Modulation of thalamo-cortical activity by the NMDA receptor antagonists ketamine and phencyclidine in the awake freely-moving rat

Neuropharmacology 158 (2019) 107745

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Modulation of thalamo-cortical activity by the NMDA receptor antagonists ketamine and phencyclidine in the awake freely-moving rat

T

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 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 b

HIGHLIGHTS

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

ABSTRACT

Keywords: NMDA receptor antagonists Thalamo-cortical networks Single unit recordings Neuronal oscillations Ketamine Phencyclidine

Non-competitive N-methyl-D-aspartate receptor antagonists mimic schizophrenia symptoms and produce immediate and persistent antidepressant effects. We investigated the effects of ketamine and phencyclidine (PCP) on thalamocortical network activity in awake, freely-moving male Wistar rats to gain new insight into the neuronal populations and brain circuits involved in the effects of NMDA-R antagonists. Single unit and local field potential (LFP) recordings were conducted in mediodorsal/centromedial thalamus and in medial prefrontal cortex (mPFC) using microelectrode arrays. Ketamine and PCP moderately increased the discharge rates of principal neurons in both areas while not 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 earlier than 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 The non-competitive N-methyl-D-aspartate receptor (NMDA-R) antagonists, phencyclidine (PCP) and ketamine are extensively used as

pharmacological tools in models for schizophrenia due to their ability to evoke effects reminiscent of the positive and negative symptoms as well as cognitive deficits in the disorder (Javitt and Zukin, 1991; 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/).

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Abbreviations RM-ANOVA repeated measures analysis of variance AMPA-R α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors AP anterior posterior AUC area under the curve CM centromedial nucleus of the thalamus CMR common median referencing dB decibel FFT fast Fourier transformation GABA gamma-Aminobutyric acid HFO high-frequency oscillations HNK (2R,6R)-hydroxynorketamine i.v intravenous

Ket L LFP MD (m)PFC NMDA-R norKet PCP RtN s.c. SD SEM V Veh

acid

(R,S)-ketamine lateral local field potential mediodorsal nucleus of the thalamus (medial) prefrontal cortex N-metyl-D-aspartate receptors (R,S)-norketamine phencyclidine reticular nucleus of the thalamus subcutaneously standard deviations standard error of means ventral vehicle

concordance with these observations, another recent study has also reported that ketamine (1.25–20 mg/kg i.v.) mediates a dose-dependent attenuation of the discharge of deep layer pyramidal neurons in the mPFC of chloral hydrate-anesthetized rats (Shen et al., 2018). The findings in the two latter ketamine studies in anesthetized rats differ from a large body of evidence from e.g. neuroimaging studies in rodent, primate and human brains supporting the notion that ketamine analogously to PCP mediates activation of cortical and thalamic areas (Chin et al., 2011; Downey et al., 2016; Maltbie et al., 2016; Vollenweider and Kometer, 2010). In the present study, we have thus conducted electrophysiological studies in awake freely-moving rats to examine the effects mediated by ketamine and PCP on neuronal discharge and neuronal oscillations in thalamo-cortical circuits (mPFC and MD/CM).

On the other hand, sub-anesthetic doses of ketamine have also been reported to mediate robust antidepressant effects in treatment-resistant unipolar and bipolar depressed patients (Monteggia and Zarate, 2015; Newport et al., 2015; Zarate et al., 2006) and antidepressant-like effects in rodents (Artigas et al., 2018a; Browne and Lucki, 2013; Mion and Villevieille, 2013). Hence, the putative neurochemical, cellular and/or circuit elements and mechanisms underlying these effects of ketamine and other NMDA-R antagonists have become extensively explored in recent years (Gould et al., 2017; Miller et al., 2016). One of the current hypotheses is that the non-competitive NMDA-R antagonists preferentially target NMDA-Rs in GABAergic neurons (Tsai and Coyle, 2002; Zanos and Gould, 2018), thereby enhancing excitatory neurotransmission in cortical areas (Furth et al., 2017; Homayoun and Moghaddam, 2007; Jackson et al., 2004; Jodo et al., 2003; Kargieman 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 reticular thalamic nucleus (RtN) and the subsequent disinhibition of thalamo-cortical afferents, such as thalamic relay neurons in the mediodorsal/centromedial (MD/CM) thalamic nuclei (Celada et al., 2013; Santana et al., 2011; Troyano-Rodriguez et al., 2014). The action of ketamine appears to also involve activation of α-amino-3-hydroxy-5methyl-4-isoxazolepropionate receptors (AMPA-Rs) (Autry et al., 2011; Fukumoto et al., 2016; Koike and Chaki, 2014; Li et al., 2010; Maeng et al., 2008; Zanos et al., 2016), an effect that subsequently may evoke biochemical/structural changes and increase mTOR signaling and synaptogenesis, thereby reshaping brain circuits involved in mood disorders (Duman and Aghajanian, 2012; Li et al., 2010; Monteggia and Zarate, 2015; Popp et al., 2016). These subsequent events have been proposed to underlie the long-lasting effects of ketamine, which outlasts the actual exposure of the drug in the central nervous system (Mion and Villevieille, 2013). Interestingly, recent contradictory observations from experiments with subanesthetic doses of PCP and ketamine in anesthetized rats challenge the “disinhibition hypothesis” as a general explanation for the effects of NMDA-R antagonists. In accordance with the hypothesis, intravenous administration of sub-anesthetic doses of PCP to chloral hydrate-anesthetized rats was found to markedly inhibit the activity of RtN GABAergic neurons, as well as to mainly activate the discharge of layer V pyramidal neurons in the medial prefrontal cortex (mPFC) and thalamo-cortical relay neurons in the MD/CM, which are reciprocally connected with mPFC (Kargieman et al., 2007; Santana et al., 2011; Troyano-Rodriguez et al., 2014). However, while intravenous administration of sub-anesthetic doses of ketamine (1–5 mg/kg i.v.) under the same experimental conditions had a similar effect on RtN GABAergic neurons, this effect did not translate into a disinhibition of MD/CM but rather a marked inhibition of the neuronal discharge of cortico-thalamic layer VI pyramidal neurons in the mPFC as well as of thalamocortical relay neurons in MD/CM (Amat-Foraster et al., 2018). In

