Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations

Neuropharmacology xxx (2016) 1e10

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Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations Arthur Bikbaev 1, Denise Manahan-Vaughan* Ruhr University Bochum, Medical Faculty, Dept. of Neurophysiology, Bochum, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 14 January 2016 Received in revised form 1 June 2016 Accepted 2 June 2016 Available online xxx

Hippocampal synaptic plasticity and learning are regulated by metabotropic glutamate receptors (mGlu) and particularly by mGlu5. In the hippocampus, synaptic plasticity is tightly linked to neuronal network oscillations in theta (5e10 Hz) and gamma (~30e100 Hz) frequency ranges, and specific changes in theta and gamma spectral power can predict for the success of patterned afferent stimulation in inducing robust long-term potentiation (LTP). In this study, we hypothesized that activation of mGlu5 mediates tetanisation-driven changes in network oscillations and thereby determines the longevity of LTP. To explore this, we applied high-frequency stimulation (HFS) to the perforant path input to the dentate gyrus (DG), in the presence of the negative allosteric modulator, 2-methyl-6-(phenylethynyl)pyridine (MPEP), or the positive allosteric modulator (S)-(4-fluorophenyl)-[3-(3-(3-(4-fluorophenyl)-[1,2,4]oxadiazol-5-yl)-piperidin-1-yl)]methanone (ADX47273). In freely behaving rats, administration of MPEP resulted in a significant impairment, whereas treatment with ADX47273 led to a significant enhancement, of LTP (>24 h) compared to vehicle-treated controls. Allosteric potentiation of mGlu5 also resulted in a significantly greater increase of the spectral power of theta and gamma oscillations within the period of 300 s after HFS, as compared to MPEP-treated animals or controls. Our findings show that the regulation of hippocampal LTP by mGlu5 is associated with modulation of network oscillatory activity in the period shortly after LTP induction. Taken together, these data demonstrate that changes in the spectral contents of local field activity that occur in response to patterned afferent stimulation require activation of mGlu5 and may be instrumental for the successful expression of persistent LTP. © 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: mGlu5 LTP Network oscillations Dentate gyrus Theta Gamma

1. Introduction Signalling through chemical synapses comprises the main interface between individual neurons, and enables electrochemical conversion of action potentials into postsynaptic potentials in a digital-to-analog manner. Depending on the strength and/or

Abbreviations: ADX47273, (S)-(4-fluorophenyl)-[3-(3-(3-(4-fluorophenyl)[1,2,4]oxadiazol-5-yl)-piperidin-1-yl]methanone; ANOVA, analysis of variance; DG, dentate gyrus; fEPSP, field excitatory postsynaptic potential; HFS, high-frequency stimulation; LFP, local field potential; LTD, long-term depression; LTP, long-term potentiation; mGlu, metabotropic glutamate receptor; mGlu5, metabotropic glutamate receptor subtype 5; MPEP, 2-methyl-6-(phenylethynyl)pyridine; PS, population spike; NMDAR, N-methyl-D-aspartate receptor. * Corresponding author. Medical Faculty, Dept. of Neurophysiology, Universitaetsstr. 150, MA4/150, Bochum 44780, Germany. E-mail address: [email protected] (D. Manahan-Vaughan). 1 Present address: Leibniz Institute for Neurobiology, Magdeburg, Germany.

temporal properties of afferent stimuli, targeted synapses can undergo bidirectional changes in the efficacy of synaptic transmission (Bi and Poo, 1998; Bliss and Collingridge, 1993; Li et al., 2004), which can last for days and weeks in rodents in vivo (Abraham, 2003; Kemp and Manahan-Vaughan, 2004; Manahan-Vaughan and Braunewell, 1999). Together with evidence as to the protein synthesisedependency of persistent forms of hippocampal synaptic plasticity (Frey et al., 1988; Manahan-Vaughan et al., 2000), and the fulfilment of a wide range of specific qualification criteria (Morris et al., 2003), including the occurrence of synaptic plasticity in association with spatial learning (Kemp and Manahan-Vaughan, 2004, 2008, 2012), this has led to the widespread belief that persistent forms of hippocampal long-term potentiation (LTP) and long-term depression (LTD) comprise the cellular basis of longterm memory (Bear, 1999; Kemp and Manahan-Vaughan, 2007; Malenka and Bear, 2004). Gamma oscillations (~30e120 Hz) in the neocortex and

http://dx.doi.org/10.1016/j.neuropharm.2016.06.004 0028-3908/© 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004

