Modulation of Thalamo-Cortical Activity by the NMDA Receptor Antagonists

Neuropharmacology 158 (2019) 107745

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Neuropharmacology

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Modulation of thalamo-cortical activity by the NMDA receptor antagonists T ketamine and phencyclidine in the awake freely-moving rat ∗ Maria Amat-Forastera,b,c,e, , Pau Celadac,d,e, Ulrike Richtera,f, Anders A. Jensenb, Niels Platha, ∗∗ Francesc Artigasc,d,e, Kjartan F. Herrika, a H. Lundbeck A/S, Translational Biology, Valby, Denmark b University of Copenhagen, Faculty of Health and Medical Sciences, Department of Drug Design and Pharmacology, Copenhagen, Denmark c Institut d'Investigacions Biomèdiques de Barcelona IIBB-CSIC, Department of Neurochemistry and Neuropharmacology, Barcelona, Spain d Centro de Investigación Biomédica en Red de Salud Mental, CIBERSAM, Barcelona, Spain e Institut d’Investigacions Biomèdiques August Pi i Sunyer, IDIBAPS, Barcelona, Spain f Lund University, Department of Experimental Medical Science, Lund, Sweden

HIGHLIGHTS

• Ketamine and PCP increase the discharge of pyramidal neurons in mPFC of awake rats. • The increase in cortical excitability is not mediated by local disinhibition. • Ketamine and PCP moderately increase the activity of MD/CM thalamo-cortical neurons. • Ketamine and PCP strongly increase the power of high-frequency oscillatory bands.

ARTICLE INFO ABSTRACT

Keywords: Non-competitive N-methyl-D-aspartate receptor antagonists mimic schizophrenia symptoms and produce immediate NMDA receptor antagonists and persistent antidepressant effects. We investigated the effects of ketamine and phencyclidine (PCP) onthalamo- Thalamo-cortical networks cortical network activity in awake, freely-moving male Wistar rats to gain new insight into the neuronal populations Single unit recordings and brain circuits involved in the effects of NMDA-R antagonists. Single unit and local field potential (LFP) recordings Neuronal oscillations were conducted in mediodorsal/centromedial thalamus and in medial prefrontal cortex (mPFC) using microelectrode Ketamine arrays. Ketamine and PCP moderately increased the discharge rates of principal neurons in both areas while not Phencyclidine attenuating the discharge of mPFC GABAergic interneurons. They also strongly affected LFP activity, reducing beta power and increasing that of gamma and high-frequency oscillation bands. These effects were short-lasting following the rapid pharmacokinetic profile of the drugs, and consequently were not present at 24 h after ketamine administration. The temporal profile of both drugs was remarkably different, with ketamine effects peaking earlierthan PCP effects. Although this study is compatible with the glutamate hypothesis for fast-acting antidepressant action,it does not support a local disinhibition mechanism as the source for the increased pyramidal neuron activity in mPFC. The short-lasting increase in thalamo-cortical activity is likely associated with the rapid psychotomimetic action of both agents but could also be part of a cascade of events ultimately leading to the persistent antidepressant effects of ketamine. Changes in spectral contents of high-frequency bands by the drugs show potential as translational biomarkers for target engagement of NMDA-R modulators.

1. Introduction pharmacological tools in models for schizophrenia due to their ability to evoke effects reminiscent of the positive and negative symptoms as The non-competitive N-methyl-D-aspartate receptor (NMDA-R) an- well as cognitive deficits in the disorder (Javitt and Zukin, 1991; tagonists, phencyclidine (PCP) and ketamine are extensively used as Krystal et al., 2003).

∗ Corresponding author. Lundbeck A/S, Department of Translational Biology, Ottiliavej 9, 2500, Valby, Denmark. ∗∗ Corresponding author. E-mail addresses: [email protected] (M. Amat-Foraster), [email protected] (P. Celada), [email protected] (U. Richter), [email protected] (A.A. Jensen), [email protected] (N. Plath), [email protected] (F. Artigas), [email protected] (K.F. Herrik). https://doi.org/10.1016/j.neuropharm.2019.107745 Received 7 June 2019; Received in revised form 19 August 2019; Accepted 20 August 2019 Available online 21 August 2019 0028-3908/ © 2019 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/). M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745

Abbreviations Ket (R,S)-ketamine L lateral RM-ANOVA repeated measures analysis of variance LFP local field potential AMPA-R α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid MD mediodorsal nucleus of the thalamus receptors (m)PFC (medial) prefrontal cortex AP anterior posterior NMDA-R N-metyl-D-aspartate receptors AUC area under the curve norKet (R,S)-norketamine CM centromedial nucleus of the thalamus PCP phencyclidine CMR common median referencing RtN reticular nucleus of the thalamus dB decibel s.c. subcutaneously FFT fast Fourier transformation SD standard deviations GABA gamma-Aminobutyric acid SEM standard error of means HFO high-frequency oscillations V ventral HNK (2R,6R)-hydroxynorketamine Veh vehicle i.v intravenous