2. Methods and materials 2.1. Animals Male albino Wistar rats (n = 28, Charles River; 250–350 g) were housed, two per cage, under standard temperature (22 ± 1.5 °C) and humidity (55–65%) in controlled laboratory conditions. Food and water was available ad libitum in a 12 h light/dark cycle (lights on at 7:00 h). After surgery, rats were single-housed and the light cycle reversed (lights off at 7:00 h). All the experimental procedures were performed in accordance with the Danish legislation regulating animal experiments; Law and Order on Animal experiments; Act No. 474 of 15/05/ 2014 and Order No. 12 of 07/01/2016, and with the specific license for this experiment issued by the National Authority. 2.2. Drugs (R,S)-Ketamine (Ketolar 50 mg/ml, Pfizer) and PCP (Lundbeck A/S, Valby, Denmark) were dissolved in 0.9% saline (2 ml/kg). Ketamine was administered at 3 and 10 mg/kg subcutaneously (s.c.), and PCP was administered at 2.5 and 5 mg/kg s.c. Doses of ketamine were chosen based on the pharmacokinetic profile of ketamine to achieve brain exposures in rats comparable to the clinical concentrations (Shaffer et al., 2014). Doses of PCP were selected based on those used in previous studies where the drug influenced cortical oscillations and based on its pharmacokinetic profile (Gastambide et al., 2013; Hiyoshi et al., 2014). Control animals received 0.9% saline s.c. 2.3. Surgical procedure Rats were anesthetized with a 1:1 Hypnorm/Dormicum mixture (0.25 mg/kg fentanyl, 12.5 mg/kg fluanisone (both Lundbeck A/S, Valby, Denmark) and 6.25 mg/kg midazolam (Roche A/S, Hvidovre, 2

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Denmark)) by s.c. injection. Under anesthesia, the fur covering the skull was shaved, iodine was applied to the skin and an eye gel was applied to the corneas. Marcain (2.5 mg/ml bupivacaine, AstraZeneca, Albertslund, Denmark) was used as local anesthesia at the incision site. Rats were placed in a stereotaxic frame (David Kopf, Tujunga, CA) and maintained at 37 °C using a heating pad. Two holes were drilled into the skull to accommodate two fixed 16-channel tungsten-microwire-arrays that came together with an additional silver ground wire (Innovative Neurophysiology, Durham, NC). The microwires (35 μm in diameter) were arranged in a 4 × 4 configuration with a spacing of 150 μm in between microelectrodes. Each array was implanted in the MD/CM (coordinates relative to bregma for center of array (Paxinos and Watson, 2007): AP (anterior posterior): 2.6 mm, L (lateral): 0.7 mm, V (ventral): 5.4 mm) and the mPFC (AP: +3.4 mm, L: 0.7 mm, V: 4.7 mm) and secured to the skull with two screws and dental cement (RelyX™ Unicem cement (3M, Copenhagen, Denmark) and GC Fuji PLUS™

cement (GC America Inc., IL)). Ground wires from each array were wrapped around an additional 2-mm screw placed over the cerebellum. After surgery, rats received 5 mg/kg carprofen analgesic (Rimadyl®, 50 mg/ml carprofen, Pfizer, NY) and 150 mg/kg amoxicillin antibiotic (Noromox Prolongatum, ScanVet, Fredensborg, Denmark) daily for 5 days. Rats were allowed 7 days of recovery before the first recording session and were habituated to the recording box during this period. 2.4. Study and experimental design Rats were divided into two groups (n = 14/group) for ketamine and PCP. All rats received three different treatments (vehicle, low dose and high dose of the drug) according to a Latin square design with a 7-day washout between injections. All recordings were carried out during the dark phase (7–19 h) and during an auditory sensory gating stimulus 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 firing rates. (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

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stimulus interval of 500 ms and an inter-trial interval of 4.5 s. The auditory stimulation paradigm was added to the study design to allow investigation of the effects of drugs on sensory gating processing. These data set will be addressed in a separate publication. On the day of the recording session, rats were transferred to a cage placed inside a sound attenuating steel box, for a 30-min habituation before the start of the experiment. Following the start of the auditory sensory gating stimulus, each recording consisted of a 20-min baseline period followed by the injection of the drug and 85 min recording post injection. For the PCP study, each recording had an additional 20-min baseline period prior to the start of the auditory sensory gating stimulus. Furthermore, each rat in the ketamine group was recorded for an additional 30-min period with the auditory sensory gating stimulus at 24 h post administration in order to explore longer-lasting effects of ketamine. Animal behavior was surveilled with a video camera during the entire recording session. At the end of the study, rats were anesthetized with chloral hydrate (450 mg/kg, intraperitoneal) and brains were marked passing 100 μA current through the arrays for 10 s using a current stimulator (model A365, World Precision Instruments, Berlin, Germany). Rats were euthanized by cervical dislocation and brains were removed and kept at −80 °C. Histological verification of the electrode placement was conducted at the end of the study.