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hippocampus are closely involved in a plethora of neuronal processes and cognitive functions (for review, see: Buzsaki and Wang, 2012; Colgin and Moser, 2010; Singer, 1993). In the hippocampus, gamma power varies as function of theta oscillations (~5e10 Hz) and is highest in the dentate gyrus (Csicsvari et al., 2003), where both extrahippocampal sensory drive from the entorhinal cortex (Bragin et al., 1995) and local mechanisms (Atallah and Scanziani, 2009; Pernia-Andrade and Jonas, 2014) contribute to the occurrence of field gamma oscillations. Parvalbumin-positive basket cells that provide perisomatic inhibition of principal cells (Freund and Buzsaki, 1996) play a pivotal role in the genesis and maintenance of hippocampal gamma oscillations (Bartos et al., 2007; Buzsaki and Wang, 2012; Whittington and Traub, 2003). Together with intrinsic sub-threshold membrane potential oscillations of hippocampal neurons at gamma (Penttonen et al., 1998; Pike et al., 2000) and theta frequencies (Sik et al., 1995; Soltesz and Deschenes, 1993; Ylinen et al., 1995), the rhythmic hyperpolarization of principal neurons results in a dynamic activity-dependent (Atallah and Scanziani, 2009) modulation of the probability of action potential generation within the gamma window (Csicsvari et al., 2003). This provides a timing mechanism for the precision of coordinated activity in a millisecond-range within spatially distributed neuronal ensembles (Buzsaki and Draguhn, 2004). From a functional perspective, intimately related theta and gamma oscillations may reflect ongoing processes within neuronal populations (Bland, 1986; Buzsaki and Watson, 2012; Canolty and Knight, 2010; Dragoi and Buzsaki, 2006) that correspond to temporal encoding (Buzsaki and Chrobak, 1995), sensory feature binding (Gray and Singer, 1989) and informational encoding and decoding (Lisman, 2005; Lisman and Idiart, 1995). Metabotropic glutamate receptors (mGlu) belong to family C (III) of the superfamily of G-protein coupled receptors and are classified into 3 groups that differ with regard to the coupling of the receptors to G-protein complexes, signalling pathways and receptor localisation (Conn and Pin, 1997; Hermans and Challiss, 2001; Nicoletti et al., 2011). Hippocampal synaptic plasticity and learning are regulated by mGlu receptors, with (group I) mGlu5 receptors being particularly striking in their intrinsic involvement in multiple forms of hippocampus-dependent memory and information processing (Mukherjee and Manahan-Vaughan, 2012). Localised in the perisynaptic annulus and predominantly at postsynaptic sites (Hainmuller et al., 2014; Lujan et al., 1996), mGlu5 receptors are activated by glutamate release, preferentially under conditions of intense or continuous neuronal activation (Ferraguti et al., 2008), thus regulating neuronal excitability (Lanneau et al., 2002) and conferring them with the ability to reinforce synaptic plasticity-related biochemical processes (Mannaioni et al., 2001; Mao and Wang, 2002a,b; Jong et al., 2009; Kato et al., 2012). In behaving rodents, pharmacological antagonism of mGlu5 causes a significant impairment of hippocampal-based spatial memory (Balschun et al., 1999), impairs late phase of LTP (ManahanVaughan and Braunewell, 2005; Naie and Manahan-Vaughan, 2004), modulates LTD-related learning processes (Goh and Manahan-Vaughan, 2013; Popkirov and Manahan-Vaughan, 2011) and determines the direction of change in synaptic strength in synaptic subcompartments of the hippocampus (Hagena and Manahan-Vaughan, 2015). A striking interdependency between neuronal oscillations and LTP is evident. We have observed that a significant increase of the gamma power shortly after tetanisation precedes successful LTP in the dentate gyrus of freely moving animals (Kalweit et al., 2015), whereas smaller, or an absence in, increase of the gamma power is associated with short-term potentiation or the failure of afferent stimulation to result in LTP (Bikbaev and Manahan-Vaughan, 2007). Importantly, high-frequency stimulation of hippocampal afferents

represents a relatively strong afferent stimulus that can be expected to activate group I mGlu receptors, which are particularly important for the induction of persistent forms of LTP such as late (L)-LTP lasting for several hours or days (Manahan-Vaughan and Braunewell, 2005; Naie and Manahan-Vaughan, 2004, 2005). Given the important role of gamma oscillations in cognitive processes, we hypothesized that the enhancement of gamma oscillations seen in the post-tetanisation period in the abovementioned studies might reflect an involvement of mGlu5 in the processing of information received through patterned activation of afferent inputs. Thus, if such tetanisation-driven changes in the network activity do not merely precede, or correlate, with LTP (Bikbaev and Manahan-Vaughan, 2007, 2008), but are necessary for the occurrence of L-LTP, then the impairment of LTP that results from mGlu5 antagonism (Manahan-Vaughan and Braunewell, 2005; Naie and Manahan-Vaughan, 2004) should be associated with little, or no, changes in the theta and gamma power. To test this hypothesis, we implemented allosteric mGlu5 modulation that allows highly selective and non-competitive activation, or inhibition, of the receptor (Hermans and Challiss, 2001; May et al., 2007; Nicoletti et al., 2011). In effect, administration of positive or negative allosteric ligands respectively boosts or reduces, mGlumediated signalling triggered by physiologically relevant sensory inputs. Using positive and negative allosteric modulation of mGlu5, we therefore explored the putative interrelationship between of late LTP and the network activity in the dentate gyrus of freely moving animals, and its dependence on mGlu5. 2. Experimental procedures 2.1. Ethics statement This 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 (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen). All efforts were made to minimize the number of animals used. 2.2. Surgery Seven-to-eight week old male Wistar rats underwent stereotaxic implantation of chronic electrodes into the dentate gyrus as described previously (Manahan-Vaughan et al., 1998). Briefly, under sodium pentobarbital anaesthesia (“Narcoren”, 52 mg/kg i.p., Merial, Halbermoos, Germany), animals underwent implantation of a monopolar recording and a bipolar stimulating electrode made from 0.1 mm diameter Teflon coated stainless steel wire. A drill hole was made (1.5 mm diameter) for the recording electrode (2.8 mm posterior to bregma, 1.8 mm lateral to the midline) and a second drill hole (1 mm diameter, 6.9 mm posterior to bregma and 4.1 mm lateral to the midline) for the stimulating electrode (Paxinos and Watson, 1998). The dura was pierced through both holes and the recording and stimulating electrode lowered into the dentate gyrus granule cell layer and the medial perforant path, respectively. To ensure the proper positioning of the electrodes, recordings of evoked field excitatory postsynaptic potentials (fEPSPs) via the implanted electrodes were made throughout surgery. Additionally, a cannula was implanted into the lateral cerebral ventricle, through which a drug solution or vehicle was administered later throughout experiments. Once verification of the location of the electrodes was complete, the entire assembly was sealed and fixed to the skull with dental acrylic (Paladur, Heraeus Kulzer GmbH, Germany). Animals were treated pre- and postoperatively with the analgesic Meloxicam (‘Metacam’, 0.2 mg/kg, i.p., Boehringer Ingelheim,

Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004

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Ingelheim am Rhein, Germany), and the skull incision was treated both with local anaesthetic (lidocaine) and a topical antibiotic. The length of recovery period was 7e10 days. After surgery, animals were housed individually under 12 h/12 h light-dark cycle with ad libitum access to water and food. 2.3. Compounds and drug treatment The positive allosteric mGlu5 modulator (S)-(4-fluorophenyl)[3-(3-(3-(4-fluorophenyl)-[1,2,4]oxadiazol-5-yl)-piperidin-1-yl)] methanone (ADX47273; kindly provided as a gift from H. Lundbeck A/S, Copenhagen, Denmark) (Liu et al., 2008) and the negative allosteric mGlu5 modulator 2-methyl-6-(phenylethynyl)pyridine (MPEP; Biotrend, Cologne, Germany) (Gasparini et al., 1999) were injected into the lateral cerebral ventricle of freely behaving rats in a 5 ml volume over a six minute period via a Hamilton syringe. ADX47273 (60 ng in 5 ml) was prepared as a stock solution in dimethylsulfoxide and then thoroughly diluted with 0.9% NaCl so that the concentration of dimethylsulfoxide in solution for injection was less than 0.05%. MPEP (1.8 mg in 5 ml) was dissolved in 0.9% NaCl. Vehicle (n ¼ 7) and MPEP (n ¼ 7) were injected 30 min, whereas ADX47273 (n ¼ 7) was injected 45 min prior to tetanisation.

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and all analyses of LFPs were carried out using Spike2 software (Cambridge Electronic Design, Cambridge, UK). 2.6. Statistical treatment and analysis of data PS and fEPSP values, as well as the data with regard to spectral power in distinct frequency bands obtained in the post-injection period, were normalized to respective mean values recorded during the baseline period (taken as 100%). For analysis of HFStriggered changes in the spectral power of the network oscillations, the data were normalized to the mean value obtained during 100-s-long interval immediately preceding the onset of HFS. The effect of allosteric mGlu5 modulation on synaptic plasticity and on the spectral power of the network oscillations, was estimated using protected one- or two-way analysis of variance with repeated measures (ANOVA) that included drug and time factors, as well as their factorial interaction. The significance of between-group differences was evaluated using Duncan’s multiple range post hoc test in the case of LTP data, or the Bonferroni test in the case of spectral power data. All statistical treatments and analyses were carried out using the STATISTICA data analysis software system (StatSoft, Inc., Tulsa, USA). The factorial effects and differences between samples were considered significant at P < 0.05. Data are presented as mean ± s.e.m.

2.4. Measurement of evoked potentials 3. Results Throughout experiments, the animals could move freely and had unrestricted access to water and food. Responses were evoked by stimulating at low frequency (0.025 Hz, 0.1 ms stimulus duration, 16 kHz sample rate). For each time-point, five evoked potentials were averaged. Both the population spike (PS) and the slope of fEPSP were monitored. The fEPSP slope was measured as the maximal slope through the five steepest points obtained on the first positive deflection of the potential. The amplitude of PS was measured from the peak of the first positive deflection of the evoked potential to the peak of the following negative potential. By means of input/output curve determination, the maximum PS was found for each individual animal, and all potentials employed as baseline criteria were evoked at a stimulus intensity that produced 40% of this maximum, with the same intensity used for tetanisation. To induce LTP, HFS (200 Hz, 0.2 ms stimulus duration, 10 bursts of 15 stimuli with 10 s interburst interval) was applied, with measurements then taken at t ¼ 5, 10, 15 min and subsequently at 15 min intervals until 4 h after HFS has elapsed. An additional 5 measurements were taken (at 15 min intervals) ca. 24 h after HFS. 2.5. Analysis of network activity The local field potential (LFP) signal was obtained in parallel with fEPSP recordings from the granule cell layer of the dentate gyrus of freely behaving animals. LFPs were sampled at 0.5 or 1 kHz and stored on a computer hard drive for further off-line analysis. To analyze the effect of mGlu5 modulation on the LFP spectra prior to HFS, 4-s long epochs were extracted 1 s after each testpulse during the 30-min long baseline period and during the 30min long interval after injection, but before LTP induction. To evaluate the tetanisation-induced changes of oscillatory activity in the theta and gamma frequency ranges, 10 equally spaced 4-s long epochs were cut for five time intervals, which comprised 100s of recordings prior to HFS, a period of 100 s during HFS, and a period of 300 s immediately after conclusion of HFS. The results for 10 epochs were averaged for each 100-s-long time interval. After visual inspection of extracted epochs, artefact-free epochs were used for Fast Fourier transform (Hamming window function, sliding window of 1.024 s with epoch overlap 75%). Acquisition, processing

