Aberrant high frequency oscillations recorded in the rat nucleus accumbens in the methylazoxymethanol acetate neurodevelopmental model of schizophrenia

Progress in Neuro-Psychopharmacology & Biological Psychiatry 61 (2015) 44–51

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Aberrant high frequency oscillations recorded in the rat nucleus accumbens in the methylazoxymethanol acetate neurodevelopmental model of schizophrenia Sailaja A. Goda a,⁎, Maciej Olszewski a, Joanna Piasecka a, Karolina Rejniak a, Miles A. Whittington b, Stefan Kasicki a, Mark J. Hunt a,b a b

Laboratory of the Limbic System, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland The Hull York Medical School, University of York, Heslington, York YO10 5DD, UK

a r t i c l e

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Article history: Received 8 January 2015 Received in revised form 30 March 2015 Accepted 30 March 2015 Available online 7 April 2015 Keywords: Clozapine High frequency oscillations MAM rat model NMDAR antagonists Schizophrenia

a b s t r a c t Background: Altered activity of the nucleus accumbens (NAc) is thought to be a core feature of schizophrenia and animal models of the disease. Abnormal high frequency oscillations (HFO) in the rat NAc have been associated with pharmacological models of schizophrenia, in particular the N-methyl-D-aspartate receptor (NMDAR) hypofunction model. Here, we tested the hypothesis that abnormal HFO are also associated with a neurodevelopmental rat model. Methods: Using prenatal administration of the mitotoxin methylazoxymethanol acetate (MAM) we obtained the offspring MAM rats. Adult MAM and Sham rats were implanted with electrodes, for local field potential recordings, in the NAc. Results: Spontaneous HFO (spHFO) in MAM rats were characterized by increased power and frequency relative to Sham rats. MK801 dose-dependently increased the power of HFO in both groups. However, the dose-dependent increase in HFO frequency found in Sham rats was occluded in MAM rats. The antipsychotic compound, clozapine reduced the frequency of HFO which was similar in both MAM and Sham rats. Further, HFO were modulated in a similar manner by delta oscillations in both MAM and Sham rats. Conclusion: Together these findings suggest that increased HFO frequency represents an important feature in certain animal models of schizophrenia. These findings support the hypothesis that altered functioning of the NAc is a core feature in animal models of schizophrenia. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Over the past 30 years, various neurodevelopmental models have been discovered and refined which are considered to hold more promise for understanding the altered neural circuitry changes taking place during development in schizophrenia (Geyer and Moghaddam, 2002; Lipska and Weinberger, 1993; Marcotte et al., 2001). In recent years the gestational rat methylazoxymethanol acetate (MAM) model has been intensely investigated (Moore et al., 2006). In this model, MAM is administered to pregnant dams at gestation day 17 which leads to disrupted development of limbic-cortical circuits. When the pups reach adulthood they display a variety of histopathological changes in

Abbreviations: NAc, Nucleus accumbens; NMDAR, N - methyl D- aspartate receptor; HFO, High Frequency oscillations; MAM, methylazoxymethanol acetate; spHFO, spontaneous HFO; LFP, Local Field Potential; PSTH, peri stimulus time histogram; E17, embryonic day 17; VTA, ventral tegmental area. ⁎ Corresponding author at: Laboratory of the Limbic System, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland. Tel.: +48 225892138.

http://dx.doi.org/10.1016/j.pnpbp.2015.03.016 0278-5846/© 2015 Elsevier Inc. All rights reserved.

cortical and subcortical regions (although ventricular enlargement is not significant) and develop behavioral changes such as deficits in prepulse and latent inhibition, working memory impairments and potentiated stimulant-induced hyperlocomotion, which validate their use as a model of schizophrenia (Braff and Geyer, 1990; Flagstad et al., 2005; Le et al., 2000, 2006; Talamini et al., 2000). Although animal models are useful for testing possible causative mechanisms, the complex symptoms that underscore a disease like schizophrenia can never be fully modeled in a less cogently developed mammal (Marcotte et al., 2001). The nucleus accumbens (NAc) integrates information from limbic regions and is involved in cognitive and psychomotor functions (Mogenson et al., 1988). Altered processing of information in limbic networks has been hypothesized to underlie some of the behavioral abnormalities in schizophrenia (Grace, 2000). We have shown previously, that spontaneous high frequency oscillations (130-180 Hz, HFO) can be recorded in local field potential (LFP) of the rodent NAc. HFO in awake rats are enhanced substantially in power following acute injection of N-methyl-D-aspartate receptor (NMDAR) antagonists, and to a lesser extent after administration of serotonergic hallucinogens (Goda et al.,