On the other hand, sub-anesthetic doses of ketamine have also been concordance with these observations, another recent study has also reported to mediate robust antidepressant effects in treatment-resistant reported that ketamine (1.25–20 mg/kg i.v.) mediates a dose-depen- unipolar and bipolar depressed patients (Monteggia and Zarate, 2015; dent attenuation of the discharge of deep layer pyramidal neurons in Newport et al., 2015; Zarate et al., 2006) and antidepressant-like effects the mPFC of chloral hydrate-anesthetized rats (Shen et al., 2018). in rodents (Artigas et al., 2018a; Browne and Lucki, 2013; Mion and The findings in the two latter ketamine studies in anesthetized rats Villevieille, 2013). Hence, the putative neurochemical, cellular and/or differ from a large body of evidence from e.g. neuroimaging studiesin circuit elements and mechanisms underlying these effects of ketamine rodent, primate and human brains supporting the notion that ketamine and other NMDA-R antagonists have become extensively explored in analogously to PCP mediates activation of cortical and thalamic areas recent years (Gould et al., 2017; Miller et al., 2016). (Chin et al., 2011; Downey et al., 2016; Maltbie et al., 2016; One of the current hypotheses is that the non-competitive NMDA-R Vollenweider and Kometer, 2010). In the present study, we have thus antagonists preferentially target NMDA-Rs in GABAergic neurons (Tsai conducted electrophysiological studies in awake freely-moving rats to and Coyle, 2002; Zanos and Gould, 2018), thereby enhancing excitatory examine the effects mediated by ketamine and PCP on neuronal dis- neurotransmission in cortical areas (Furth et al., 2017; Homayoun and charge and neuronal oscillations in thalamo-cortical circuits (mPFC and Moghaddam, 2007; Jackson et al., 2004; Jodo et al., 2003; Kargieman MD/CM). et al., 2007; Moghaddam et al., 1997; Suzuki et al., 2002). In the case of PCP, these actions involve the blockade of NMDA-Rs in the GABAergic 2. Methods and materials reticular thalamic nucleus (RtN) and the subsequent disinhibition of thalamo-cortical afferents, such as thalamic relay neurons in the med- 2.1. Animals iodorsal/centromedial (MD/CM) thalamic nuclei (Celada et al., 2013; Santana et al., 2011; Troyano-Rodriguez et al., 2014). The action of Male albino Wistar rats (n = 28, Charles River; 250–350 g) were ketamine appears to also involve activation of α-amino-3-hydroxy-5- housed, two per cage, under standard temperature (22 ± 1.5 °C) and methyl-4-isoxazolepropionate receptors (AMPA-Rs) (Autry et al., 2011; humidity (55–65%) in controlled laboratory conditions. Food and water Fukumoto et al., 2016; Koike and Chaki, 2014; Li et al., 2010; Maeng was available ad libitum in a 12 h light/dark cycle (lights on at 7:00 h). et al., 2008; Zanos et al., 2016), an effect that subsequently may evoke After surgery, rats were single-housed and the light cycle reversed biochemical/structural changes and increase mTOR signaling and sy- (lights off at 7:00 h). All the experimental procedures were performed naptogenesis, thereby reshaping brain circuits involved in mood dis- in accordance with the Danish legislation regulating animal experi- orders (Duman and Aghajanian, 2012; Li et al., 2010; Monteggia and ments; Law and Order on Animal experiments; Act No. 474 of 15/05/ Zarate, 2015; Popp et al., 2016). These subsequent events have been 2014 and Order No. 12 of 07/01/2016, and with the specific license for proposed to underlie the long-lasting effects of ketamine, which outlasts this experiment issued by the National Authority. the actual exposure of the drug in the central nervous system (Mion and Villevieille, 2013). 2.2. Drugs Interestingly, recent contradictory observations from experiments with subanesthetic doses of PCP and ketamine in anesthetized rats (R,S)-Ketamine (Ketolar 50 mg/ml, Pfizer) and PCP (Lundbeck A/S, challenge the “disinhibition hypothesis” as a general explanation for Valby, Denmark) were dissolved in 0.9% saline (2 ml/kg). Ketamine the effects of NMDA-R antagonists. In accordance with the hypothesis, was administered at 3 and 10 mg/kg subcutaneously (s.c.), and PCP was intravenous administration of sub-anesthetic doses of PCP to chloral administered at 2.5 and 5 mg/kg s.c. Doses of ketamine were chosen hydrate-anesthetized rats was found to markedly inhibit the activity of based on the pharmacokinetic profile of ketamine to achieve brain RtN GABAergic neurons, as well as to mainly activate the discharge of exposures in rats comparable to the clinical concentrations (Shaffer layer V pyramidal neurons in the medial prefrontal cortex (mPFC) and et al., 2014). Doses of PCP were selected based on those used in pre- thalamo-cortical relay neurons in the MD/CM, which are reciprocally vious studies where the drug influenced cortical oscillations and based connected with mPFC (Kargieman et al., 2007; Santana et al., 2011; on its pharmacokinetic profile (Gastambide et al., 2013; Hiyoshi et al., Troyano-Rodriguez et al., 2014). However, while intravenous admin- 2014). Control animals received 0.9% saline s.c. istration of sub-anesthetic doses of ketamine (1–5 mg/kg i.v.) under the same experimental conditions had a similar effect on RtN GABAergic 2.3. Surgical procedure neurons, this effect did not translate into a disinhibition of MD/CM but rather a marked inhibition of the neuronal discharge of cortico-tha- Rats were anesthetized with a 1:1 Hypnorm/Dormicum mixture lamic layer VI pyramidal neurons in the mPFC as well as of thalamo- (0.25 mg/kg fentanyl, 12.5 mg/kg fluanisone (both Lundbeck A/S, cortical relay neurons in MD/CM (Amat-Foraster et al., 2018). In Valby, Denmark) and 6.25 mg/kg midazolam (Roche A/S, Hvidovre,

2 M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745

Denmark)) by s.c. injection. Under anesthesia, the fur covering the skull cement (GC America Inc., IL)). Ground wires from each array were was shaved, iodine was applied to the skin and an eye gel was applied wrapped around an additional 2-mm screw placed over the cerebellum. to the corneas. Marcain (2.5 mg/ml bupivacaine, AstraZeneca, After surgery, rats received 5 mg/kg carprofen analgesic (Rimadyl®, Albertslund, Denmark) was used as local anesthesia at the incision site. 50 mg/ml carprofen, Pfizer, NY) and 150 mg/kg amoxicillin antibiotic Rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA) and (Noromox Prolongatum, ScanVet, Fredensborg, Denmark) daily for 5 maintained at 37 °C using a heating pad. Two holes were drilled into the days. Rats were allowed 7 days of recovery before the first recording skull to accommodate two fixed 16-channel tungsten-microwire-arrays session and were habituated to the recording box during this period. that came together with an additional silver ground wire (Innovative Neurophysiology, Durham, NC). The microwires (35 μm in diameter) 2.4. Study and experimental design were arranged in a 4 × 4 configuration with a spacing of 150 μmin between microelectrodes. Each array was implanted in the MD/CM Rats were divided into two groups (n = 14/group) for ketamine and (coordinates relative to bregma for center of array (Paxinos and PCP. All rats received three different treatments (vehicle, low dose and Watson, 2007): AP (anterior posterior): 2.6 mm, L (lateral): 0.7 mm, V high dose of the drug) according to a Latin square design with a 7-day (ventral): 5.4 mm) and the mPFC (AP: +3.4 mm, L: 0.7 mm, V: 4.7 mm) washout between injections. All recordings were carried out during the and secured to the skull with two screws and dental cement (RelyX™ dark phase (7–19 h) and during an auditory sensory gating stimulus Unicem cement (3M, Copenhagen, Denmark) and GC Fuji PLUS™ consisting in double click pairs (10 ms white noise) with an inter-

Fig. 1. Classification of single units in the mPFC into putative pyramidal neurons and putative interneurons and their basal firingrates.(A) Scatter plot of single units over the half-amplitude width and the time from trough to peak, both calculated from the average waveform of each isolated unit. (B1, B2) Representative example of the average waveform of a putative pyramidal neuron and a putative interneuron, respectively. (C1) Scatter plot of the single units over the time from trough to peak vs the basal firing rate (averaged 20-min period before injection). (C2) Distribution of single units over the basal firing rate (averaged 20-min period before injection). Single units with a probability of < 0.9 to belong to a cluster remained unclassified (grey).