all units were treated as “new units” for each recording session. For single units recorded in the mPFC, the time from trough to peak and the half-amplitude width were computed for the mean action potential waveform of each unit. These features have been widely used in the literature to classify cortical neurons into putative pyramidal neurons and putative interneurons, and the approach has been validated with optogenetic tagging and monosynaptic interactions (Bartho et al., 2004; Stark et al., 2013; Watson et al., 2016). Here, a Gaussian mixture model with two components was fitted to the distribution of the trough-topeak time by using MATLAB's fitgmdist function, which is based on the Expectation-Maximization algorithm. The model will assign each unit to one of the two components, which correspond to the two putative cell types, by maximizing the posterior probability for the unit to belong to its assigned component. Single units with a posterior probability of < 0.9 to be from either distribution remained unclassified, while single units with a posterior probability of > 0.9 were classified as either putative interneurons or pyramidal neurons (Fig. 1). 2.7. Electrophysiological data analysis Single unit and LFP analysis was carried out in MATLAB (MathWorks, Natick, MA). For each LFP signal, a spectrogram with time and frequency resolution of 5 s and 0.5 Hz, respectively, was obtained employing the Fast Fourier transform (FFT). A spectrogram is a time series of power spectral densities and allows assessing the spectral content of a signal, such as the presence of oscillatory activity in certain frequency bands, over time. Within every spectrogram, each power spectral density was normalized to the average power spectral density during the stable 20-min baseline period immediately prior to injection. In this study, the spectrograms were then analyzed in 6 main frequency bands: delta 0.5–4 Hz, theta 4–8 Hz, alpha 8–12 Hz, beta 12–30 Hz, gamma 30–100 Hz, and high-frequency oscillations (HFO) 130–180 Hz. Prior to the analysis of the spectral content, artefact detection was performed in all channels, and 5-s segments in which data points exceeded ± 6 standard deviations (SD) from the mean were not included in the analysis. Rejection of a 75-s period before any pauses in the recording was also performed. The mean spectral content in the mPFC and the MD/CM in one recording was then obtained as the average over all 16 baseline-normalized spectrograms of the corresponding array. Subsequently, also the overall averages for each treatment were calculated. Due to the baseline-normalization the spectral content is presented either as percentage change or in decibel (dB). Similar to the LFP analysis, the analysis of the single units began with analyzing each single unit separately. For each unit, the firing rate was computed for consecutive 1-min bins and normalized to the average firing rate during the stable 20-min baseline period immediately prior to injection. The normalization step was only conducted for the acute studies but not for the 24 h study since neurons could not be tracked across recordings and thus had to be considered separate units. The mean firing rate in the mPFC and the MD/CM in one recording was then obtained as the average over all single units from the corresponding array and recording. Subsequently, also the overall averages for each treatment were calculated. Due to the baseline-normalization the firing rates are presented as percentage change. Comparison between treatments were then performed separately for the ketamine and the PCP group. For this comparison, a 10-min window was chosen based on the drugs’ pharmacokinetic profiles (Gastambide et al., 2013). Repeated measures analysis of variance (RM-ANOVA) was used to assess differences in firing rate (average 10-min bin, period 0–60 min post administration for the acute studies and 30-min period 24 h post administration for the 24 h study) and in baseline-normalized LFP power (average 10-min bin, period 0–80 min post administration for the acute studies and 30-min period 24 h post administration for the 24 h study). In more detail, MATLABs fitlme function was used to fit each tested dataset with a linear mixed-effect model that had the fixed-effect predictors Subject, Treatment, Time, and the interaction between Treatment and Time. The

2.5. Electrophysiological recordings Rats were connected to a 16-channel connector headstage (Plexon, Dallas, TX) via a commutator (Plexon, Dallas, TX) that did not restrict the rat's movement during the recording. Both single units and local field potentials (LFPs) were recorded simultaneously in the MD/CM and the mPFC. Recordings were paused shortly at the time of the injection and during occasional disconnections of the headstage connector from the array due to the animal's movement. Neuronal activity was recorded by using a preamplifier (Plexon, Dallas, TX), and data were acquired using Plexon's OmniPlex A Neural Data Acquisition System. A screw placed in the skull served as hardware reference, and the common median within each 16-wire array served as a digital reference (common median referencing (CMR), Plexon, Dallas, TX). Spike channels were bandpass filtered between 0.3 and 9 kHz (2-poles Bessel filter), preamplified 1000× and digitalized at 40 kHz. LFP channels were bandpass filtered between 0.5 and 200 Hz (4-pole Butterworth filter), preamplified 1000×, digitalized at 40 kHz and downsampled to 2 kHz. Continuous data files were saved and analyzed offline. 2.6. Spike sorting and cell classification Spikes were automatically sorted using the Kilosort spike sorting software (Pachitariu et al., 2016) followed by a manual curation step with the ‘phy’ graphical user interface (https://github.com/kwikteam/ phy). During the manual curation step, the automatically determined groups of spikes could be reviewed and split, as well as merged. In more detail, the cross-correlogram features, spike waveform similarity and spike drift patterns of each group of spikes detected by a specific template were compared to those from similar templates to determine whether or not they should be merged. Furthermore, autocorrelogram analysis was performed to obtain an indication of multi-unit activity and to determine whether a group of spikes should be split. Importantly, a group of spikes was only considered as single unit activity if it was well-isolated in feature space, showed no indication of multi-unit activity (lack of an absolute refractory period), had no atypical waveform and was stable throughout the recording session. As a final step following manual curation, groups of spikes with > 1% of inter-spike intervals shorter than 1.1 ms were excluded (this step was performed in MATLAB (MathWorks, Natick, MA)). With this procedure, an average of two putative single units were isolated from each electrode (Supplementary Fig. 1). Although some single units may have been occasionally recorded repeatedly over the weeks of recording sessions, 4

