003), suppressed LTP by means of a disruption of actin filament assemblies mediated by inhibition of BDNF signaling. The impairments in action potential properties may, thus, have directly resulted from the chronic inflammation processes that accompanied the late phase of EAE: Chronic inflammation debilitates neuronal activity and disrupts both synaptic plasticity and memory formation (Kotilinek et al., 2008; Belarbi et al., 2012). Various correlative studies have proposed a link between specific aspects of spatial learning and LTP (Moser et al., 1998; Li et al., 2003; Kemp and Manahan-Vaughan, 2007). Our spatial learning task made minimal demands on the motor or movement ability of the mice. Nonetheless, we found that in late EAE mice were impaired in spatial memory, although their movement speed was unaffected compared to control animals. Our findings are consistent with studies in which the water maze or the T-maze tests were used to evaluate spatial memory in EAE in rats (HaussWegrzyniak et al., 2000; Min et al., 2009) and mice (Ziehn et al., 2010). These deficits were associated with reduced synaptic density and increased apoptosis of neurons, interneurons and astrocytes in the late phase of EAE in mice (Ziehn et al., 2010). In contrast, a single transfer of autoimmune T cells, resembling a relapse of MS, led to microglia activation and neuronal injury in the hippocampus, but did not affect spatial memory that was evaluated 30 and 90 days post-induction (KurkowskaJastrzebska et al., 2013). However, methodological variations – most notably rats in contrast to mice, and EAE induction by a single transfer of encephalitogenic T cells in contrast to EAE induction by MOG35–55 injection – may explain this discrepancy. The finding that both LTP and spatial learning were impaired in the late phase of EAE indicates, nonetheless, that EAE potently affects hippocampal function. By contrast, object recognition memory was unaffected by EAE. Although this form of memory depends on the joint efforts of several brain structures, including sensory cortices, and temporal lobe structures such as the perirhinal cortex (Brown et al., 2012), the specific paradigm used requires hippocampal information processing (Eichenbaum et al., 1999; Manns and Eichenbaum, 2009; Goh and Manahan-Vaughan, 2013a), specifically because the relationship between the objects is learned within a spatial and temporal context (Brown et al., 2012). Hippocampus-dependent object recognition memory is tightly associated with hippocampal LTD (Kemp and Manahan-Vaughan, 2004, 2007, 2012), and most particularly, with context-dependent object recognition in mice (Goh and Manahan-Vaughan, 2013a,b,c; Etkin et al., 2006). Given the observation that we observed no alterations in object recognition memory in EAE mice, we explored whether LTD is affected. We observed that intact object recognition memory during the late phase of EAE is paralleled by intact LTD. This finding aligns with reports that context-dependent object recognition memory may be mediated by hippocampal LTD (Goh and Manahan-Vaughan, 2013a). This latter possibility is also corroborated by the fact that

manipulations that prevent hippocampal LTD resulting from novel object recognition, also prevent object recognition memory from being formed (Goh and ManahanVaughan, 2013b,c). Our findings with regard to object recognition memory are in line with another study that reported that this form of memory was preserved in mice at day 60 after the induction of EAE (Rodrigues et al., 2011). It could be that the impairment of object-recognition memory and LTD are dependent on the severity of axonal and neuronal loss in the hippocampus. This view is consistent with a prior study (Broadbent et al., 2004), showing that small hippocampal lesions mainly affect spatial learning, whereas only vast lesions impair objectrecognition memory. On the other side, decreased exploratory behavior and increased anxious behavior are revealed in the late phase (32–50 days after immunization) of mild EAE (Peruga et al., 2011). These behavioral changes correlated with perihippocampal neuroinflammation and a significant reduction of hippocampal CA1 neurons (Peruga et al., 2011). Taken together, this suggests that deficits in LTD and object recognition might emerge at a much later stage of EAE. This study assessed hippocampus-dependent synaptic plasticity, in conjunction with learning and memory, in the early and the late phases of MOG35–55 peptide-induced EAE. We observed that the ability to express hippocampal LTP is not affected in the early phase of EAE, whereas in the late phase of EAE, both LTP and spatial learning are impaired. The reduction in action potential firing frequencies that we observed in late-phase EAE will have contributed to this effect, by mediating impaired information transfer in, or from, the hippocampus (Staff et al., 2000; Jung et al., 2001). Context-dependent object recognition memory and hippocampal LTD were unaffected in the late phase of EAE, suggesting on the one hand, that brain structures that support object recognition memory were less affected by EAE, and that not all cognitive processes that are supported by hippocampal plasticity were affected. Taken together, our data suggest that the debilitation of hippocampus-dependent spatial memory that occurs in MS may derive from specific deficits in hippocampal function that lead to impairments of LTP.

CONFLICT OF INTEREST The authors have declared that no conflicting interests exist. Acknowledgments—This work was supported by a grant from the German Research Foundation (Deutsche Forsch ungsgemeinschaft, www.dfg.de) to D. Manahan-Vaughan (GRK736). We gratefully acknowledge the technical support of Christiane Reick, Jens Colitti-Klausnitzer and Beate Krenzek. We thank Petra Ku¨sener for animal care.

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Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

T. Novkovic et al. / Neuroscience xxx (2015) xxx–xxx Ziehn MO, Avedisian AA, Tiwari-Woodruff S, Voskuhl RR (2010) Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE. Lab Invest 90:774–786. Zorzella-Pezavento SF, Chiuso-Minicucci F, Franc¸a TG, Ishikawa LL, da Rosa LC, Marques C, Ikoma MR, Sartori A (2013) Persistent

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inflammation in the CNS during chronic EAE despite local absence of IL-17 production. Mediators Inflammation 2013:519627.

(Accepted 4 March 2015) (Available online xxxx)

Please cite this article in press as: Novkovic T et al. Hippocampal function is compromised in an animal model of multiple sclerosis. Neuroscience (2015), http://dx.doi.org/10.1016/j.neuroscience.2015.03.008

Hippocampal function is compromised in an animal model of multiple sclerosis.

Multiple sclerosis (MS) is a progressive inflammatory autoimmune disease that is characterized by demyelination and axonal damage in the nervous syste...
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