3 M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745 stimulus interval of 500 ms and an inter-trial interval of 4.5 s. The au- all units were treated as “new units” for each recording session. For ditory stimulation paradigm was added to the study design to allow single units recorded in the mPFC, the time from trough to peak and the investigation of the effects of drugs on sensory gating processing. These half-amplitude width were computed for the mean action potential data set will be addressed in a separate publication. On the day of the waveform of each unit. These features have been widely used in the recording session, rats were transferred to a cage placed inside a sound literature to classify cortical neurons into putative pyramidal neurons attenuating steel box, for a 30-min habituation before the start of the and putative interneurons, and the approach has been validated with experiment. Following the start of the auditory sensory gating stimulus, optogenetic tagging and monosynaptic interactions (Bartho et al., 2004; each recording consisted of a 20-min baseline period followed by the Stark et al., 2013; Watson et al., 2016). Here, a Gaussian mixture model injection of the drug and 85 min recording post injection. For the PCP with two components was fitted to the distribution of the trough-to- study, each recording had an additional 20-min baseline period prior to peak time by using MATLAB's fitgmdist function, which is based on the the start of the auditory sensory gating stimulus. Furthermore, each rat Expectation-Maximization algorithm. The model will assign each unit in the ketamine group was recorded for an additional 30-min period to one of the two components, which correspond to the two putative with the auditory sensory gating stimulus at 24 h post administration in cell types, by maximizing the posterior probability for the unit to be- order to explore longer-lasting effects of ketamine. Animal behavior long to its assigned component. Single units with a posterior probability was surveilled with a video camera during the entire recording session. of < 0.9 to be from either distribution remained unclassified, while At the end of the study, rats were anesthetized with chloral hydrate single units with a posterior probability of > 0.9 were classified as ei- (450 mg/kg, intraperitoneal) and brains were marked passing 100 μA ther putative interneurons or pyramidal neurons (Fig. 1). current through the arrays for 10 s using a current stimulator (model A365, World Precision Instruments, Berlin, Germany). Rats were eu- 2.7. Electrophysiological data analysis thanized by cervical dislocation and brains were removed and kept at −80 °C. Histological verification of the electrode placement was con- Single unit and LFP analysis was carried out in MATLAB ducted at the end of the study. (MathWorks, Natick, MA). For each LFP signal, a spectrogram with time and frequency resolution of 5 s and 0.5 Hz, respectively, was obtained 2.5. Electrophysiological recordings employing the Fast Fourier transform (FFT). A spectrogram is a time series of power spectral densities and allows assessing the spectral Rats were connected to a 16-channel connector headstage (Plexon, content of a signal, such as the presence of oscillatory activity in certain Dallas, TX) via a commutator (Plexon, Dallas, TX) that did not restrict frequency bands, over time. Within every spectrogram, each power the rat's movement during the recording. Both single units and local spectral density was normalized to the average power spectral density field potentials (LFPs) were recorded simultaneously in the MD/CMand during the stable 20-min baseline period immediately prior to injection. the mPFC. Recordings were paused shortly at the time of the injection In this study, the spectrograms were then analyzed in 6 main frequency and during occasional disconnections of the headstage connector from bands: delta 0.5–4 Hz, theta 4–8 Hz, alpha 8–12 Hz, beta 12–30 Hz, the array due to the animal's movement. Neuronal activity was re- gamma 30–100 Hz, and high-frequency oscillations (HFO) 130–180 Hz. corded by using a preamplifier (Plexon, Dallas, TX), and data were Prior to the analysis of the spectral content, artefact detection was acquired using Plexon's OmniPlex A Neural Data Acquisition System. A performed in all channels, and 5-s segments in which data points ex- screw placed in the skull served as hardware reference, and the ceeded ± 6 standard deviations (SD) from the mean were not included common median within each 16-wire array served as a digital reference in the analysis. Rejection of a 75-s period before any pauses in the re- (common median referencing (CMR), Plexon, Dallas, TX). Spike chan- cording was also performed. The mean spectral content in the mPFC nels were bandpass filtered between 0.3 and 9 kHz (2-poles Bessel and the MD/CM in one recording was then obtained as the average over filter), preamplified 1000× and digitalized at 40 kHz. LFP channels all 16 baseline-normalized spectrograms of the corresponding array. were bandpass filtered between 0.5 and 200 Hz (4-pole Butterworth Subsequently, also the overall averages for each treatment were cal- filter), preamplified 1000×, digitalized at 40 kHz and downsampled to culated. Due to the baseline-normalization the spectral content is pre- 2 kHz. Continuous data files were saved and analyzed offline. sented either as percentage change or in decibel (dB). Similar to the LFP analysis, the analysis of the single units began with analyzing each 2.6. Spike sorting and cell classification single unit separately. For each unit, the firing rate was computed for consecutive 1-min bins and normalized to the average firing rate during Spikes were automatically sorted using the Kilosort spike sorting the stable 20-min baseline period immediately prior to injection. The software (Pachitariu et al., 2016) followed by a manual curation step normalization step was only conducted for the acute studies but not for with the ‘phy’ graphical user interface (https://github.com/kwikteam/ the 24 h study since neurons could not be tracked across recordings and phy). During the manual curation step, the automatically determined thus had to be considered separate units. The mean firing rate in the groups of spikes could be reviewed and split, as well as merged. In more mPFC and the MD/CM in one recording was then obtained as the detail, the cross-correlogram features, spike waveform similarity and average over all single units from the corresponding array and re- spike drift patterns of each group of spikes detected by a specific cording. Subsequently, also the overall averages for each treatment template were compared to those from similar templates to determine were calculated. Due to the baseline-normalization the firing rates are whether or not they should be merged. Furthermore, autocorrelogram presented as percentage change. Comparison between treatments were analysis was performed to obtain an indication of multi-unit activity then performed separately for the ketamine and the PCP group. For this and to determine whether a group of spikes should be split. Im- comparison, a 10-min window was chosen based on the drugs’ phar- portantly, a group of spikes was only considered as single unit activity if macokinetic profiles (Gastambide et al., 2013). Repeated measures it was well-isolated in feature space, showed no indication of multi-unit analysis of variance (RM-ANOVA) was used to assess differences in activity (lack of an absolute refractory period), had no atypical wave- firing rate (average 10-min bin, period 0–60 min post administration for form and was stable throughout the recording session. As a final step the acute studies and 30-min period 24 h post administration for the following manual curation, groups of spikes with > 1% of inter-spike 24 h study) and in baseline-normalized LFP power (average 10-min bin, intervals shorter than 1.1 ms were excluded (this step was performed in period 0–80 min post administration for the acute studies and 30-min MATLAB (MathWorks, Natick, MA)). With this procedure, an average of period 24 h post administration for the 24 h study). In more detail, two putative single units were isolated from each electrode MATLABs fitlme function was used to fit each tested dataset with a (Supplementary Fig. 1). Although some single units may have been linear mixed-effect model that had the fixed-effect predictors Subject, occasionally recorded repeatedly over the weeks of recording sessions, Treatment, Time, and the interaction between Treatment and Time. The