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overall interaction between Treatment and Time for the fitted model was evaluated with MATLABs anova method. The p-values for the individual interaction coefficients between Treatment and Time, which were returned by the fitlme function, were Bonferroni-corrected before interpretation. For the firing rate, the area under the curve (AUC) for the period 0–60 min post administration was also calculated for each treatment, and differences between treatments were assessed using oneway ANOVA in SigmaPlot (version 11.0). Evoked activity in response to the auditory stimuli was not studied in detail here. A possible impact of the auditory sensory gating stimulus 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 animals of the PCP study (n = 14). One-way RM ANOVA was used to assess changes during the period -40-0 min prior administration (average 10-min bin) (SigmaPlot, version 11.0). Data are presented as mean ± standard error of the mean (SEM) and a p-value of < 0.05 was considered significant. When multiple comparisons were conducted using the same data, the p-value was adjusted using a Bonferroni correction.

electrospray ionization mode. Parent and daughter ion mass to charge ratios (m/z) of 238 > 220, 224 > 125, 240 > 125 and 244 > 86 were used for quantification of (R,S)-ketamine, (R,S)-norketamine, HNK and PCP, respectively. Nitrogen was used for the nebulizer and collision gases. The peak area correlated linearly with the concentration in the range of 5–5000 ng/g in brain for all analytes. Data acquisition and analysis was performed using Masslynx software, version 4.1 (Waters Denmark, Taastrup, Denmark). 3. Results 3.1. Classification and basal firing rates of mPFC and MD/CM neurons Neurons recorded in the mPFC were separated into putative pyramidal neurons and putative GABAergic interneurons based on the criteria outlined in Section 2.6 (Fig. 1 A). In the mPFC, a total of 2075 single units were identified as putative pyramidal neurons (78%), 322 single units were unclassified (12%) and 267 single units were identified as putative interneurons (10%), which is consistent with previous reports on the percentage of GABAergic interneurons in the cortex (DeFelipe et al., 2013). A representative example of the average waveform of a putative pyramidal neuron and a putative interneuron is shown in Fig. 1B. The respective firing rates of putative pyramidal neurons and putative interneurons in the mPFC were not used as classification criteria for these neurons as there was no clear clustering (Fig. 1 C). In the MD/CM, which is mainly composed of thalamic relay neurons, 3053 single units were identified (see total number of neurons recorded per treatment in Table 1). The mean firing rates of the recorded neurons during the 20-min baseline immediately prior to injection were: 4.51 ± 0.06 spikes/s for putative pyramidal neurons in the mPFC (n = 2075), 9.90 ± 0.37 spikes/s for putative interneurons (n = 267) and 5.19 ± 0.07 spikes/s for thalamic relay neurons in the MD/CM (n = 3053). The firing rate properties for the respective neurons were in good agreement with those observed in previous studies (Bartho et al., 2004; Furth et al., 2017). The auditory stimulus did not significantly affect the overall firing rate (Supplementary Fig. 2 A). Further analyses of the effects of NMDA-R antagonists on sensory gating will be published elsewhere.

2.8. Locomotor activity test Levels of basal activity after administration of ketamine (3 and 10 mg/kg), PCP (2.5 and 5 mg/kg) and vehicle (0.9% saline) s.c. were assessed in a separate group of animals (n = 9/treatment). The test was performed during the light phase in macrolon type III (high model) cages equipped with two rings of infrared light beams to count motility and rearings. The rats were habituated to the cages, in which they were placed individually, the day before the test. For the test, the rats were habituated for 1 h, followed by a 1-h baseline period, the injection of drug or vehicle, and a 3-h post administration period. The total number of activity counts was calculated every 5 min during the entire recording session. The time interval between 20 min and 85 min post administration was chosen to compare treatment effects (averaged 5 min bin). Two-way RM-ANOVA was utilized to assess the differences between treatments (SigmaPlot, version 11.0). 2.9. Tissue collection A random subset of the animals used in the locomotor activity test was used for a pharmacokinetics study after a one-week washout (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 (n = 3/treatment). Brain concentrations of (R,S)-ketamine, PCP, (R,S)norketamine and (2R, 6R)-hydroxynorketamine (HNK) were measured at 15 and 60 min post administration. Samples were stored at −80 °C until analysis.

3.2. Effect of ketamine on single unit activity in the mPFC and MD/CM The effect of ketamine (3 and 10 mg/kg s.c.) on the discharge of mPFC and MD/CM neurons was studied in a group of 14 rats. One recording session with ketamine 3 mg/kg is missing from one subject due to hardware malfunction. Overall, both 3 and 10 mg/kg ketamine significantly increased the firing rate of putative pyramidal neurons in the mPFC when compared to vehicle treatment in the 60 min after administration (AUC, F2,39 = 23.381, p < 0.001). This effect was significant at 10–20 min post administration for the low dose and up to 50 min post administration for the high dose (RM-ANOVA, F[treatment]2,244 = 0.24, p = 0.79; F[time]6,244 = 0.17, p = 0.64; F

2.10. Quantitative analysis Brain concentrations of (R,S)-ketamine, (R,S)-norketamine, HNK and PCP were determined using high performance liquid chromatography coupled to tandem mass spectrometry (MS/MS). Brain homogenate samples were prepared by homogenizing the whole brain with 70% acetonitrile (1:4, v/v) followed by centrifugation and collection of the supernatant. Brain samples (25 μl) were precipitated with 150 μl acetonitrile containing internal standard. After centrifugation, 100 μl supernatant from each sample was transferred to a new plate and mixed with 100 μl 0.1% formic acid. After a quick centrifugation, the samples were placed in the auto-sampler. Chromatography was performed on a Waters Acquity UPLC HSS T3 column (2.1 × 50 mm 1.8 μm) using a 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 retention times were 1.28, 1.26, 1.08 and 1.38 min for (R,S)-ketamine, (R,S)-norketamine, HNK and PCP respectively. MS/MS detection was performed with a Waters XevoTQS instrument in positive-ion

Table 1 Scheme showing the total number of neurons recorded in each acute study per treatment. Ketamine (Ket), Phencyclidine (PCP).