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2013); indicating that altered HFO are associated with pharmacological models of schizophrenia. Although increased HFO power can be recorded in many brain regions in response to NMDAR antagonists (Hiyoshi et al., 2014; Hunt et al., 2011; Kulikova et al., 2012; Nicolas et al., 2011; Phillips et al., 2012b) the NAc appears to be an important locus of this activity (Olszewski et al., 2013a). In MAM rats, Phillips et al. (2012b) found increased cortical HFO power and reduced gamma power (30-80 Hz) in response to NMDAR antagonists compared to Sham rats. Spontaneous HFO in the hippocampus, also known as ripples does not appear to be altered in the MAM model, since the intrinsic ripple characteristics are not significantly different from controls, although, spike-ripple synchronization is disrupted in MAM rats (Phillips et al., 2012a). Within the NAc, no major spectral differences, up to and including the gamma band, have been reported in MAM rats (Ewing and Grace, 2013). However, it is currently not known whether higher frequencies in the NAc are altered in MAM rats. Given that LFP oscillations in the NAc are fundamentally altered in pharmacological models of schizophrenia (most notably NMDAR antagonism) we tested the hypothesis that HFO would also be altered in the MAM model of schizophrenia. We also examined changes in HFO after administration of the NMDAR antagonist, MK801 and antipsychotic, clozapine. 2. Materials and methods 2.1. Animals Experiments were performed on the offspring of timed pregnant female Wistar rats, N = 7 that were isolated and received an intraperitoneal (i.p.) injection of 25 mg/kg MAM (methylazoxymethanol acetate, MRIGlobal, USA) or saline at embryonic day 17 (E17) to generate two groups - MAM rats and their controls/Sham rats. They were bred and maintained in the animal house facility at the Nencki Institute. The offspring born to MAM and Sham/saline injected dams were weaned at postnatal day P21 (at par with the standard procedure used for in house animals and in accordance with the protocols of the ethics committee) and as per the description in the previous report (Lodge, 2013). Only the male animals were taken for further experiments. A total of 17 MAM and 17 Sham rats were used in this study. One animal in the MAM group that had misplaced electrodes was excluded from analysis. All animal experiments were carried out according to the European Communities Council Directive of 24 November 1986 (86/ 609/EEC) and accepted by the local ethics committee. All efforts were made to minimize animal suffering, to reduce the number of animals used and to utilize alternatives to in vivo techniques if available. 2.2. Surgery Male offspring (280-350 g) were anesthetized with isoflurane and implanted unilaterally with twisted stainless steel electrodes (110 μm, Science Products, Germany), insulated except at the tip and targeted to NAc according to the coordinates of the stereotaxic atlas (Paxinos and Watson, 1998) (AP: +1.6 mm; ML: +1.2 mm; DV:-7.1 mm). A silver wire connected to a skull screw posterior to the bregma was used as the ground/reference. Additional stainless steel hooks and screws were also implanted to hold the socket firmly on the skull. The animals were then transferred to their home cage where they were provided with unlimited access to food and water. 500 mg of paracetamol dissolved in 300 ml of bottled drinking water was provided to the animals for a period of 3-4 days after surgery to ease the surgical pain and facilitate speedy recovery. 2.3. LFP recordings One week after surgery, the animals were handled for approximately 30 min per day, for 2 days. Following this, LFPs were recorded for

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20 min for at least 2 days, to check the signal quality, habituate the animals to the experimental chamber (dimensions: 50 × 44 × 40 cm), and also to the process of connecting/disconnecting to the recording cable. All experiments were performed from 8 AM to 6 PM in a quiet room with dim light and not in darkness. LFPs were recorded using a JFET preamplifier at the head stage, the signals were relayed through a commutator (Crist Instruments, USA), amplified ×1000, filtered 0.1–1000 Hz (A-M Systems, USA), digitized 4 kHz (Micro1401, Cambridge Electronic Design, Cambridge, UK) and stored on a computer for offline analysis. 2.4. Experiments All animals were recorded for 20 min and then given i.p. injection of either the drug or its vehicle. All experiments were performed according to a Latin square design, whereby every animal received each dose of the drug in a pseudo-randomized order to minimize the number of animals used. The drug washout between consecutive experiments was at least 3 days. For the first set of experiments (N = 8 MAM and N = 11 Sham) after baseline recording for 20 min the animals were given (i.p.) injection of MK801 (0.05, 0.15, 0.3 mg/kg; Sigma Aldrich, Poznan, Poland) or its vehicle (saline) and were recorded for an hour post injection. For experiments using antipsychotics (N = 9 MAM and N = 6 Sham), after 20 min baseline, the animals were first injected (i.p.) with MK801 (0.15 mg/kg) or its vehicle (saline) and then after 30 min they were injected (i.p.) with clozapine (15 mg/kg) or its vehicle – DMSO (Sigma Aldrich, Poznan, Poland) – and recorded for at least an hour. Motor activity of the animal was also measured using beam breaks system; consisting of 15 infrared photocells at a distance of 2.54 cm between each (Columbus Instruments, USA) both during baseline habituation period and also for the duration of the experiments. The device measured the horizontal motor activity and the sum of total activity was analyzed as movement across the chamber. 2.5. Histology Electrolytic lesions were made and the brains dissected. Electrode placement was identified on 40 μm thick Cresyl violet stained sections. The animals which had the electrodes located in the NAc alone were included for offline analysis. 2.6. Data analysis Raw LFPs were inspected and large movement artifacts were removed from the signal. From the power spectral analysis using FFT, total power of frequency bands and dominant frequency for HFO typically 130–180 Hz were analyzed in 60 s data bins using FFT of 4096 points (Spike2). The total power of delta (0-4 Hz) and gamma (30-100 Hz) bands were also analyzed. For modulation analysis the accumbal LFPs were digitally filtered (delta: 0.1-4 Hz and HFO: 130–180 Hz). The mean amplitude and standard deviation (SD) of the filtered signals were calculated at baseline for each rat. Events corresponding to a peak, when the trough-to-peak amplitude exceeded 3 and 5 SD, were extracted from the filtered waveforms (Spike2 script) to create event channels. The event channel corresponding to delta 3 SD was used as a trigger channel for peristimulus time histogram (PSTH) of HFO event channel (5 SD) and for calculating the averaged potential of the filtered delta waveform channel. These thresholds were chosen as they correspond to high amplitude delta oscillations and the highest amplitude HFO band. Thus, the threshold values for extracting events were selected on the basis of analysis of amplitudes of delta and HFO bands in baseline and after drugs. PSTHs and averaged potentials were obtained for the last 1000-s period during baseline (BL) and after MK801 injection. The post injection clozapine period was analyzed starting 15 min after injection. Before averaging PSTHs were normalized (i.e. the number of events per bin was divided by the total number of events divided by the number of bins). Normalization was