4 M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745 overall interaction between Treatment and Time for the fitted model electrospray ionization mode. Parent and daughter ion mass to charge was evaluated with MATLABs anova method. The p-values for the in- ratios (m/z) of 238 > 220, 224 > 125, 240 > 125 and 244 > 86 dividual interaction coefficients between Treatment and Time, which were used for quantification of (R,S)-ketamine, (R,S)-norketamine, were returned by the fitlme function, were Bonferroni-corrected before HNK and PCP, respectively. Nitrogen was used for the nebulizer and interpretation. For the firing rate, the area under the curve (AUC) for collision gases. The peak area correlated linearly with the concentration the period 0–60 min post administration was also calculated for each in the range of 5–5000 ng/g in brain for all analytes. Data acquisition treatment, and differences between treatments were assessed using one- and analysis was performed using Masslynx software, version 4.1 way ANOVA in SigmaPlot (version 11.0). (Waters Denmark, Taastrup, Denmark). Evoked activity in response to the auditory stimuli was not studied in detail here. A possible impact of the auditory sensory gating stimulus 3. Results was studied by comparing the basal activity levels 20 min before and 20 min after the start of the stimulation (still prior to injection) in the 3.1. Classification and basal firing rates of mPFC and MD/CM neurons animals of the PCP study (n = 14). One-way RM ANOVA was used to assess changes during the period -40-0 min prior administration Neurons recorded in the mPFC were separated into putative pyr- (average 10-min bin) (SigmaPlot, version 11.0). Data are presented as amidal neurons and putative GABAergic interneurons based on the mean ± standard error of the mean (SEM) and a p-value of < 0.05 was criteria outlined in Section 2.6 (Fig. 1 A). In the mPFC, a total of 2075 considered significant. When multiple comparisons were conducted single units were identified as putative pyramidal neurons (78%), 322 using the same data, the p-value was adjusted using a Bonferroni cor- single units were unclassified (12%) and 267 single units were identi- rection. fied as putative interneurons (10%), which is consistent with previous reports on the percentage of GABAergic interneurons in the cortex 2.8. Locomotor activity test (DeFelipe et al., 2013). A representative example of the average waveform of a putative pyramidal neuron and a putative interneuron is Levels of basal activity after administration of ketamine (3 and shown in Fig. 1B. The respective firing rates of putative pyramidal 10 mg/kg), PCP (2.5 and 5 mg/kg) and vehicle (0.9% saline) s.c. were neurons and putative interneurons in the mPFC were not used as clas- assessed in a separate group of animals (n = 9/treatment). The test was sification criteria for these neurons as there was no clear clustering performed during the light phase in macrolon type III (high model) (Fig. 1 C). In the MD/CM, which is mainly composed of thalamic relay cages equipped with two rings of infrared light beams to count motility neurons, 3053 single units were identified (see total number of neurons and rearings. The rats were habituated to the cages, in which they were recorded per treatment in Table 1). The mean firing rates of the re- placed individually, the day before the test. For the test, the rats were corded neurons during the 20-min baseline immediately prior to in- habituated for 1 h, followed by a 1-h baseline period, the injection of jection were: 4.51 ± 0.06 spikes/s for putative pyramidal neurons in drug or vehicle, and a 3-h post administration period. The total number the mPFC (n = 2075), 9.90 ± 0.37 spikes/s for putative interneurons of activity counts was calculated every 5 min during the entire re- (n = 267) and 5.19 ± 0.07 spikes/s for thalamic relay neurons in the cording session. The time interval between 20 min and 85 min post MD/CM (n = 3053). The firing rate properties for the respective neu- administration was chosen to compare treatment effects (averaged rons were in good agreement with those observed in previous studies 5 min bin). Two-way RM-ANOVA was utilized to assess the differences (Bartho et al., 2004; Furth et al., 2017). The auditory stimulus did not between treatments (SigmaPlot, version 11.0). significantly affect the overall firing rate(Supplementary Fig. 2 A). Further analyses of the effects of NMDA-R antagonists on sensory gating 2.9. Tissue collection will be published elsewhere.

A random subset of the animals used in the locomotor activity test was used for a pharmacokinetics study after a one-week washout 3.2. Effect of ketamine on single unit activity in the mPFC andMD/CM (n = 6–8/treatment). Ketamine was administered at the dose of 10 mg/ kg s.c. and PCP was administered at the dose 5 mg/kg s.c. in awake rats The effect of ketamine (3 and 10 mg/kg s.c.) on the discharge of (n = 3/treatment). Brain concentrations of (R,S)-ketamine, PCP, (R,S)- mPFC and MD/CM neurons was studied in a group of 14 rats. One re- norketamine and (2R, 6R)-hydroxynorketamine (HNK) were measured cording session with ketamine 3 mg/kg is missing from one subject due at 15 and 60 min post administration. Samples were stored at −80 °C to hardware malfunction. Overall, both 3 and 10 mg/kg ketamine sig- until analysis. nificantly increased the firing rate of putative pyramidal neurons inthe mPFC when compared to vehicle treatment in the 60 min after ad- 2.10. Quantitative analysis ministration (AUC, F2,39 = 23.381, p < 0.001). This effect was significant at 10–20 min post administration for the low dose andupto Brain concentrations of (R,S)-ketamine, (R,S)-norketamine, HNK 50 min post administration for the high dose (RM-ANOVA, F[treat- and PCP were determined using high performance liquid chromato- ment]2,244 = 0.24, p = 0.79; F[time]6,244 = 0.17, p = 0.64; F graphy coupled to tandem mass spectrometry (MS/MS). Brain homo- genate samples were prepared by homogenizing the whole brain with Table 1 70% acetonitrile (1:4, v/v) followed by centrifugation and collection of Scheme showing the total number of neurons recorded in each acute study per the supernatant. Brain samples (25 μl) were precipitated with 150 μl treatment. Ketamine (Ket), Phencyclidine (PCP). acetonitrile containing internal standard. After centrifugation, 100 μl Putative pyramidal Putative Thalamic relay supernatant from each sample was transferred to a new plate and mixed neurons interneurons neurons with 100 μl 0.1% formic acid. After a quick centrifugation, the samples Vehicle 278 63 658 were placed in the auto-sampler. Chromatography was performed on a Ket 3 mg/kg s.c. 359 36 628 Waters Acquity UPLC HSS T3 column (2.1 × 50 mm 1.8 μm) using a Ket 10 mg/kg s.c. 324 36 638 mobile phase gradient of 0.1% formic acid in water and acetonitrile pumped through the column with a flow rate of 0.6 ml/min. The re- Vehicle 339 56 364 PCP 2.5 mg/kg 372 44 412 tention times were 1.28, 1.26, 1.08 and 1.38 min for (R,S)-ketamine, s.c. (R,S)-norketamine, HNK and PCP respectively. MS/MS detection was PCP 5 mg/kg s.c. 403 32 353 performed with a Waters XevoTQS instrument in positive-ion

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Fig. 2. Effect of ketamine (Ket; 3and 10 mg/kg s.c.) (A, B, C) and phencyclidine (PCP; 2.5 and 5 mg/kg s.c.) (D, E, F) treatment on the firing rates of putative pyramidal neurons in the mPFC (A1, A2, D1, D2), putative interneurons in the mPFC (B1, B2, E1, E2) and thalamic relay neurons in the MD/CM (C1, C2, F1, F2). Line plots show the percentage change in firing rate relative to the 20-min baseline before injection. Displayed are the mean (lines) and mean ± SEM (shaded areas) per 1-min bin. Bar his- tograms show the corresponding mean percentage change in firing rate during a 10-min period post administration (10–20 min post administration for ketamine and 30–40 min post administration for PCP, indicated by a red line in the corresponding line plot; error bars correspond to SEM). *p < 0.05 vs. vehicle (Veh).