5

Putative pyramidal neurons

Putative interneurons

Thalamic relay neurons

Vehicle Ket 3 mg/kg s.c. Ket 10 mg/kg s.c.

278 359 324

63 36 36

658 628 638

Vehicle PCP 2.5 mg/kg s.c. PCP 5 mg/kg s.c.

339 372

56 44

364 412

403

32

353

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Fig. 2. Effect of ketamine (Ket; 3 and 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 histograms 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 LFPs from mPFC (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 the averaged peak effect (10–20 min post administration) furthermore revealed a dose-dependency of the response (Fig. 2 A2). For putative interneurons in the mPFC, ketamine showed a clear tendency towards an increase in the firing rate at both doses during the first 30 min following administration (Fig. 2 B1). However, in contrast to the increase of firing rate in putative pyramidal neurons, these effects did not reach statistical significance when compared to vehicle treatment during the period 0–60 min post administration (AUC, F2,39 = 1.94, p = 0.172; 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). In the MD/CM, ketamine did not show a significant effect of treatment when considering AUC firing values of thalamic relay neurons (AUC, F2,39 = 2.85, p = 0.08). However, at the low dose, ketamine significantly increased the firing rate of thalamic relay neurons as compared to vehicle treatment during 10–20 min post administration (RM-ANOVA, F[treatment]2,244 = 1.17, p = 0.31; F[time]6,244 = 2.71, p = 0.014; F[treatment*time]12,244 = 2.42, p = 0.005) (Fig. 2 C1 and C2). Furthermore, the distribution of changes in firing rate for each treatment group and neuronal subpopulation was studied to assess whether different neuronal responses could be detected. Since there were no clear indications for multi-modal distributions (Supplementary Fig. 3 A), the neuronal subpopulations were not further sub-divided into different types of responses. We also investigated potential longerlasting effects of 3 and 10 mg/kg ketamine and found that neither dose of the drug significantly modulated the firing rates of mPFC and MD/ CM neurons at 24 h post administration (Supplementary Fig. 4).

[treatment]2,236 = 0.07, p = 0.93; F[time]6,236 = 1.95, p = 0.07; F [treatment*time]12,236 = 1.86, p = 0.040) (Fig. 2 F1). Analogously to ketamine the averaged peak effect was only significant for the low dose (Fig. 2 F2). Similar to ketamine, the distribution of changes in firing rate for each treatment group and neuronal subpopulation revealed only one type of neuronal response (Supplementary Fig. 3). Therefore, neuronal subpopulations were not further sub-divided into different types of response. 3.4. Effect of ketamine on LFP activity in the mPFC and MD/CM The effect of ketamine (3 and 10 mg/kg s.c.) on LFP activity in the thalamo-cortical circuits was also studied (n = 14 for vehicle and the high dose, n = 13 for the low dose). Fig. 3 A1, B1 and C1 shows the evolution of the spectral content in the mPFC over time for each treatment, and the derived changes in power in the six investigated frequency bands are given in Fig. 3 A2, B2 and C2. The average spectra over 0–20 min post administration in Fig. 3 D further illustrate the nature of the changes in the different frequency bands. Ketamine was found to dose-dependently modulate LFP activity in the mPFC by decreasing beta power and increasing that of the gamma and HFO bands. Ketamine significantly decreased beta power (F[treatment]2,327 = 0.51, p = 0.59; F[time]8,327 = 1.68, p = 0.10; F[treatment*time]16,327 = 2.54, p = 0.001) and increased gamma and HFO power as compared to vehicle treatment (gamma: F[treatment]2,327 = 0.37, p = 0.69; F[time]8,327 = 3.84, p < 0.001; F[treatment*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 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 10 mg/kg on beta was significant at 50–60 min post administration. For gamma and HFO, ketamine 10 mg/kg produced a more prolonged effect that persisted up to 40 min post administration for gamma and until the end of the recording session (85 min post administration) for HFO. Ketamine also dose-dependently modulated the LFP activity in the MD/CM by decreasing beta power and increasing the power in the gamma and HFO bands (Fig. 3 E, F, G and H). Ketamine significantly decreased beta power (F[treatment]2,327 = 0.37, p = 0.68; F [time]8,327 = 2.09, p = 0.036; F[treatment*time]16,327 = 2.31, p = 0.003) and increased gamma and HFO power as compared to vehicle treatment (gamma: F[treatment]2,327 = 0.36, p = 0.69; F [time]8,327 = 3.33, p = 0.001; F[treatment*time]16,327 = 7.07, p < 0.001; HFO: F[treatment]2,327 = 0.16, p = 0.84; F [time]8,327 = 2.05, p = 0.04; F[treatment*time]16,327 = 10.48, p < 0.001). Similar to the observations for the drug in the mPFC, the effects of ketamine 3 mg/kg on beta and gamma lasted for up to 20 min post administration, while its effects at the 10 mg/kg dose persisted for up to 40 min post administration. The effect of ketamine 3 mg/kg on HFO lasted for up to 30 min post administration, while the 10 mg/kg dose produced a more prolonged effect that persisted up to 70 min post administration. Longer-lasting effects of ketamine on the spectral content were also investigated. However, neither dose of the drug significantly modulated the spectral content of LFPs in the mPFC and the MD/CM at 24 h post administration (Supplementary Fig. 5).