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done to emphasize the population response by minimizing the contribution of particular rats with especially strong responses. The interval histogram was calculated for the delta event (3 SD) channel. 2.7. Statistics Data were expressed either as absolute values or as percentage change of the baseline values. The majority of the data passed the Kolmogorov-Smirnov normality test and were therefore treated as parametric. Further analyses were done using Student's t-test’s, oneway ANOVA, and repeated measures two-way ANOVA as appropriate (GraphPad Software, San Diego California, USA). Bonferroni post hoc test was used to find significant differences between MAM and Sham groups. In all the cases, values were expressed as mean ± SEM and were considered to be significant if p b 0.05. 3. Results 3.1. Spontaneous HFO in the NAc is enhanced in MAM rats Consistent with our previous findings, inspection of accumbal power spectra revealed the presence of a small peak in the HFO (130-180 Hz) range. In MAM rats the power and frequency of the HFO band appeared greater compared to those of the Sham controls. Mean power spectra for MAM (n = 16) and Sham (n = 17) rats at baseline are shown in Fig. 1A. One rat was excluded from the MAM group due to misplaced electrodes. Integrated power for the HFO (130-180 Hz) band at baseline revealed significantly greater power (t(31) = 3.6, p = 0.001) and a higher dominant frequency (t(31) = 7.1, p b 0.0001) in MAM compared to Sham rats (B, C). In contrast, integrated power for delta (b 4 Hz) and gamma (30-100 Hz) were not different between the groups (t(31) b 0.8, p b 0.42; 1D, E). 3.2. MK801-induced increases in HFO frequency, but not power, are occluded in MAM rats Representative spectrograms showing the effect of 0.15 mg/kg MK801 on HFO in MAM and Sham rats are shown in Fig. 2A. Mean power spectra of all the rats are shown in 2B. MK801 dosedependently increased the frequency of HFO in Sham (n = 11) but not MAM rats (n = 7). The effect of injection of MK801 on the frequency of HFO is shown in 2C. Repeated measures two way ANOVA of the change in frequency after MK801 revealed a significant effect of dose