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Fig. 3. Effect of ketamine (Ket; 3 and 10 mg/kg s.c.) treatment on the spectral content of LFPsfrommPFC(A, B, C, D) and MD/CM (E, F, G, H) (n = 14 for vehicle (Veh), n = 13 for Ket3 and n = 14 for Ket10). The spectral content is always normalized to the average power spectral density during the 20-min baseline period before injection, and shown in decibel (dB) in (A1, B1, C1, E1, F1, G1) and as percentage change otherwise. Line plots show the mean (lines) and mean ± SEM (shaded areas) of the spectral content averaged per 1-min bin over different frequency bands for Veh (A2, E2), Ket3 (B2, F2) and Ket10 (C2, G2), as well as the spectral content averaged over the first 20-min period post administration (D, H). Color-coded spectrograms plotted with 0.5 Hz resolution (y-axis) and 5-s time bin (x-axis) represent the average spectral content for Veh (A1, E1), Ket3 (B1, F1) and Ket10 (C1, G1). *p < 0.05 vs. Veh.

[treatment*time]12,244 = 5.62, p < 0.001) (Fig. 2 A1). An analysis of [treatment]2,236 = 0.07, p = 0.93; F[time]6,236 = 1.95, p = 0.07; F the averaged peak effect (10–20 min post administration) furthermore [treatment*time]12,236 = 1.86, p = 0.040) (Fig. 2 F1). Analogously to revealed a dose-dependency of the response (Fig. 2 A2). For putative ketamine the averaged peak effect was only significant for the low dose interneurons in the mPFC, ketamine showed a clear tendency towards (Fig. 2 F2). an increase in the firing rate at both doses during the first 30 minfol- Similar to ketamine, the distribution of changes in firing rate for lowing administration (Fig. 2 B1). However, in contrast to the increase each treatment group and neuronal subpopulation revealed only one of firing rate in putative pyramidal neurons, these effects did notreach type of neuronal response (Supplementary Fig. 3). Therefore, neuronal statistical significance when compared to vehicle treatment during the subpopulations were not further sub-divided into different types of re- period 0–60 min post administration (AUC, F2,39 = 1.94, p = 0.172; sponse. RM-ANOVA, F[treatment]2,203 = 0.30, p = 0.74; F[time]6,203 = 1.59, p = 0.15; F[treatment*time]12,203 = 2.42, p = 0.05) (Fig. 2 B1 and B2). 3.4. Effect of ketamine on LFP activity in the mPFC and MD/CM In the MD/CM, ketamine did not show a significant effect of treatment when considering AUC firing values of thalamic relay neurons The effect of ketamine (3 and 10 mg/kg s.c.) on LFP activity inthe

(AUC, F2,39 = 2.85, p = 0.08). However, at the low dose, ketamine thalamo-cortical circuits was also studied (n = 14 for vehicle and the significantly increased the firing rate of thalamic relay neuronsas high dose, n = 13 for the low dose). Fig. 3 A1, B1 and C1 shows the compared to vehicle treatment during 10–20 min post administration evolution of the spectral content in the mPFC over time for each

(RM-ANOVA, F[treatment]2,244 = 1.17, p = 0.31; F[time]6,244 = 2.71, treatment, and the derived changes in power in the six investigated p = 0.014; F[treatment*time]12,244 = 2.42, p = 0.005) (Fig. 2 C1 and frequency bands are given in Fig. 3 A2, B2 and C2. The average spectra C2). over 0–20 min post administration in Fig. 3 D further illustrate the Furthermore, the distribution of changes in firing rate for each nature of the changes in the different frequency bands. Ketamine was treatment group and neuronal subpopulation was studied to assess found to dose-dependently modulate LFP activity in the mPFC by de- whether different neuronal responses could be detected. Since there creasing beta power and increasing that of the gamma and HFO bands. were no clear indications for multi-modal distributions (Supplementary Ketamine significantly decreased beta power (F[treatment]2,327 = 0.51, Fig. 3 A), the neuronal subpopulations were not further sub-divided p = 0.59; F[time]8,327 = 1.68, p = 0.10; F[treatment*- into different types of responses. We also investigated potential longer- time]16,327 = 2.54, p = 0.001) and increased gamma and HFO power as lasting effects of 3 and 10 mg/kg ketamine and found that neither dose compared to vehicle treatment (gamma: F[treatment]2,327 = 0.37, of the drug significantly modulated the firing rates of mPFC andMD/ p = 0.69; F[time]8,327 = 3.84, p < 0.001; F[treatment*- CM neurons at 24 h post administration (Supplementary Fig. 4). time]16,327 = 6.37, p < 0.001; HFO: F[treatment]2,327 = 0.06, p = 0.94; F[time]8,327 = 1.44, p = 0.18; F[treatment*- time]16,327 = 11.39, p < 0.001). The effect of ketamine 3 mg/kg lasted 3.3. Effect of PCP on single unit activity in the mPFC andMD/CM for up to 20 min post administration for beta and gamma, and up to 30 min post administration for HFO. The effect produced by ketamine The effect of PCP (2.5 and 5 mg/kg s.c.) on the discharge ofmPFC 10 mg/kg on beta was significant at 50–60 min post administration. For and MD/CM neurons was studied in a different group of 14 rats. One gamma and HFO, ketamine 10 mg/kg produced a more prolonged effect recording session each with PCP 2.5 and 5 mg/kg from one subject is that persisted up to 40 min post administration for gamma and until the missing due to hardware malfunction and/or excessive noise. As ob- end of the recording session (85 min post administration) for HFO. served with ketamine, PCP overall significantly increased the firing rate Ketamine also dose-dependently modulated the LFP activity in the of putative pyramidal neurons in the mPFC when compared to vehicle MD/CM by decreasing beta power and increasing the power in the treatment in the 60 min after administration (AUC, F2,39 = 6.87, gamma and HFO bands (Fig. 3 E, F, G and H). Ketamine significantly p = 0.004). For the higher dose, this effect was significant at 10–60 min decreased beta power (F[treatment]2,327 = 0.37, p = 0.68; F post administration, while for the lower dose the increase was only [time]8,327 = 2.09, p = 0.036; F[treatment*time]16,327 = 2.31, significant at 40–50 min post administration (RM-ANOVA, F[treat- p = 0.003) and increased gamma and HFO power as compared to ve- ment]2,243 = 0.18, p = 0.84; F[time]6,243 = 0.61, p = 0.72; F[treat- hicle treatment (gamma: F[treatment]2,327 = 0.36, p = 0.69; F ment*time]12,243 = 3.67, p < 0.001) (Fig. 2 D1). The analysis of the [time]8,327 = 3.33, p = 0.001; F[treatment*time]16,327 = 7.07, averaged peak effect furthermore indicates a dose-dependency ofthe p < 0.001; HFO: F[treatment]2,327 = 0.16, p = 0.84; F response, however, only the high dose was significantly different from [time]8,327 = 2.05, p = 0.04; F[treatment*time]16,327 = 10.48, vehicle (Fig. 2 D2). For putative interneurons, PCP showed, similar to p < 0.001). Similar to the observations for the drug in the mPFC, the ketamine, a tendency towards an increase in the firing rate of putative effects of ketamine 3 mg/kg on beta and gamma lasted for upto20min interneurons at both doses, but the overall effect on AUC did not reach post administration, while its effects at the 10 mg/kg dose persisted for statistical significance (AUC,2,39 F = 3.10, p = 0.07; RM-ANOVA, F up to 40 min post administration. The effect of ketamine 3 mg/kg on [treatment]2,180 = 0.93, p = 0.40; F[time]6,180 = 0.44, p = 0.85; F HFO lasted for up to 30 min post administration, while the 10 mg/kg [treatment*time]12,180 = 1.51, p = 0.12) (Fig. 2 E). dose produced a more prolonged effect that persisted up to 70 min post Both doses of PCP significantly increased the firing rate of thalamic administration. relay neurons as compared to vehicle treatment in the 60 min post Longer-lasting effects of ketamine on the spectral content were also administration (AUC, F2,39 = 3.93, p = 0.034). For the lower dose, the investigated. However, neither dose of the drug significantly modulated effect was significant at 20–40 min post administration, while forthe the spectral content of LFPs in the mPFC and the MD/CM at 24 h post higher dose the increase was only significant at the isolated time period administration (Supplementary Fig. 5). of 10–20 min post administration (RM-ANOVA, F