3.1. Allosteric modulation of mGlu5 affects LTP in the dentate gyrus Application of HFS to the control group of animals induced robust LTP of both population spike and fEPSP slope that remained stable over the recording period (25 h) (Fig. 1). Consistent with previous reports (Naie and Manahan-Vaughan, 2004), administration of MPEP led to a marked impairment of the late phase of LTP, which endured for maximally 3 h after HFS. In contrast to both MPEP-treated animals and controls, allosteric mGlu5 potentiation by ADX47273 resulted in an enhancement of both the early and late phases of LTP in the dentate gyrus (Fig. 1B). ADX47273, MPEP or vehicle had no effect on basal synaptic transmission: fEPSPs evoked by test-pulse stimulation were equivalent in all test groups in comparison with pre-injection baseline values. By contrast, in the period after HFS, allosteric mGlu5 modulation significantly affected the PS (drug P < 0.001; time P < 0.01; 2-way ANOVA) and fEPSP (both P < 0.001; 2-way ANOVA), although factorial interaction effects were not significant. In rats treated with MPEP, LTP was significantly impaired compared to LTP responses recorded in vehicle or ADX47273treated animals (both P < 0.001; Duncan test). In contrast, positive allosteric modulation of mGlu5 by ADX47273 led to a significant enhancement of LTP, reflected by an increase of PS and fEPSP slope values above those seen in tetanised controls (both P < 0.001; Duncan test). Thus, negative and positive allosteric modulation of mGlu5 led to opposing effects with regard to synaptic plasticity in the DG of freely behaving rats: MPEP impaired LTP and ADX47273 enhanced it. 3.2. ADX47273, but not MPEP, prevents injection-induced enhancements of theta and gamma oscillations The advantage of allosteric modulators of mGlu receptors is their non-competitive binding to the receptor complex. This provides the possibility to either inhibit or potentiate mGlu-mediated signalling triggered by orthosteric binding, which typically requires rather high concentrations of glutamate that are released upon intense electrical (afferent) activity (Nicoletti et al., 2011). Neither

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Fig. 1. Positive and negative allosteric modulation of mGlu5 elicits opposite effects on HFS-induced LTP in the dentate gyrus of freely behaving rats. A. Analog traces represent perforant path-dentate gyrus (DG)- evoked field potentials (i) 5 min prior to, (ii) 5 min after, and (iii) 24 h after induction of LTP. Scale bars correspond to 5 mV and 5 ms. B. Allosteric mGlu5 antagonism by MPEP impairs, whereas allosteric potentiation by ADX47273 enhances LTP in the DG as evidenced by respective changes in the mean PS (left) and fEPSP values (right). The arrows signify the time-points of ligand or vehicle injection, and of high-frequency stimulation (HFS) of the perforant path. Line-breaks in the X-axis indicate breaks in continuous recording of fEPSPs and LFPs. Data are shown as mean ± s.e.m.

MPEP or ADX47273 affected the baseline in the current study. However, previous studies have shown that mGlu ligands used in doses that ostensibly have no effect on synaptic transmission or strength potently influence the outcome of a subsequent attempt to induce synaptic plasticity (Mukherjee and Manahan-Vaughan, 2012). This suggests that mGlu ligands are exercising “behind the scenes” effects that are not manifested as changes in evoked potentials, but may be detectable at the level of neuronal oscillations. Thus, to examine whether allosteric mGlu5 modulation, under conditions of basal synaptic transmission (baseline), is associated with changes in network activity, we evaluated the spectral power of theta and gamma oscillations that were recorded during the period after drug or vehicle injection, but prior to HFS. Visual inspection of the field recordings revealed no apparent changes in the network activity induced by the injection of vehicle, ADX47273 or MPEP. However, quantification of the mean spectral power in the baseline and post-injection periods, revealed a treatment-dependent significant difference for frequencies below 20 Hz or above 60 Hz (Fig. 2). In control animals, injection of vehicle led to significant increases of both theta and gamma spectral power to 138.2 ± 9.1% and 111.5 ± 1.0%, respectively, when compared to baseline values (both P < 0.001; Bonferroni test). Similarly, in animals that received MPEP, a less prominent, but nonetheless, significant increase of the spectral power of theta 111.3 ± 3.2% (P < 0.01; Bonferroni test) and gamma oscillations to 112.2 ± 0.8% (P < 0.001; Bonferroni test) was evident. In contrast, no marked change of the theta power (95.4 ± 3.5%) and significant decrease of the gamma spectral power to 93.5 ± 0.8% (P < 0.001; Bonferroni test) was found after injection of ADX47273 in comparison with respective baseline values. As a consequence, the effect of mGlu5 modulation on the mean theta power was significant in the post-injection period (P < 0.001; ANOVA), reflecting lower spectral power upon either positive (P < 0.001; Bonferroni test) or negative (P < 0.01; Bonferroni test) modulation of mGlu5 in comparison with controls. This effect was also significant for gamma spectral power (P < 0.001; ANOVA), but only the values in ADX47273 group in post-injection period were markedly different in comparison with those obtained from control or MPEP-treated rats (both P < 0.001; Bonferroni test). Thus, we found that increases of the theta oscillatory power that are triggered by vehicle injection of the animals were partially, or