(F(3,48) = 6.1, p = 0.0013), group (F(1,16) = 7.11, p = 0.017) and dose × group interaction (F(3,48) = 3.46, p = 0.023). Bonferroni post hoc test revealed 0.15 mg/kg (p b 0.05) and 0.3 mg/kg (p b 0.001) doses of MK801 increased HFO frequency in Sham but not MAM rats. Significant differences were also found between 0.05 mg/kg and 0.3 mg/kg doses (p b 0.01) in Sham but not MAM rats. Complete time courses showing the effect of MK801 on HFO frequency are shown in Fig. 2D. Due to the difference in power of spHFO we expressed power as a percentage of baseline values (for absolute values see Supplementary Fig. 1). MK801 dose-dependently increased the power of HFO in both MAM and Sham rats (Fig. 2E). Repeated measures two way ANOVA revealed significant effect of dose (F(3,48) = 58.95, p b 0.0001) but no group effect (F(1,16) = 0.00, p = 0.99) or dose × group interaction (F(3,51) = 0.28, p = 0.83). Bonferroni post hoc test revealed a significant difference between vehicle and 0.15 and 0.3 mg/kg doses for both MAM and Sham (p b 0.05), but not between vehicle and the 0.05 mg/kg dose. Complete time courses for all doses of MK801 are shown in Fig. 2F. We also analyzed the absolute values, insert Fig. 2E, repeated measures two way ANOVA revealed a significant effect of dose (F(3,48) = 46.33, p b 0.0001) but not group (F(1,16) = 1.09, p = 0.28) and dose × group interaction (F(3,48) = 1.3, p = 0.62). However, pair-wise comparison of the absolute values, using Student's t-test, revealed a significant difference in the power of HFO between MAM and Sham for vehicle (t(16) = 4.75, p = 0.0002) and also for lowest dose (t(16) = 2.521, p = 0.022), but not for 0.15 mg/kg (t(16) = 2.1, p = 0.057) or the highest dose 0.3 mg/kg (t(16) = 0.02, p = 0.98). MK801 dose-dependently produced locomotor hyperactivity (repeated measures two way ANOVA, dose (F(3,17) = 41.8, p b 0.0001)) but no effect for group (F(1,17) = 1.66, p = 0.3) or dose × group interaction (F(3,48) = 0.52, p = 0.84) (Fig. 3). We also evaluated locomotor activity during the 2 baseline days (20 min). ANOVA did reveal a significant effect of day (F1,17 = 5.04, p = 0.038), but no differences were found for group (F1,17 = 0.09, p = 0.77), and group × time interaction (F1,17 = 0.00, p = 0.96). 3.3. Clozapine produces comparable reductions in HFO frequency in MAM and Sham rats Changes in spHFO frequency and power for individual rats 60 min after injection of clozapine 15 mg/kg with respect to baseline are shown in Fig. 4A. Clozapine reduced the frequency of spHFO in both

Fig. 1. Spontaneous accumbal HFO are enhanced in MAM rats. A, The mean power spectra of MAM (n = 16) and Sham (n = 17) rats calculated for the 1200 s at baseline. B, C, Power and frequency of HFO (130-180 Hz) during baseline was significantly higher in MAM compared to Sham rats. D, E, Power of delta (b4 Hz) and gamma (30-100 Hz) was not significantly different between MAM and Sham rats. **p b 0.01, ***p b 0.001 indicate significant differences between MAM and Sham rats in unpaired t-test.

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Fig. 2. MK801 dose-dependently increases the power but not frequency of accumbal HFO in MAM rats. A, Representative spectrograms showing the effect of intraperitoneal injection of 0.15 mg/kg MK801 on the HFO band. B, Mean power spectra after injection of MK801 from MAM (n = 7) and Sham rats (n = 11). C, MK801 dose-dependently increases the frequency of HFO in Sham, but not MAM, rats. D, Complete time courses showing the frequency of HFO in both MAM and Sham rats after different doses of MK801. E, Bar chart showing the average change in HFO power for the post injection period relative to baseline. MK801 produces comparable dose-dependent increases in the power of HFO in both MAM and Sham rats. Insert shows the absolute power of HFO post injection of MK801 (asterisks are ***p b 0.001 and *p b 0.05, Student's t-test). F, Complete time courses showing the changes in power of HFO for MAM and Sham rats.

Fig. 3. MK801 dose-dependently increases locomotor activity in MAM and Sham rats. Total numbers of beam breaks post injection of MK801 (0, 0.05, 0.15 and 0.3 mg/kg) in MAM (n = 7) and Sham rats (n = 11) are shown. MK801 dose-dependently increased the total number of beam breaks, but there were no significant differences between MAM and Sham rats.

MAM (p b 0.001, paired t-test, N = 9) and Sham (p b 0.05, N = 6) (Fig. 4A1). HFO frequency post clozapine was 128.3 ± 2.9 Hz and 125.8 ± 3.8 Hz, for Sham and MAM respectively. The frequency shift between the groups approached but did not reach statistical significance (p = 0.075, unpaired t-test). Since the power of spHFO was significantly larger in MAM rats (p b 0.05), we analyzed percentage change relative to baseline values. In general, clozapine increased the power of spHFO in the NAc, however there was no significant difference between the groups (p = 0.96, Fig. 4A2). The absolute values for frequency and power are shown for all rats at baseline and 60-min post clozapine (Fig. 4B1, B2). Mean power spectra, in MAM and Sham rats, 60-min post injection of clozapine 15 mg/kg are shown in C. We also examined the effect of clozapine on MK801-induced enhancement of HFO in MAM and Sham rats. Repeated measures two-way ANOVA revealed a significant effect of clozapine (F(1,13) = 71.32, p b 0.0001) but no group effect (F(1,13) = 0.0, p = 0.99) or clozapine × group interaction (F(1,13) = 0.27, p = 0.61) (Supplementary Fig. 2). Bonferroni post hoc test revealed that clozapine significantly reduced the frequency of HFO in both MAM and Sham rats (p b 0.001, for both).

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Fig. 4. Clozapine produces comparable reductions in the frequency of HFO in MAM and Sham rats. A, Bar chart showing the change in HFO frequency (A1) and power (A2) in MAM (n = 9) and Sham (n = 6) rats. Values are taken 60-min after injection and are with respect to baseline. B, Plots showing the individual changes in frequency and power (*p b 0.05, ***p b 0.001 indicate significant differences between baseline and post clozapine; paired t-test). C, Mean power spectra approx 60 min post injection of clozapine for MAM and Sham rats.