8 M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745

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Fig. 4. Effect of phencyclidine (PCP; 2.5 and 5 mg/kg s.c.) treatment on the spectral content of LFPsfrommPFC(A, B, C, D) and MD/CM (E, F, G, H) (n = 14/ treatment). The spectral content is always normalized to the average power spectral density during the 20-min baseline period before injection, and shown in decibel (dB) in (A1, B1, C1, E1, F1, G1) and as percentage change otherwise. Line plots show the mean (lines) and mean ± SEM (shaded areas) of the spectral content averaged per 1-min bin over different frequency bands for vehicle (Veh) (A2, E2), PCP2.5 (B2, F2) and PCP5 (C2, G2), as well as the spectral content averaged over 20–40 min post administration (D, H). Color-coded spectrograms plotted with 0.5 Hz resolution (y-axis) and 5 s time bin (x-axis) represent the average spectral content for Veh (A1, E1), PCP2.5 (B1, F1) and PCP5 (C1, G1). *p < 0.05 vs. Veh.

3.5. Effect of PCP on LFP activity in the mPFC and MD/CM of PCP resulted in a significant increase in activity counts at both doses (F[treatment]4,480 = 5.37, p = 0.001; F[time]12,480 = 0.69, p = 0.76; The effects of PCP (2.5 and 5 mg/kg s.c.) on LFP activity in thetwo F[treatment*time]48,480 = 2.30, p < 0.001) (Fig. 5 A). Seen over time, brain regions were also studied (n = 14/treatment). The data obtained the increase in locomotor activity induced by ketamine became first for the mPFC are presented in Fig. 4 in a similar manner as for ketamine significant at ~20 min post administration while the increase mediated (Fig. 3), with the only difference being that the average spectra in Fig. 4 by PCP was first significant at ~65–70 min post administration (Fig. 5 D and 4 H are based on the time period 20–40 min post administration B). due to the different pharmacokinetic properties of PCP. As observed for ketamine, PCP modulated the LFP activity in the mPFC by dose-de- 3.7. Pharmacokinetics of ketamine and PCP pendently increasing gamma and HFO power (gamma: F[treatment]2,356 = 0.09, p = 0.91; F[time]8,356 = 0.70, p = 0.67; F[treat- Brain concentrations of ketamine, its major metabolites, norketa- ment*time]16,356 = 7.84, p < 0.001; HFO: F[treatment]2,356 = 0.003, mine and HNK, and PCP were determined after administration of ke- p = 0.99; F[time]8,356 = 0.20, p = 0.99; F[treatment*- tamine 10 mg/kg s.c. and PCP 5 mg/kg s.c. in awake rats at 15 and time]16,356 = 9.69, p < 0.001). However, unlike ketamine, PCP did 60 min post administration (Fig. 6). Maximal brain concentrations of not significantly change beta power in the mPFC. The effect ofPCP ketamine were noted at 15 min post administration while maximal followed a different temporal profile than ketamine, with its effect brain concentrations of PCP were noted at 60 min post administration. being sustained for a longer period of time. This is consistent with the In contrast to ketamine, the brain concentrations of norketamine and different pharmacokinetics of the two compounds (Gastambide et al., HNK were higher at 60 min than at 15 min post administration. 2013). Both the low and high doses of PCP increased gamma and HFO as compared to vehicle until the end of the recording session (85 min 4. Discussion post administration). Analogously to its effects in the mPFC, PCP modulated the LFPac- NMDA-R antagonists -particularly, ketamine- produce a rapid and tivity in the MD/CM in a dose-dependent manner, mediating significant persistent antidepressant response as well as acute psychotomimetic increases on gamma and HFO power (gamma: F[treat- effects. The development of novel and improved antidepressant drugs ment]2,356 = 0.13, p = 0.87; F[time]8,356 = 1.22, p = 0.28; F[treat- urgently requires an increased mechanistic understanding of these ac- ment*time]16,356 = 6.92, p < 0.001; HFO: F[treatment]2,356 = 0.05, tions. In the present study, we thus investigated the modulatory effects p = 0.95; F[time]8,356 = 0.42, p = 0.91; F[treatment*- of PCP and ketamine on neuronal discharge and LFP activity in time]16,356 = 8.78, p < 0.001). These effects lasted until the end of the recording period (85 min post administration) at both doses of PCP. Both doses also significantly decreased beta power in the MD/CM as compared to vehicle (F[treatment]2,356 = 0.02, p = 0.98; F [time]8,356 = 1.44, p = 0.18; F[treatment*time]16,356 = 2.13, p = 0.007). Moreover, PCP decreased delta power at the high dose (5 mg/kg) but not at the low dose (2.5 mg/kg) when compared to vehicle (F[treatment]2,356 = 0.02, p = 0.97; F[time]8,356 = 3.19, p = 0.001; F[treatment*time]16,356 = 2.23, p = 0.004). In the PCP treatment group of animals, the 20-min baseline period with the auditory stimulus was preceded by an additional 20-min baseline period without the stimulus, thereby allowing to assess its possible impact on LFP activity. The auditory stimulus was not found to significantly affect the overall band power with the exception ofthe delta band, which was significantly increased at the time period of 10–20 min after the start of the auditory stimulus as compared to the time period prior the start of the auditory stimulus (delta in the mPFC:

F3,129 = 5.51, p < 0.001; delta in the MD/CM: F3,129 = 3.12, p = 0.026) (Supplementary Fig. 1 B and C).