completely, prevented by negative, or positive, allosteric modulation of mGlu5, respectively. Additionally, potentiation of mGlu5 by ADX47273 effectively prevented an enhancement of the gamma oscillations after ligand injection, as was the case in MPEP-treated and control animals. 3.3. Positive and negative modulation of mGlu5 have opposite effects on HFS-triggered enhancements of theta and gamma oscillations Next, we tested our hypothesis that impairment of LTP as a result of allosteric mGlu5 antagonism is associated with a suppression of HFS-driven changes in theta and gamma oscillations. Given our finding that allosteric mGlu5 potentiation enhanced LTP in freely moving rats, we also explored whether HFS triggers a more pronounced enhancement of the network oscillations if given in the presence of ADX47273. First, we found that application of HFS significantly affected the relative spectral power of both theta and gamma oscillations in all groups (all P < 0.001; ANOVA). In vehicle-treated control and ADXtreated animals that showed robust LTP at later time-points, no change of theta and a significant increase in gamma power (both P < 0.001; Bonferroni test) were observed during the HFS period (Fig. 3). However, animals in these two groups also showed strong enhancements of both theta and gamma oscillatory activity within the first 5 min after LTP induction, as compared with pretetanisation values (P < 0.001 for all time points after HFS). The effect of HFS in MPEP-treated rats, all of which exhibited impaired LTP, was strikingly different. In this group, HFS led to marked decreases in theta and gamma power as compared to pre-HFS values (both P < 0.001; Bonferroni test). Furthermore, only moderate enhancements of gamma oscillations were observed in the post-HFS period (Fig. 3B, C). Thus, we observed strikingly different spectral changes as a result of HFS in the presence of MPEP as opposed to vehicle or ADX47273. Two-way ANOVA revealed that both factorial effects and their interactions were significant for the theta and gamma frequency ranges (all P < 0.001; two-way ANOVA). The relative spectral power of theta oscillations in rats treated with MPEP was markedly lower during HFS and within the first 200 s after HFS compared to controls (P < 0.01, 0.001 and 0.05, respectively;

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Fig. 2. The time-period after injection of either vehicle or MPEP, is associated with increased spectral power for frequencies below 20 Hz and above 60 Hz. Treatment with ADX47273 suppresses this effect. A. Examples of local field potential (LFP) epochs recorded in a freely moving rat during the baseline period (black trace) and after vehicle injection (grey trace). The asterisk indicates the application of a test-pulse stimulus (stimulus artefact is truncated). B. Graphs of the mean values of absolute spectral power (left) and relative spectral power (right) (normalized to baseline values) for all epochs recorded prior to and after vehicle injection (same rat as in A; frequencies above 150 Hz are not shown). C. Graph of the mean spectral power for 4-s long epochs recorded in the period after injection, but prior to HFS application in control (grey), ADX47273 (red) and MPEP (blue) groups (normalized to respective baseline means, not shown). D. Potentiation of mGlu5 by ADX47273 prevents injection-induced increase in the theta (5e10 Hz) and gamma (30e100) power that was evident in control and MPEP-treated rats within 30 min after administration of either vehicle, or ligand. Data are shown as mean ± s.e.m. #P < 0.001: (Bonferroni test for within-group differences between post-injection period and respective baseline), **P < 0.01, ***P < 0.001: (Bonferroni test for between-group differences in post-injection period).

Bonferroni test), or compared to ADX47273-treated rats (all P < 0.001; Bonferroni test). The effect of allosteric mGlu5 antagonism was even more pronounced in the case of relative gamma power, which was markedly lower in MPEP-treated animals both during and within 300 s after HFS, compared to rats treated with either vehicle, or ADX47273 (all P < 0.001; Bonferroni test). Moreover, we found that the enhancement of LTP after administration of ADX47273 (Fig. 1) was preceded by a significantly greater HFS-triggered increase of theta and gamma spectral power compared to that observed in vehicle-treated control rats. Specifically, the relative spectral power of theta oscillations was markedly higher in ADX47273-treated rats both 200 s and 300 s after HFS, compared to controls (P < 0.01 and P < 0.001, respectively; Bonferroni test). In comparison with controls, the increase of gamma spectral power in animals treated with ADX47273 was significantly greater as early as 100 s after HFS (P < 0.01; Bonferroni test), and remained significantly enhanced at both the 200 s and 300 s time points (both P < 0.001; Bonferroni test). Furthermore, we found that the profiles of the spectral power of high gamma (Canolty et al., 2006; Sullivan et al., 2011) or epsilon (Freeman, 2007) oscillations (ca. 100e150 Hz) in vehicle, ADX47273-or MPEP-treated rats in the post-tetanisation period matched their respective profiles with regard to the gamma oscillations recorded under each specific condition. Thus, these findings confirmed our hypothesis that activation of mGlu5 is required for distinct changes in the spectral contents of the network activity, in response to HFS, that precede and determine the robustness and longevity of subsequent LTP.