3.3. Effect of PCP on single unit activity in the mPFC and MD/CM The effect of PCP (2.5 and 5 mg/kg s.c.) on the discharge of mPFC and MD/CM neurons was studied in a different group of 14 rats. One recording session each with PCP 2.5 and 5 mg/kg from one subject is missing due to hardware malfunction and/or excessive noise. As observed with ketamine, PCP overall significantly increased the firing rate of putative pyramidal neurons in the mPFC when compared to vehicle treatment in the 60 min after administration (AUC, F2,39 = 6.87, p = 0.004). For the higher dose, this effect was significant at 10–60 min post administration, while for the lower dose the increase was only significant at 40–50 min post administration (RM-ANOVA, F[treatment]2,243 = 0.18, p = 0.84; F[time]6,243 = 0.61, p = 0.72; F[treatment*time]12,243 = 3.67, p < 0.001) (Fig. 2 D1). The analysis of the averaged peak effect furthermore indicates a dose-dependency of the response, however, only the high dose was significantly different from vehicle (Fig. 2 D2). For putative interneurons, PCP showed, similar to ketamine, a tendency towards an increase in the firing rate of putative interneurons at both doses, but the overall effect on AUC did not reach statistical significance (AUC, F2,39 = 3.10, p = 0.07; RM-ANOVA, F [treatment]2,180 = 0.93, p = 0.40; F[time]6,180 = 0.44, p = 0.85; F [treatment*time]12,180 = 1.51, p = 0.12) (Fig. 2 E). Both doses of PCP significantly increased the firing rate of thalamic relay neurons as compared to vehicle treatment in the 60 min post administration (AUC, F2,39 = 3.93, p = 0.034). For the lower dose, the effect was significant at 20–40 min post administration, while for the higher dose the increase was only significant at the isolated time period of 10–20 min post administration (RM-ANOVA, F 8

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Fig. 4. Effect of phencyclidine (PCP; 2.5 and 5 mg/kg s.c.) treatment on the spectral content of LFPs from mPFC (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; F[treatment*time]48,480 = 2.30, p < 0.001) (Fig. 5 A). Seen over time, the increase in locomotor activity induced by ketamine became first significant at ~20 min post administration while the increase mediated by PCP was first significant at ~65–70 min post administration (Fig. 5 B).

The effects of PCP (2.5 and 5 mg/kg s.c.) on LFP activity in the two brain regions were also studied (n = 14/treatment). The data obtained for the mPFC are presented in Fig. 4 in a similar manner as for ketamine (Fig. 3), with the only difference being that the average spectra in Fig. 4 D and 4 H are based on the time period 20–40 min post administration due to the different pharmacokinetic properties of PCP. As observed for ketamine, PCP modulated the LFP activity in the mPFC by dose-dependently 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[treatment*time]16,356 = 7.84, p < 0.001; HFO: F[treatment]2,356 = 0.003, p = 0.99; F[time]8,356 = 0.20, p = 0.99; F[treatment*time]16,356 = 9.69, p < 0.001). However, unlike ketamine, PCP did not significantly change beta power in the mPFC. The effect of PCP followed a different temporal profile than ketamine, with its effect being sustained for a longer period of time. This is consistent with the different pharmacokinetics of the two compounds (Gastambide et al., 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 post administration). Analogously to its effects in the mPFC, PCP modulated the LFP activity in the MD/CM in a dose-dependent manner, mediating significant increases on gamma and HFO power (gamma: F[treatment]2,356 = 0.13, p = 0.87; F[time]8,356 = 1.22, p = 0.28; F[treatment*time]16,356 = 6.92, p < 0.001; HFO: F[treatment]2,356 = 0.05, p = 0.95; F[time]8,356 = 0.42, p = 0.91; F[treatment*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 of the 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).

3.7. Pharmacokinetics of ketamine and PCP Brain concentrations of ketamine, its major metabolites, norketamine and HNK, and PCP were determined after administration of ketamine 10 mg/kg s.c. and PCP 5 mg/kg s.c. in awake rats at 15 and 60 min post administration (Fig. 6). Maximal brain concentrations of ketamine were noted at 15 min post administration while maximal brain concentrations of PCP were noted at 60 min post administration. In contrast to ketamine, the brain concentrations of norketamine and HNK were higher at 60 min than at 15 min post administration. 4. Discussion NMDA-R antagonists -particularly, ketamine- produce a rapid and persistent antidepressant response as well as acute psychotomimetic effects. The development of novel and improved antidepressant drugs urgently requires an increased mechanistic understanding of these actions. In the present study, we thus investigated the modulatory effects of PCP and ketamine on neuronal discharge and LFP activity in

3.6. Effects of ketamine and PCP on locomotor activity The effects of ketamine (3 and 10 mg/kg s.c.) and PCP (2.5 and 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 increased activity counts as compared to vehicle, while animals administered with the low dose (3 mg/kg) did not exhibit significantly different locomotor activity than vehicle-treated animals (F[treatment]4,480 = 5.37, p = 0.001; F[time]12,480 = 0.69, p = 0.76; F [treatment*time]48,480 = 2.30, p < 0.001) (Fig. 5 A1). Administration

Fig. 5. Effect of ketamine (Ket; 3 and 10 mg/kg s.c.) and phencyclidine (PCP; 2.5 and 5 mg/kg s.c) on locomotor activity (n = 9/treatment). (A) Bar histogram representing the mean total activity count of the period between 20 and 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 mean activity counts ± SEM. *p < 0.05 vs. vehicle (Veh). 10