3.6. Effects of ketamine and PCP on locomotor activity

The effects of ketamine (3 and 10 mg/kg s.c.) and PCP (2.5and 5 mg/kg s.c.) on locomotion were assessed in groups of 9 rats per treatment. Administration of ketamine resulted in a dose-dependent increase in total activity counts. Ketamine 10 mg/kg significantly in- Fig. 5. Effect of ketamine (Ket; 3 and 10 mg/kg s.c.) and phencyclidine (PCP; creased activity counts as compared to vehicle, while animals ad- 2.5 and 5 mg/kg s.c) on locomotor activity (n = 9/treatment). (A) Bar histo- ministered with the low dose (3 mg/kg) did not exhibit significantly gram representing the mean total activity count of the period between 20 and different locomotor activity than vehicle-treated animals (F[treat- 85 min post administration. (B) Line plots representing the time courses for changes in locomotor activity with a 5-min time bin. Data are presented as ment]4,480 = 5.37, p = 0.001; F[time]12,480 = 0.69, p = 0.76; F mean activity counts ± SEM. *p < 0.05 vs. vehicle (Veh). [treatment*time]48,480 = 2.30, p < 0.001) (Fig. 5 A1). Administration

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GABAergic projection neurons such as RtN neurons. The former mechanism would lead to a disinhibition of pyramidal neurons and the latter to an overall increase of the activity in the cortex via disinhibition of MD/CM thalamo-cortical neurons (Artigas et al., 2018b; Homayoun and Moghaddam, 2007; Santana et al., 2011; Troyano-Rodriguez et al., 2014; Zanos et al., 2018). However, according to our findings neither PCP nor ketamine reduced local inhibitory input in the mPFC, since we rather observed a trend towards increased firing rates in putative fast-spiking interneurons for both drugs. Moreover, our findings neither provide strong support for the hypothesis that the dose-dependent increase in mPFC pyramidal cell firing is entirely driven by disinhibition of thalamic MD/ CM neurons, as these neurons did not show a dose-dependent increase in firing – in fact, for both ketamine and PCP, only the low dose showed a significant increase in firing at peak effect. Together, this suggests that the increased pyramidal activity may instead be caused by disinhibition of long-range GABAergic input or by increased excitatory input from other subcortical and cortical regions innervating the mPFC, such as the Fig. 6. Pharmacokinetic profile of ketamine (Ket; 10 mg/kg s.c.) and phency- hippocampus and the amygdala. clidine (PCP; 5 mg/kg s.c) in awake rats (n = 3/treatment). Bar plots show the NMDA-R antagonists have previously been found to modulate LFP levels of (R,S)-ketamine (Ket), (R,S)-norketamine (norKet), (2R, 6R)-hydro- activity in multiple frequency bands in anesthetized rats (Amat-Foraster xynorketamine (HNK) and PCP. Brain concentrations (ng/g) are presented as mean ± SD. et al., 2018; Llado-Pelfort et al., 2016), freely-moving rats (Hakami et al., 2009; Hiyoshi et al., 2014; Hunt and Kasicki, 2013), primates (Ma et al., 2018) and humans (Rivolta et al., 2015; Sanacora et al., 2014; thalamo-cortical circuits. Shaw et al., 2015). In the delta band, decreases in power induced by The effects of PCP and ketamine on neuronal discharge andLFP PCP and several other psychotomimetic drugs in anesthetized rats have activity during peak effect are summarized in Table 2. We found that been shown to be reversed by antipsychotic treatment (Celada et al., both drugs dose-dependently increased the discharge of putative mPFC 2013; Llado-Pelfort et al., 2016). Here, delta power was only decreased pyramidal neurons. Remarkably, this effect did not coincide with a by PCP in the MD/CM, suggesting that this effect is not a useful bio- reduced discharge from local putative fast-spiking interneurons or a marker of psychotomimetic activity in awake rats. dose-dependent increase in the firing of thalamic MD/CM neurons, as Decreases in beta power induced by ketamine have also been found would be expected from the disinhibition hypothesis. Furthermore, in humans (de la Salle et al., 2016) and in non-human primates where it both ketamine and PCP affected LFP activity by increasing the band was shown to correlate with working memory deficits (Ma et al., 2018). power at higher frequencies (gamma and HFO) and decreasing the band Other psychotomimetic agents with different modes of action have also power at lower frequencies (beta), except for PCP in mPFC. The effects been reported to decrease beta oscillations in rodents (Hiyoshi et al., on both firing and LFP activity followed the pharmacokinetic profile of 2014). Importantly, inhibition of mPFC-MD projections has been shown the drugs. In the case of ketamine, these effects were also observed ata to impair working memory as measured by beta frequency synchroni- dose that did not induce statistically significant hyperlocomotor activity zation (Parnaudeau et al. 2013, 2015, 2017). Hence, together with the in the rats. decreases in beta power observed in the present study, decreases in beta For PCP, the observed increase in discharge of putative mPFC pyr- power may represent a potential translational biomarker for cognitive amidal neurons and thalamic relay neurons is consistent with the impairments. overall effect of the drug in chloral hydrate-anesthetized rats Increases in gamma power induced by NMDA-R antagonists have (Kargieman et al., 2007; Santana et al., 2011). In contrast, for ketamine been attributed to the psychotomimetic actions of these drugs (Hakami two recent studies in chloral hydrate-anesthetized rats reported a re- et al., 2009; Hong et al., 2010). However, these increases could not be duced discharge in these neuronal populations (Amat-Foraster et al., reversed by antipsychotic drugs such as clozapine or haloperidol (Jones 2018; Shen et al., 2018). These observations clearly suggest a differ- et al., 2012). Moreover, while other psychotomimetic drugs such as DOI ential interaction of anesthesia with the neurotransmitter systems tar- and methamphetamine did not amplify gamma oscillations (Hiyoshi geted by PCP and ketamine and question the value of anesthetized et al., 2014; Wood et al., 2012), lanicemine and HNK produced rodents for exploration of ketamine's modulation of these brain circuits. The observed acute effects of ketamine and PCP on the discharge of excitatory mPFC pyramidal neurons are in agreement with previous Table 2 studies in awake rodents, in which several different NMDA-R antago- Effects of ketamine (Ket) (3 and 10 mg/kg s.c.) and phencyclidine (PCP)(2.5 nists have been found to increase firing of pyramidal neurons (Furth and 5 mg/kg s.c.) on firing rate and oscillatory activity in the mPFC andMD/ CM. Percentage change relative to baseline during the peak effect: 10-30%↓, no et al., 2017; Homayoun and Moghaddam, 2007; Jodo et al., 2003; significant change ↔, 10–25%↑, 25–50% ↑↑, 50–100% ↑↑↑. Molina et al., 2014; Suzuki et al., 2002). These effects are thus compatible with the glutamate hypothesis for fast-acting antidepressants, Ket 3 Ket 10 PCP 2.5 PCP 5 which suggests that a transient increase in glutamate release in the PFC Singe unit Pyramidal neurons ↑ ↑↑ ↔ ↑↑↑ is a key proximal event that leads to the activation of AMPA-Rs, thereby Interneurons ↔ ↔ ↔ ↔ evoking a cascade of intracellular signaling pathways involved in sy- Thalamic relay neurons ↑ ↔ ↑↑ ↔ naptogenesis, ultimately leading to the antidepressant effects (Duman LFP mPFC Delta ↔ ↔ ↔ ↔ and Aghajanian, 2012; Moghaddam et al., 1997; Monteggia and Zarate, Beta ↓ ↓ ↔ ↔ Gamma ↑ ↑↑ ↑ ↑↑ 2015). HFO ↑↑ ↑↑↑ ↑↑ ↑↑↑ Direct blockade of NMDA-Rs on pyramidal neurons in the mPFC LFP MD/CM Delta ↔ ↔ ↔ ↓ would be expected to result in a reduction of their firing rate (Rotaru Beta ↓ ↓ ↓ ↓ et al., 2011). Hence, it has been hypothesized that NMDA-R antagonists Gamma ↑ ↑↑ ↔ ↑↑ HFO ↑↑ ↑↑↑ ↑↑ ↑↑↑ preferentially block NMDA-Rs on cortical GABAergic interneurons or