4. Discussion The metabotropic glutamate receptor mGlu5 is remarkable in its involvement in a multitude of cognition-relevant hippocampal

processes. On the behavioural level, it is involved in hippocampusdependent spatial learning and memory (Balschun et al., 1999, 2006). At the level of synaptic plasticity, it determines the longevity of both LTP (Manahan-Vaughan and Braunewell, 2005; Naie and Manahan-Vaughan, 2004) and LTD, and is required for spatial learning-induced forms of hippocampal plasticity (Goh and Manahan-Vaughan, 2013; Popkirov and Manahan-Vaughan, 2011). It is therefore not surprising that mGlu5 is also implicated in hippocampus-associated cognitive disorders and several diseases such as Angelman syndrome (Pignatelli et al., 2014), Fragile-X syndrome (Huber et al., 2002; Bear et al., 2004), Alzheimer’s disease (Hu et al., 2014), epilepsy (Wong et al., 2005) and psychosis (Nicoletti et al., 2015). In this study, we first confirmed that negative allosteric modulation of mGlu5 prevents persistent (>24 h) LTP, as reported previously (Manahan-Vaughan and Braunewell, 2005; Naie and Manahan-Vaughan, 2004). We additionally found that positive allosteric modulation of mGlu5 strengthens weak synaptic potentiation in freely behaving rats, as was previously reported in acute hippocampal slices (Kroker et al., 2011). Furthermore, we show that positive allosteric modulation of mGlu5 strongly reinforces LTP in behaving rats and increases the magnitude of LTP that endures for over 24 h.

4.1. Allosteric modulation of mGlu5 affects neuronal oscillations in the absence of changes in synaptic transmission During the recordings of basal synaptic transmission prior to and after administration of vehicle or allosteric modulators, the animals were awake, resting and could move freely in the recording chamber. In control animals, the period after injection of vehicle was associated with a significant increase of both theta and gamma power in comparison with baseline period. The injection procedure

Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004

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Fig. 3. Allosteric potentiation of mGlu5 enhances, whereas allosteric inhibition of mGlu5 suppresses network oscillations in the period immediately after LTP induction. A. Examples of two HFS trains delivered in the presence of vehicle (grey trace), ADX47273 (red trace) or MPEP (blue trace). Dashed rectangles denote the LFP epochs used for spectral analysis. B. Mean values of spectral power averaged per group for all LFP epochs and normalized to respective means obtained during the 100 s immediately preceding the HFS. Note the HFStriggered increase of spectral power of high-frequency (>40 Hz) oscillations in control and ADX47273-treated animals. C. Allosteric potentiation of mGlu5 (red trace) is associated with a sustained enhancement of network oscillations in the theta (5e10 Hz), gamma (30e100 Hz) and high gamma/epsilon (100e150 Hz) frequency ranges, whereas mGlu5 antagonism (blue trace) leads to significantly lower spectral power in periods both during and after HFS, compared to vehicle-treated controls (grey trace). Data are shown as mean ± s.e.m.

involves an interaction between the experimenter and the animals (see Methods), and the enhancement of the oscillatory activity could reflect a certain level of arousal caused by this. Although no effect of the treatments was evident with regard to the evoked fEPSPs, we found that administration of MPEP led to a less prominent increase of theta power as compared with controls, although gamma power was unaffected. Group I mGlu receptors engage in constitutive activity that occurs in the absence of glutamate and is regulated by Homer protein (Ango et al., 2001). As an inverse agonist (full antagonist), MPEP prevents both constitutive and ligand-dependent activity (Gasparini et al., 1999). This could have contributed to a subtle reduction of neuronal excitability in the current study, that was reflected by a relative reduction of theta power in MPEP-treated rats. Treatment of rodents with MPEP impairs attention to and learning of subtle changes in the context or circumstances of a  et al., 2015). Thus, the effect of mGlu5 test environment (Andre antagonism on the expected increase in theta power may also reflect an impoverishment of transfer of sensory information to, and from, the dentate gyrus. This interpretation is corroborated by

observations that pharmacological manipulations that disrupt attention impair the induction of dentate gyrus LTP and associated spatial learning processes (Hansen and Manahan-Vaughan, 2015). Although activation of mGlu receptors is typically expected to occur upon conditions of glutamate spillover, mGlu5 can also be activated during baseline activity, as reported for pyramidal neurons of the rat sensorimotor cortex (Bandrowski et al., 2003). In this scenario, allosteric potentiation of mGlu5 would be likely to increase neuronal excitability and could be expected to enhance theta oscillations. This was not the case, however, in our study. Rather, we observed that allosteric potentiation of mGlu5 by ADX47273 prevented the injection-induced enhancement of theta and gamma oscillations. Importantly, the period after injection of ADX47273 was also associated with a reduction of gamma power. Parvalbumin-positive interneurons that provide perisomatic inhibition of principal cells play a crucial role in the genesis of gamma oscillations (Bartos et al., 2007). Parvalbumin-positive interneurons (putative basket cells) express mGlu5 in the dentate gyrus (Hainmuller et al., 2014) and in the CA1 region (van Hooft et al., 2000). Interestingly, although

Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004

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activation of group I mGlu receptors does not affect the activity of basket cells, it increases the firing rate of mGlu1-and mGlu5expressing somatostatin-positive interneurons (van Hooft et al., 2000) that inhibit distal dendrites and reduce the excitability of principal cells (Katona et al., 1999). Thus, suppression of the injection-induced enhancements of network oscillations by ADX47273, as seen in the present study might have been mediated by similar mechanisms involving dendritic feedback inhibition. From a functional perspective, the reduced theta-gamma activity following allosteric mGlu5 potentiation may reflect an optimization of neuronal oscillations that, in turn, serves as a fundament for the subsequent successful potentiation of synaptic transmission. The changes in neuronal oscillations that we detected may also explain why doses of mGlu modulators, that do not ostensibly change synaptic efficacy, exert such a potent impact on the direction of change in synaptic strength when plasticity-inducing protocols are applied (see Mukherjee and Manahan-Vaughan, 2012 for review). 4.2. Allosteric modulation of mGlu5 alters neuronal oscillations that predict whether LTP results from afferent stimulation, and regulates the longevity of LTP The time-period immediately after HFS is critical for LTP persistence and reflects a vulnerable and decisive state for the establishment of subsequent LTP (Wigstrom and Gustafsson, 1983). In this particular period, the spectral contents of the population activity of neuronal ensembles demonstrates transient HFSinduced changes (Bikbaev and Manahan-Vaughan, 2007). In fact, the profiles of theta and gamma power, within the few minutes immediately after patterned afferent stimulation, predict the success (or failure) of LTP induction (Bikbaev and Manahan-Vaughan, 2008). In the present study, the theta and gamma profiles observed in both control and ADX47273-treated animals matched those previously reported as being characteristic for successful LTP, whereas in MPEP-treated animals they closely resembled profiles associated with short-term potentiation that lasts for less than 4 h (Bikbaev and Manahan-Vaughan, 2007, 2008; Kalweit et al., 2015). These results demonstrate that network oscillatory responses to tetanisation require activation of mGlu5. Foremost, these findings indicate that the abovementioned changes in theta and gamma oscillations can serve as a predictor of the degree of change in hippocampal synaptic efficacy and are intrinsically associated with the successful occurrence of hippocampal LTP. We propose that mGlu5 modulates network oscillations in the theta and gamma frequency ranges, after sensory (afferent) activation of the hippocampus, thereby optimising the conditions for information processing and encoding of memory traces. N-methylD-aspartate receptors (NMDARs) are thought to act as coincidence detectors for synchronized activity (i.e. properly timed presynaptic inputs), and their activation is crucial for LTP and spike timingdependent plasticity phenomena (Bi and Poo, 1998; Bliss and Collingridge, 1993). Importantly, it has been reported that mGlu5 can anchor to NMDARs via PSD95 (Tu et al., 1999) and regulates NMDAR-mediated signalling (Jia et al., 1998). Furthermore, activation of mGlu5 potentiates NMDAR-mediated calcium currents (Mannaioni et al., 2001), triggers phosphorylation of the GluN1 (Takagi et al., 2010) and GluN2A subunits of NMDARs (Takagi et al., 2012), and also regulates NMDAR subunit composition (Matta et al., 2011). In this context, mGlu receptors that are structurally and functionally coupled to NMDARs can function as “heavy artillery” that is recruited upon strong synaptic activation, and serves to reinforce the likelihood that the resultant LTP is robust and persistent. This study was conducted in the dentate gyrus that represents the main gateway to hippocampal trisynaptic loop. However, the

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dentate gyrus is not just a relay station for the neocortical information it transmits to the CA regions, but performs initial processing of information related to phenomena such as pattern separation (Kesner et al., 2004; Leutgeb et al., 2007; Kesner, 2013), regulates LTP thresholds (Wigstrom and Gustafsson, 1983), and contributes to the determination of informational saliency for hippocampus-dependent encoding (Hansen and ManahanVaughan, 2014; Hagena et al., 2016). In the intact rat brain, local gamma activity in the dentate gyrus has a peak frequency of ~80 Hz and is strongly driven by oscillatory inputs from the entorhinal cortex (Bragin et al., 1995), where it is characterized by a dominant frequency of ~90 Hz (Chrobak and Buzsaki, 1998). Furthermore, fast (~65e140 Hz) gamma oscillations in the hippocampus are strongly coupled to gamma activity in the medial entorhinal cortex, whereas slow (~25e50 Hz) gamma oscillations have been attributed to the activation of the commissural/associational pathway from CA3 to CA1 (Colgin et al., 2009). On the other hand, deafferentation of the entorhinal cortex causes a suppression of the gamma activity in the dentate gyrus, followed by activation of an intrahippocampal gamma generator in the CA3/CA1 regions and the appearance of slower gamma oscillations with a dominant frequency of ca. 40 Hz (Bragin et al., 1995). Additionally, recordings from the CA3 region in freely behaving mice revealed a broad power distribution in the range of 30e80 Hz, whereas clear peaks between 30 and 40 Hz were characteristic for both spontaneous and kainate-induced gamma oscillations in CA3 slice recordings (Lu et al., 2011). Taken together, these data indicate functionally distinct roles for low (30e50 Hz) and high (50e100 Hz or higher) gamma sub-ranges. Low gamma frequencies can reflect the activation of rather local intrahippocampal circuitry, while high gamma activity seems to be more related to long-range connectivity, activation of extrahippocampal inputs and processing of sensory information. Analysis of HFS-induced changes of the relative gamma power in rats treated either with vehicle, or with ADX47273, in the present study, revealed a strong enhancement of gamma/epsilon oscillations in a wide frequency range of 30e150 Hz. However, gamma oscillations, in the period after HFS, in rats treated with ADX47273, were characterized by a narrow peak at ~85 Hz, hence suggesting that allosteric mGlu5 potentiation optimizes the transfer from and/or processing of neocortical information within the dentate gyrus. Granule cells in the dentate gyrus are characterized by relatively low firing rates, but their majority fire series (bursts) of action potentials that are phase-locked to the ongoing theta and gamma cycle (Pernia-Andrade and Jonas, 2014). This confers granule cells with the ability to act as high-pass filters of neocortical sensory inputs, and modulation of their excitability is likely to influence the transfer of information to downstream neurons in CA3 and CA1 areas. Intriguingly, a selective impairment of single spike transmission in Synaptotagmin 1 knock-out mice led to impaired performance in a variety of behavioural paradigms, but did not affect hippocampus-dependent tasks (Xu et al., 2012). This finding led to the suggestion that neurotransmission in bursts of action potentials, as opposed to regular spiking, is necessary and sufficient for hippocampal function (Buzsaki, 2012). Bursts of action potentials were proposed to increase the reliability of synaptic transmission (Lisman, 1997) or represent the distinct neural code based upon frequency resonance (Izhikevich, 2002; Izhikevich et al., 2003). Supporting the latter suggestion, the somatodendritic membrane of hippocampal cells, characterized by a spatially nonuniform distribution of active conductances, was shown recently to be optimized for the selective filtering and amplification of inputs in the theta and gamma frequency ranges (Vaidya and Johnston, 2013). Thus, enhancements of theta oscillations driven by patterned afferent stimulation is likely to promote bursting of granule cells at gamma frequencies, leading to an increase of local

Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004

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gamma power. Our results show that such enhancements of theta and gamma network oscillations require activation of mGlu5. 5. Conclusion This study expands the impressive portfolio of key roles for mGlu5 in hippocampal function and shows that the predictive coupling of, and characteristic changes in, hippocampal network oscillations that occur shortly after LTP induction intrinsically depend on mGlu5. Specifically, our results demonstrate that changes in theta and gamma oscillations that occur as a consequence of HFS not only serve as a predictor of the magnitude and persistency of HFS-driven changes in hippocampal synaptic efficacy, but are intrinsically associated with the successful occurrence of hippocampal LTP. Furthermore, activation of mGlu5 can serve as a decisive factor in the optimization of information transfer that occurs as a result of strong afferent stimulation of cortical inputs to hippocampus. Conflict of interest statement The authors declare that they do not have any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. Acknowledgments This work was supported by a Deutsche Forschungsgemeinschaft grant (SFB 874/B1) to D. Manahan-Vaughan. We are grateful to Jens Colitti-Klausnitzer for technical assistance, and to H. Lundbeck A/S, Copenhagen, Denmark for their generous gift of ADX47273. References Abraham, W.C., 2003. How long will long-term potentiation last? Philos. Trans. R. Soc. Lond. B Biol. Sci. 358, 735e744. , M.E., Güntürkün, O., Manahan-Vaughan, D., 2015. The metabotropic glutaAndre mate receptor, mGlu5 is required for extinction learning that occurs in the absence of a context change. Hippocampus 25, 149e158. Ango, F., Prezeau, L., Muller, T., Tu, J.C., Xiao, B., Worley, P.F., Pin, J.P., Bockaert, J., Fagni, L., 2001. Agonist-independent activation of metabotropic glutamate receptors by the intracellular protein Homer. Nature 411, 962e965. Atallah, B.V., Scanziani, M., 2009. Instantaneous modulation of gamma oscillation frequency by balancing excitation with inhibition. Neuron 62, 566e577. Balschun, D., Manahan-Vaughan, D., Wagner, T., Behnisch, T., Reymann, K.G., Wetzel, W., 1999. A specific role for group I mGluRs in hippocampal LTP and hippocampus-dependent spatial learning. Learn. Mem. 6, 138e152. Balschun, D., Zuschratter, W., Wetzel, W., 2006. Allosteric enhancement of metabotropic glutamate receptor 5 function promotes spatial memory. Neuroscience 142, 691e702. Bandrowski, A.E., Huguenard, J.R., Prince, D.A., 2003. Baseline glutamate levels affect group I and II mGluRs in layer V pyramidal neurons of rat sensorimotor cortex. J. Neurophysiol. 89, 1308e1316. Bartos, M., Vida, I., Jonas, P., 2007. Synaptic mechanisms of synchronized gamma oscillations in inhibitory interneuron networks. Nat. Rev. Neurosci. 8, 45e56. Bear, M.F., 1999. Homosynaptic long-term depression: a mechanism for memory? Proc. Natl. Acad. Sci. U. S. A. 96, 9457e9458. Bear, M.F., Huber, K.M., Warren, S.T., 2004. The mGluR theory of fragile X mental retardation. Trends Neurosci. 27, 370e377. Bi, G.Q., Poo, M.M., 1998. Synaptic modifications in cultured hippocampal neurons: dependence on spike timing, synaptic strength, and postsynaptic cell type. J. Neurosci. 18, 10464e10472. Bikbaev, A., Manahan-Vaughan, D., 2007. Hippocampal network activity is transiently altered by induction of long-term potentiation in the dentate gyrus of freely behaving rats. Front. Behav. Neurosci. 1, 7. Bikbaev, A., Manahan-Vaughan, D., 2008. Relationship of hippocampal theta and gamma oscillations to potentiation of synaptic transmission. Front. Neurosci. 2, 56e63. Bland, B.H., 1986. The physiology and pharmacology of hippocampal formation theta rhythms. Prog. Neurobiol. 26, 1e54.

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Please cite this article in press as: Bikbaev, A., Manahan-Vaughan, D., Metabotropic glutamate receptor, mGlu5, regulates hippocampal synaptic plasticity and is required for tetanisation-triggered changes in theta and gamma oscillations, Neuropharmacology (2016), http://dx.doi.org/ 10.1016/j.neuropharm.2016.06.004