3.4. Modulation of HFO activity by delta band oscillations Bouts of spHFO could be clearly seen in the spectrograms of the raw LFPs of both MAM and Sham rats (Fig. 5). Visual inspection indicated that these bursts were modulated by slow oscillations. Crosscorrelation of HFO events triggered on delta (b 4 Hz) oscillations revealed powerful modulation by delta in Sham and MAM group animals in all tested situations: baseline (A, B), and after MK801 (C, D) and clozapine injection (E, F). Further analyses were performed to validate this result. Both PSTHs and averaged potentials were overlapped, as shown on panels in the right column of Fig. 5. From the plots it is clear that the decreased delta amplitude is accompanied by a reduced number of HFO events, while the increasing phase is accompanied by an increase of the number of high amplitude of events. Both the PSTHs and the averaged delta potential after clozapine administration showed deeper oscillations which might occur in bouts. Paired t-test analysis of dominating delta frequency in Sham group showed significant difference (p = 0.0229, t = 2.642, df = 11) between baseline and after clozapine treatment (2.02 Hz vs 2.36 Hz) (Fig. 5G). In MAM group the same test did not show significant difference (p = 0.064, t = 2.11, df = 9) for the delta band frequency between baseline and after clozapine treatment (1.97 Hz vs 2.33 Hz) (Fig. 5H).

4. Discussion We have shown previously that spontaneous HFO (spHFO) can be recorded as a low amplitude oscillation in the NAc of freely moving rats (Hunt et al., 2006, 2009). In this study, spHFO were clearly visible in the power spectra of MAM and Sham rats. In MAM rats we found that spHFO were greater in power (~ 2-fold) and frequency (~ 20 Hz) compared to Sham rats. Notably, we did not find changes in the integrated power of delta or gamma frequency bands. In line with this, Ewing and Grace (2013) did not report changes for spontaneous LFP oscillations b 100 Hz in the NAc, although auditory evoked potential activity is altered in the core of the NAc. Increased cortical gamma power has been reported in MAM rats, using EEG skull screws, which detect changes over a much larger area

than LFP electrodes (Kocsis et al., 2014). In an earlier study, Phillips et al (2012b) found that under control conditions (vehicle injection) the integrated power for the HFO band was not statistically greater in MAM compared to Sham rats indicating differences in this frequency band between the NAc and cortical regions. However, inspection of cortical power spectra revealed the presence of a small peak, at approx. 170 Hz, in MAM but not Sham rats (see Fig. 4 in (Phillips et al., 2012b)) indicating that although much larger increases in HFO occur in the NAc, nevertheless much weaker, but parallel changes can be observed in the cortex. This situation is reminiscent of a recent study by our group showing that administration of antagonists produces changes in frequency and power of HFO in the NAc that are paralleled to a much weaker extent in cortical areas (Olszewski et al., 2013a). Our studies, and those of others, have shown that the mechanism enhancing HFO amplitude in acute conditions is, at least in part, mediated by NMDAR (Hiyoshi et al., 2014; Hunt et al., 2006; Kulikova et al., 2012; Nicolas et al., 2011; Phillips et al., 2012b). In our study, we found that MK801 produced dose dependent increases in the power of HFO in the NAc which were comparable for both MAM and Sham rats. Due to the larger power in MAM rats, prior to injection, we also analyzed data relative to baseline values. As expected, analysis of the absolute power revealed greater values in MAM rats (Fig. 2E, inset). It is important to point out that although the power of spHFO in MAM rats was greater than in Sham rats, HFO power post NMDAR antagonism was far more substantial than the changes in spHFO in MAM rats. Phillips et al. (2012b) also showed that NMDAR antagonists increase the power of HFO in cortical areas, however, in their study they found that the power of HFO was generally larger in MAM compared to Sham rats. The difference between this and our studies may be related to the different regions examined (we recorded LFPs in the NAc while Phillips et al. recorded cortical EEGs), type of electrode (we used small tips versus screws), or associated with the time points taken for analysis as Phillips et al. reported changes in power for a specific 10-min bin (15–25 min post MK801) while we analyzed the time course for the first hour post MK801. With respect to frequency, MK801 produced a dose-dependent increase in the frequency of HFO in Sham rats reaching approx. 150 Hz for the highest dose. In contrast, in MAM rats which already displayed faster HFO, around 160 Hz, injection of MK801 did not typically increase