11 M. Amat-Foraster, et al. Neuropharmacology 158 (2019) 107745 antidepressant-like effects and increased gamma oscillations similar to antidepressant effects of ketamine. Even though further studies are the effects of ketamine but at doses without psychotomimetic effects necessary to understand to which extent the effects presented in this (Sanacora et al., 2014; Zanos et al., 2016). Therefore, more studies are study contribute to the antidepressant actions and psychotomimetic needed to correlate changes in gamma power with any of the clinical effects of both drugs, the presented acute effects may be highly relevant features of NMDA-R antagonists. Nevertheless, the marked gamma ef- as markers of target engagement and aid in the understanding of the fects could be used as a measure of target engagement of NMDA-R long-lasting therapeutic benefits of ketamine as well as its short-term antagonists. psychotomimetic effects. Collectively, the findings presented herein The most pronounced effects observed for ketamine and PCP in this expand the knowledge of the neuronal populations, brain areas and study were the robust and dose-dependent increases in HFO power in circuits involved in the actions of non-competitive NMDA-R antagonists both mPFC and MD/CM, as also observed in previous studies in awake and may help to elucidate the mechanisms of action of ketamine and to rodents (Flores et al., 2015; Hansen et al., 2019; Hiyoshi et al., 2014; Hunt identify valid biomarkers of its antidepressant action. et al., 2006; Nicolas et al., 2011). In agreement with the observation that an awake state is required for NMDA-R antagonists to enhance HFO Financial disclosures power (Hunt et al., 2009), this effect could not be observed in anesthetized rats (Supplementary Fig. 6). The translational value of HFO between The work leading to these results has received funding from rodents and humans is currently unknown, since only few human EEG Lundbeck A/S, the Innovation Fund Denmark and SAF2015-68346-P studies have reported oscillatory activity above 100 Hz (Nottage et al., (Spanish Ministry of Economy and Competitiveness, co-financed by 2015). In any case, based on its marked preclinical effects, the power European Regional Development Fund (ERDF)) and PI12/00156 and increases in HFO may potentially be a useful translational biomarker for PI16/00287 Instituto de Salud Carlos III, co-financed by European target engagement in the case of NMDA-R modulators. Regional Development Fund (ERDF)). Support from the Centro de Whether any certain frequency band can be used as a reliable bio- Investigación Biomédica en Red de Salud Mental (CIBERSAM) and marker for the antidepressant properties of NMDA-R modulating com- Generalitat de Catalunya Grup de Recerca Consolidat, 2017SGR717 is pounds is still unresolved, and firm conclusions will depend on a better also acknowledged. Ulrike Richter is funded by Sweden's Innovation translation between rodent and human studies by considering beha- Agency VINNOVA. vioral states and widening the frequency range studied in humans to include HFO. Conflicts of interest The low dose of ketamine used in this study did not produce significant locomotor activity changes yet modulated neuronal activity MAF, KFH and NP are Lundbeck A/S employees. AAJ and UR re- significantly. This indicates that measures of neuronal activity aremore ported no potential conflicts of interest. FA has also received con- sensitive than behavioral measures, and that they are potentially in- sultation fees from Lundbeck A/S and is scientific advisor to Neurolixis. dependent. Our PK/PD study showed that the acute effects induced by FA and PC are PI and co-PI of a research contract with Lundbeck A/S. ketamine and PCP on neuronal activity and locomotor activity correlate well with the pharmacokinetic profiles of these compounds in the brain. Acknowledgements Interestingly, these effects do not follow the profile of ketamine's major metabolites norketamine and HNK in the brain, as changes in neuronal A partial account of the present study was presented at the Society activity would be expected to peak at later timepoints if driven by the for Neuroscience 2017 Annual Meeting. We would like to thank Claus metabolites. On the other hand, PCP increased neuronal firing and the Agerskov for his skillful assistance in data processing. We would also power of gamma and HFO at a brain concentration much smaller than like to thank Christopher Jones for his assistance with the quantitative that of ketamine and metabolites, which suggests a more efficient in bioanalysis and Lars Arvastson for his help on statistics. vivo blockade of NMDA-R by PCP, at least of those receptors controlling thalamo-cortical activity. Appendix A. Supplementary data A main limitation of the present study is the use of naïve animals. Future studies employing animal models for depression would allow for Supplementary data to this article can be found online at https:// an even better assessment of the potential of certain reported effects as doi.org/10.1016/j.neuropharm.2019.107745. biomarkers of the antidepressant actions of the drugs. Another limitation is the use of an auditory stimulation during the recordings. We References thoroughly assessed possible effects of the stimulation and found that it did not affect the overall absolute firing rate of the recorded neurons Amat-Foraster, M., Jensen, A.A., Plath, N., Herrik, K.F., Celada, P., Artigas, F., 2018. during baseline and only affected LFP activity in the delta band. Thus, Temporally dissociable effects of ketamine on neuronal discharge and gamma oscillations in rat thalamo-cortical networks. Neuropharmacology 137, 13–23. the effects of NMDA-R antagonists on lower frequency bands should be Artigas, F., Bortolozzi, A., Celada, P., 2018a. Can we increase speed and efficacy of an- interpreted with caution. tidepressant treatments? Part I: general aspects and monoamine-based strategies. Eur. In summary, the present study shows that ketamine and PCP simi- Neuropsychopharmacol. 28, 445–456. Artigas, F., Celada, P., Bortolozzi, A., 2018b. Can we increase the speed and efficacy of larly modulate thalamo-cortical circuits in awake freely-moving rats by antidepressant treatments? Part II. Glutamatergic and RNA interference strategies. increasing the firing of both excitatory mPFC pyramidal neurons and Eur. Neuropsychopharmacol. 28, 457–482. MD/CM thalamic relay neurons together with an increase in band Autry, A.E., Adachi, M., Nosyreva, E., Na, E.S., Los, M.F., Cheng, P.F., Kavalali, E.T., power at higher frequencies (gamma and HFO) and a decrease in beta Monteggia, L.M., 2011. NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475, 91–95. power at comparable doses. The increased pyramidal neuron activity in Bartho, P., Hirase, H., Monconduit, L., Zugaro, M., Harris, K.D., Buzsaki, G., 2004. mPFC is not likely to be mediated by local disinhibition since no at- Characterization of neocortical principal cells and interneurons by network interac- tenuation of the discharge of GABAergic neurons was observed in PFC. tions and extracellular features. J. Neurophysiol. 92, 600–608. Browne, C.A., Lucki, I., 2013. Antidepressant effects of ketamine: mechanisms underlying Since the emergence of psychotomimetic effects and the increase in fast-acting novel antidepressants. Front. Pharmacol. 4, 161. thalamo-cortical activity by the drugs exhibit the same temporal pro- Celada, P., Llado-Pelfort, L., Santana, N., Kargieman, L., Troyano-Rodriguez, E., Riga, file, the short-lasting effects induced by ketamine and PCP on thalamo- M.S., Artigas, F., 2013. Disruption of thalamocortical activity in schizophrenia models: relevance to antipsychotic drug action. Int. J. 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