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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 hippocampus and the amygdala. NMDA-R antagonists have previously been found to modulate LFP activity in multiple frequency bands in anesthetized rats (Amat-Foraster 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; Shaw et al., 2015). In the delta band, decreases in power induced by PCP and several other psychotomimetic drugs in anesthetized rats have been shown to be reversed by antipsychotic treatment (Celada et al., 2013; Llado-Pelfort et al., 2016). Here, delta power was only decreased by PCP in the MD/CM, suggesting that this effect is not a useful biomarker of psychotomimetic activity in awake rats. Decreases in beta power induced by ketamine have also been found in humans (de la Salle et al., 2016) and in non-human primates where it was shown to correlate with working memory deficits (Ma et al., 2018). Other psychotomimetic agents with different modes of action have also been reported to decrease beta oscillations in rodents (Hiyoshi et al., 2014). Importantly, inhibition of mPFC-MD projections has been shown to impair working memory as measured by beta frequency synchronization (Parnaudeau et al. 2013, 2015, 2017). Hence, together with the decreases in beta power observed in the present study, decreases in beta power may represent a potential translational biomarker for cognitive impairments. Increases in gamma power induced by NMDA-R antagonists have been attributed to the psychotomimetic actions of these drugs (Hakami et al., 2009; Hong et al., 2010). However, these increases could not be reversed by antipsychotic drugs such as clozapine or haloperidol (Jones et al., 2012). Moreover, while other psychotomimetic drugs such as DOI and methamphetamine did not amplify gamma oscillations (Hiyoshi et al., 2014; Wood et al., 2012), lanicemine and HNK produced

Fig. 6. Pharmacokinetic profile of ketamine (Ket; 10 mg/kg s.c.) and phencyclidine (PCP; 5 mg/kg s.c) in awake rats (n = 3/treatment). Bar plots show the levels of (R,S)-ketamine (Ket), (R,S)-norketamine (norKet), (2R, 6R)-hydroxynorketamine (HNK) and PCP. Brain concentrations (ng/g) are presented as mean ± SD.

thalamo-cortical circuits. The effects of PCP and ketamine on neuronal discharge and LFP activity during peak effect are summarized in Table 2. We found that both drugs dose-dependently increased the discharge of putative mPFC pyramidal neurons. Remarkably, this effect did not coincide with a reduced discharge from local putative fast-spiking interneurons or a dose-dependent increase in the firing of thalamic MD/CM neurons, as would be expected from the disinhibition hypothesis. Furthermore, both ketamine and PCP affected LFP activity by increasing the band power at higher frequencies (gamma and HFO) and decreasing the band power at lower frequencies (beta), except for PCP in mPFC. The effects on both firing and LFP activity followed the pharmacokinetic profile of the drugs. In the case of ketamine, these effects were also observed at a dose that did not induce statistically significant hyperlocomotor activity in the rats. For PCP, the observed increase in discharge of putative mPFC pyramidal neurons and thalamic relay neurons is consistent with the overall effect of the drug in chloral hydrate-anesthetized rats (Kargieman et al., 2007; Santana et al., 2011). In contrast, for ketamine two recent studies in chloral hydrate-anesthetized rats reported a reduced discharge in these neuronal populations (Amat-Foraster et al., 2018; Shen et al., 2018). These observations clearly suggest a differential interaction of anesthesia with the neurotransmitter systems targeted by PCP and ketamine and question the value of anesthetized 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 studies in awake rodents, in which several different NMDA-R antagonists have been found to increase firing of pyramidal neurons (Furth et al., 2017; Homayoun and Moghaddam, 2007; Jodo et al., 2003; Molina et al., 2014; Suzuki et al., 2002). These effects are thus compatible with the glutamate hypothesis for fast-acting antidepressants, which suggests that a transient increase in glutamate release in the PFC is a key proximal event that leads to the activation of AMPA-Rs, thereby evoking a cascade of intracellular signaling pathways involved in synaptogenesis, ultimately leading to the antidepressant effects (Duman and Aghajanian, 2012; Moghaddam et al., 1997; Monteggia and Zarate, 2015). Direct blockade of NMDA-Rs on pyramidal neurons in the mPFC would be expected to result in a reduction of their firing rate (Rotaru et al., 2011). Hence, it has been hypothesized that NMDA-R antagonists preferentially block NMDA-Rs on cortical GABAergic interneurons or

Table 2 Effects of ketamine (Ket) (3 and 10 mg/kg s.c.) and phencyclidine (PCP) (2.5 and 5 mg/kg s.c.) on firing rate and oscillatory activity in the mPFC and MD/ CM. Percentage change relative to baseline during the peak effect: 10-30%↓, no significant change ↔, 10–25%↑, 25–50% ↑↑, 50–100% ↑↑↑.

Singe unit LFP mPFC

LFP MD/CM

11

Pyramidal neurons Interneurons Thalamic relay neurons Delta Beta Gamma HFO Delta Beta Gamma HFO