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HFO frequency further. Increased frequency of spHFO is a fundamental feature of MAM and the acute NMDAR hypofunction model and it is possible that the mechanism underlying the increases in frequency may be common for both models. In contrast, power changes are additive. One explanation for the dissociation between MK801-induced increases in power and frequency in MAM rats is that the HFO network is not capable of operating at frequencies above a given threshold (i.e. 160 Hz). Alternatively, it is possible that two different networks mediate changes in HFO power and frequency. There is some evidence that NMDAR hypofunction may underlie certain neurophysiological changes in MAM rats. For example, NMDAR hypofunction has been shown to parallel the reduction in the inhibitory drive of the amygdala on ventral hippocampalevoked firing of cortical neurons found in MAM rats (Esmaeili and Grace, 2013). In MAM rats, reduced NR2B protein level has been shown and altered NMDA current in hippocampal neurons is characterized by a decrease in the decay time of the second excitatory postsynaptic current (Snyder et al., 2013). In anesthetized rats, abnormalities have been noted in the membrane potential properties of NAc and cortical neurons in MAM rats, in particular a lower proportion of neurons exhibit bistable membrane properties (transitions to UP and DOWN states) (Moore et al., 2006). Whether deficits in NMDAR function contribute to this is not known, however, NMDAR certainly play an important role in the bistability of medium spiny neurons (Vergara et al., 2003; Wolf et al., 2005). Given that NMDAR hypofunction partly reproduces the changes we found in MAM rats it remains possible that the impaired function of NMDA receptors may contribute to the enhanced power and frequency of HFO in MAM rats. Clozapine is a second generation antipsychotic drug that is used to treat some of the symptoms of schizophrenic patients. In an earlier study we found that clozapine and risperidone, and to a lesser extent first generation drugs, reduce the frequency of spontaneous and NMDAR-antagonist enhanced HFO (Olszewski et al., 2013b). These drugs also produce small but significant increases in their power. We observed equivalent reductions in HFO frequency in MAM and Sham rats which would point toward a similar mechanism underlying the effect of clozapine. MAM rats have a hyperdopaminergic phenotype (Lodge and Grace, 2007), however, the altered firing of ventral tegmental area (VTA) dopaminergic neurons is unlikely to account for the clozapineinduced reduction in frequency we observed. This is because acute administration of antipsychotic compounds produces opposite effects on the firing of VTA dopaminergic neurons in MAM and Sham rats (Sham - increased firing, MAM - decreased firing). Additionally, baseline dopamine levels in NAc in MAM rats are not increased (Flagstad et al., 2004; Lena et al., 2007) suggesting that increased dopamine efflux does not underlie this effect. As yet, the mechanisms responsible for the generation of spHFO and the underlying changes produced by psychotic and antipsychotic compounds on this oscillation are unclear. Analyses of high amplitude HFO entrainment to delta showed that the modulation pattern was preserved in awake rats and occurred both in Sham and MAM animals. Examples of such association between delta and membrane potential were presented in several papers when LFPs were recorded simultaneously with intracellular unit activity in anesthetized and awake animals (Leung and Yim, 1993; Poulet and Petersen, 2008; Rudolph et al., 2007). Such data show that negative deflection in delta band oscillations corresponds to positive (depolarized) state of membrane potential, while positive deflection corresponds to hyperpolarized membrane. The number of high amplitude HFO events

was associated with a positive deflection of the delta wave and the number of entrained events increased after injection of MK801. The mechanism responsible for the entrainment of the HFO amplitude to delta band was preserved after injection of MK801 and clozapine. This suggests that although NMDAR blockade increases the number of HFO events, modulation of HFO by delta does not appear to be dependent on NMDA receptors. It is important to point out that delta activity per se seems to be insufficient to induce HFO as these oscillations are not present in various types of anesthesia even after ketamine administration (Hunt et al., 2009). In humans, far less is known about changes in the HFO band, sometimes referred to as ‘high-gamma band oscillations (60-200 Hz)’. This is mainly due to the fact that high frequencies tend to get filtered out or at the limit of detection of EEG recordings. However, MEG and ECoG studies have identified HFO frequencies in the HFO range in the human brain associated with cognitive functions such as perception and working memory (see (Uhlhaas, 2011)). Ketamine administration increases the power of cortical gamma (40-85 Hz) in humans (Hong et al., 2010) and experimental, although unlike rat findings there were no significant elevations of frequencies above 85 Hz. As yet few human studies have examined HFO in subcortical regions. HFO (80–150 Hz) have been recorded in deep electrodes in the human NAc in patients suffering from major depression (Cohen et al., 2009), however, whether this oscillation is altered in SZ remains to be determined. In conclusion, our findings show that both spHFO in the MAM model and NMDAR antagonist-enhanced HFO are qualitatively similar – they are characterized by increased frequency and power compared to control animals. These findings point to a fundamental change in the neural circuitry of the NAc that may converge in the NMDAR hypofunction and MAM animal models of schizophrenia. Quantitatively, however, they are different as the power of HFO is roughly two-fold greater in the MAM model compared with Sham rats whilst NMDAR antagonists can increase the power of HFO by several orders of magnitude, depending on dose. Altered HFO frequency therefore appears to be a feature common to pharmacological and MAM models of schizophrenia. These findings are in line with a number of other studies which support the hypothesis that abnormal neurophysiology of the NAc is a fundamental part of schizophrenia. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.pnpbp.2015.03.016. Ethical statement All animal experiments were carried out according to the European Communities Council Directive of 24 November 1986 (86/609/EEC) and accepted by the local ethics committee. All efforts were made to minimize animal suffering, to reduce the number of animals used, and to utilize alternatives to in vivo techniques if available. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by the EU and European Regional Development Fund (Polish Science Foundation grant MPD/2009/4), NCS DEC2011/03/B/NZ4/03053 and the Wellcome Trust.

Fig. 5. HFO in both MAM and Sham rats are modulated by delta oscillations. Modulation of accumbal HFO activity by delta band oscillations in Sham and MAM rats is shown in the left column of A-F panels. The upper part of each panel shows filtered delta oscillations, the bottom part shows the bouts of HFO as a spectrogram. The right columns of A-F panels show overlapped the averaged delta potentials and high amplitude HFO activity. The HFO activity is shown as a peristimulus time histogram and the averaged delta potential was triggered by the peaks of delta oscillations with amplitudes bigger than the 3 SD of delta activity (see Materials and methods). Delta frequency after injection of clozapine was higher than during baseline in Sham rats, in other situations there were no significant differences (G, H). All means presented as mean ± SEM, BL – baseline, CLOZ – after injection of clozapine, MK801 – after injection of MK801, statistical significance: * p b 0.05.

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References Braff DL, Geyer MA. Sensorimotor gating and schizophrenia. Human and animal model studies. Arch Gen Psychiatry 1990;47:181–8. Cohen MX, Axmacher N, Lenartz D, Elger CE, Sturm V, Schlaepfer TE. Good vibrations: cross-frequency coupling in the human nucleus accumbens during reward processing. J Cogn Neurosci 2009;21:875–89. Esmaeili B, Grace AA. Afferent drive of medial prefrontal cortex by hippocampus and amygdala is altered in MAM-treated rats: evidence for interneuron dysfunction. Neuropsychopharmacology 2013;38:1871–80. Ewing SG, Grace AA. Deep brain stimulation of the ventral hippocampus restores deficits in processing of auditory evoked potentials in a rodent developmental disruption model of schizophrenia. Schizophr Res 2013;143:377–83. Flagstad P, Mork A, Glenthoj BY, van BJ, Michael-Titus AT, Didriksen M. Disruption of neurogenesis on gestational day 17 in the rat causes behavioral changes relevant to positive and negative schizophrenia symptoms and alters amphetamine-induced dopamine release in nucleus accumbens. Neuropsychopharmacology 2004;29: 2052–64. Flagstad P, Glenthoj BY, Didriksen M. Cognitive deficits caused by late gestational disruption of neurogenesis in rats: a preclinical model of schizophrenia. Neuropsychopharmacology 2005;30:250–60. Geyer MA, Moghaddam B. Animal models relevant to schizophrenia disorders. Neuropsychopharmacology: the fifth generation of progress. American College of Neuropsychopharmacology; 2002. p. 689–701. Goda SA, Piasecka J, Olszewski M, Kasicki S, Hunt MJ. Serotonergic hallucinogens differentially modify gamma and high frequency oscillations in the rat nucleus accumbens. Psychopharmacology (Berl) 2013;228:271–82. Grace AA. Gating of information flow within the limbic system and the pathophysiology of schizophrenia. Brain Res Brain Res Rev 2000;31(2-3):330–41. [2000 Mar]. Hiyoshi T, Kambe D, Karasawa J, Chaki S. Differential effects of NMDA receptor antagonists at lower and higher doses on basal gamma band oscillation power in rat cortical electroencephalograms. Neuropharmacology 2014;85:384–96. http://dx.doi.org/10. 1016/j.neuropharm.2014.05.037. [Epub@2014 Jun 5.:384-396]. Hong LE, Summerfelt A, Buchanan RW, O'Donnell P, Thaker GK, Weiler MA, et al. Gamma and delta neural oscillations and association with clinical symptoms under subanesthetic ketamine. Neuropsychopharmacology 2010;35:632–40. Hunt MJ, Raynaud B, Garcia R. Ketamine dose-dependently induces high-frequency oscillations in the nucleus accumbens in freely moving rats. Biol Psychiatry 2006;60: 1206–14. Hunt MJ, Matulewicz P, Gottesmann C, Kasicki S. State-dependent changes in highfrequency oscillations recorded in the rat nucleus accumbens. Neuroscience 2009; 164:380–6. Hunt MJ, Falinska M, Leski S, Wojcik DK, Kasicki S. Differential effects produced by ketamine on oscillatory activity recorded in the rat hippocampus, dorsal striatum and nucleus accumbens. J Psychopharmacol 2011;25:808–21. Kocsis B, Lee P, Deth R. Enhancement of gamma activity after selective activation of dopamine D4 receptors in freely moving rats and in a neurodevelopmental model of schizophrenia. Brain Struct Funct 2014;219:2173–80. Kulikova SP, Tolmacheva EA, Anderson P, Gaudias J, Adams BE, Zheng T, et al. Opposite effects of ketamine and deep brain stimulation on rat thalamocortical information processing. Eur J Neurosci 2012;36:3407–19. Le PG, Grottick AJ, Higgins GA, Martin JR, Jenck F, Moreau JL. Spatial and associative learning deficits induced by neonatal excitotoxic hippocampal damage in rats: further evaluation of an animal model of schizophrenia. Behav Pharmacol 2000;11:257–68. Le PG, Gourevitch R, Hazane F, Hoareau C, Jay TM, Krebs MO. Peri-pubertal maturation after developmental disturbance: a model for psychosis onset in the rat. Neuroscience 2006;143:395–405.

51

Lena I, Chessel A, Le PG, Krebs MO, Garcia R. Alterations in prefrontal glutamatergic and noradrenergic systems following MK-801 administration in rats prenatally exposed to methylazoxymethanol at gestational day 17. Psychopharmacology (Berl) 2007; 192:373–83. Leung LS, Yim CY. Rhythmic delta-frequency activities in the nucleus accumbens of anesthetized and freely moving rats. Can J Physiol Pharmacol 1993;71:311–20. Lipska BK, Weinberger DR. Delayed effects of neonatal hippocampal damage on haloperidol-induced catalepsy and apomorphine-induced stereotypic behaviors in the rat. Brain Res Dev Brain Res 1993;75:213–22. Lodge DJ. The MAM rodent model of schizophrenia. Curr Protoc Neurosci 2013. [Chapter 9:Unit9.43]. Lodge DJ, Grace AA. Aberrant hippocampal activity underlies the dopamine dysregulation in an animal model of schizophrenia. J Neurosci 2007;27:11424–30. Marcotte ER, Pearson DM, Srivastava LK. Animal models of schizophrenia: a critical review. J Psychiatry Neurosci 2001;26:395–410. Mogenson GJ, Yang CR, Yim CY. Influence of dopamine on limbic inputs to the nucleus accumbens. Ann N Y Acad Sci 1988;537:86–100. Moore H, Jentsch JD, Ghajarnia M, Geyer MA, Grace AA. A neurobehavioral systems analysis of adult rats exposed to methylazoxymethanol acetate on E17: implications for the neuropathology of schizophrenia. Biol Psychiatry 2006;60:253–64. Nicolas MJ, Lopez-Azcarate J, Valencia M, Alegre M, Perez-Alcazar M, Iriarte J, et al. Ketamine-induced oscillations in the motor circuit of the rat basal ganglia. PLoS One 2011;6:e21814. Olszewski M, Dolowa W, Matulewicz P, Kasicki S, Hunt MJ. NMDA receptor antagonistenhanced high frequency oscillations: are they generated broadly or regionally specific? Eur Neuropsychopharmacol 2013a;23:1795–805. Olszewski M, Piasecka J, Goda SA, Kasicki S, Hunt MJ. Antipsychotic compounds differentially modulate high-frequency oscillations in the rat nucleus accumbens: a comparison of first- and second-generation drugs. Int J Neuropsychopharmacol 2013b;16: 1009–20. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1998. Phillips KG, Bartsch U, McCarthy AP, Edgar DM, Tricklebank MD, Wafford KA, et al. Decoupling of sleep-dependent cortical and hippocampal interactions in a neurodevelopmental model of schizophrenia. Neuron 2012a;76:526–33. Phillips KG, Cotel MC, McCarthy AP, Edgar DM, Tricklebank M, O'Neill MJ, et al. Differential effects of NMDA antagonists on high frequency and gamma EEG oscillations in a neurodevelopmental model of schizophrenia. Neuropharmacology 2012b;62: 1359–70. Poulet JF, Petersen CC. Internal brain state regulates membrane potential synchrony in barrel cortex of behaving mice. Nature 2008;454:881–5. Rudolph M, Pospischil M, Timofeev I, Destexhe A. Inhibition determines membrane potential dynamics and controls action potential generation in awake and sleeping cat cortex. J Neurosci 2007;27:5280–90. Snyder MA, Adelman AE, Gao WJ. Gestational methylazoxymethanol exposure leads to NMDAR dysfunction in hippocampus during early development and lasting deficits in learning. Neuropsychopharmacology 2013;38:328–40. Talamini LM, Ellenbroek B, Koch T, Korf J. Impaired sensory gating and attention in rats with developmental abnormalities of the mesocortex. Implications for schizophrenia. Ann N Y Acad Sci 2000;911(486-94):486–94. Uhlhaas PJ. High-frequency oscillations in schizophrenia. Clin EEG Neurosci 2011;42: 77–82. Vergara R, Rick C, Hernandez-Lopez S, Laville JA, Guzman JN, Galarraga E, et al. Spontaneous voltage oscillations in striatal projection neurons in a rat corticostriatal slice. J Physiol 2003;553:169–82. Wolf JA, Moyer JT, Finkel LH. The role of NMDA currents in state transitions of the nucleus accumbens medium spiny neuron. Neurocomputing 2005;65–66:565–70.