Ket 3

Ket 10

PCP 2.5

PCP 5

↑ ↔ ↑ ↔ ↓ ↑ ↑↑ ↔ ↓ ↑ ↑↑

↑↑ ↔ ↔ ↔ ↓ ↑↑ ↑↑↑ ↔ ↓ ↑↑ ↑↑↑

↔ ↔ ↑↑ ↔ ↔ ↑ ↑↑ ↔ ↓ ↔ ↑↑

↑↑↑ ↔ ↔ ↔ ↔ ↑↑ ↑↑↑ ↓ ↓ ↑↑ ↑↑↑

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antidepressant-like effects and increased gamma oscillations similar to the effects of ketamine but at doses without psychotomimetic effects (Sanacora et al., 2014; Zanos et al., 2016). Therefore, more studies are needed to correlate changes in gamma power with any of the clinical features of NMDA-R antagonists. Nevertheless, the marked gamma effects could be used as a measure of target engagement of NMDA-R antagonists. The most pronounced effects observed for ketamine and PCP in this study were the robust and dose-dependent increases in HFO power in both mPFC and MD/CM, as also observed in previous studies in awake rodents (Flores et al., 2015; Hansen et al., 2019; Hiyoshi et al., 2014; Hunt 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 power (Hunt et al., 2009), this effect could not be observed in anesthetized rats (Supplementary Fig. 6). The translational value of HFO between rodents and humans is currently unknown, since only few human EEG studies have reported oscillatory activity above 100 Hz (Nottage et al., 2015). In any case, based on its marked preclinical effects, the power increases in HFO may potentially be a useful translational biomarker for target engagement in the case of NMDA-R modulators. Whether any certain frequency band can be used as a reliable biomarker for the antidepressant properties of NMDA-R modulating compounds is still unresolved, and firm conclusions will depend on a better translation between rodent and human studies by considering behavioral states and widening the frequency range studied in humans to include HFO. The low dose of ketamine used in this study did not produce significant locomotor activity changes yet modulated neuronal activity significantly. This indicates that measures of neuronal activity are more sensitive than behavioral measures, and that they are potentially independent. Our PK/PD study showed that the acute effects induced by ketamine and PCP on neuronal activity and locomotor activity correlate well with the pharmacokinetic profiles of these compounds in the brain. Interestingly, these effects do not follow the profile of ketamine's major metabolites norketamine and HNK in the brain, as changes in neuronal activity would be expected to peak at later timepoints if driven by the metabolites. On the other hand, PCP increased neuronal firing and the power of gamma and HFO at a brain concentration much smaller than that of ketamine and metabolites, which suggests a more efficient in vivo blockade of NMDA-R by PCP, at least of those receptors controlling thalamo-cortical activity. A main limitation of the present study is the use of naïve animals. Future studies employing animal models for depression would allow for an even better assessment of the potential of certain reported effects as biomarkers of the antidepressant actions of the drugs. Another limitation is the use of an auditory stimulation during the recordings. We thoroughly assessed possible effects of the stimulation and found that it did not affect the overall absolute firing rate of the recorded neurons during baseline and only affected LFP activity in the delta band. Thus, the effects of NMDA-R antagonists on lower frequency bands should be interpreted with caution. In summary, the present study shows that ketamine and PCP similarly modulate thalamo-cortical circuits in awake freely-moving rats by increasing the firing of both excitatory mPFC pyramidal neurons and MD/CM thalamic relay neurons together with an increase in band power at higher frequencies (gamma and HFO) and a decrease in beta power at comparable doses. The increased pyramidal neuron activity in mPFC is not likely to be mediated by local disinhibition since no attenuation of the discharge of GABAergic neurons was observed in PFC. Since the emergence of psychotomimetic effects and the increase in thalamo-cortical activity by the drugs exhibit the same temporal profile, the short-lasting effects induced by ketamine and PCP on thalamocortical activity could be associated with the psychotomimetic-like effects observed with the drugs. Furthermore, these acute effects could also be responsible for triggering a cascade of events involving increased synaptogenesis and could ultimately lead to the persistent

antidepressant effects of ketamine. Even though further studies are necessary to understand to which extent the effects presented in this study contribute to the antidepressant actions and psychotomimetic effects of both drugs, the presented acute effects may be highly relevant as markers of target engagement and aid in the understanding of the long-lasting therapeutic benefits of ketamine as well as its short-term psychotomimetic effects. Collectively, the findings presented herein expand the knowledge of the neuronal populations, brain areas and circuits involved in the actions of non-competitive NMDA-R antagonists and may help to elucidate the mechanisms of action of ketamine and to identify valid biomarkers of its antidepressant action. Financial disclosures The work leading to these results has received funding from Lundbeck A/S, the Innovation Fund Denmark and SAF2015-68346-P (Spanish Ministry of Economy and Competitiveness, co-financed by European Regional Development Fund (ERDF)) and PI12/00156 and PI16/00287 Instituto de Salud Carlos III, co-financed by European Regional Development Fund (ERDF)). Support from the Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM) and Generalitat de Catalunya Grup de Recerca Consolidat, 2017SGR717 is also acknowledged. Ulrike Richter is funded by Sweden's Innovation Agency VINNOVA. Conflicts of interest MAF, KFH and NP are Lundbeck A/S employees. AAJ and UR reported no potential conflicts of interest. FA has also received consultation fees from Lundbeck A/S and is scientific advisor to Neurolixis. FA and PC are PI and co-PI of a research contract with Lundbeck A/S. Acknowledgements A partial account of the present study was presented at the Society for Neuroscience 2017 Annual Meeting. We would like to thank Claus Agerskov for his skillful assistance in data processing. We would also like to thank Christopher Jones for his assistance with the quantitative bioanalysis and Lars Arvastson for his help on statistics. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.neuropharm.2019.107745. References Amat-Foraster, M., Jensen, A.A., Plath, N., Herrik, K.F., Celada, P., Artigas, F., 2018. Temporally dissociable effects of ketamine on neuronal discharge and gamma oscillations in rat thalamo-cortical networks. Neuropharmacology 137, 13–23. Artigas, F., Bortolozzi, A., Celada, P., 2018a. Can we increase speed and efficacy of antidepressant treatments? Part I: general aspects and monoamine-based strategies. Eur. Neuropsychopharmacol. 28, 445–456. Artigas, F., Celada, P., Bortolozzi, A., 2018b. Can we increase the speed and efficacy of antidepressant treatments? Part II. Glutamatergic and RNA interference strategies. Eur. Neuropsychopharmacol. 28, 457–482. Autry, A.E., Adachi, M., Nosyreva, E., Na, E.S., Los, M.F., Cheng, P.F., Kavalali, E.T., Monteggia, L.M., 2011. NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses. Nature 475, 91–95. Bartho, P., Hirase, H., Monconduit, L., Zugaro, M., Harris, K.D., Buzsaki, G., 2004. Characterization of neocortical principal cells and interneurons by network interactions and extracellular features. J. Neurophysiol. 92, 600–608. Browne, C.A., Lucki, I., 2013. Antidepressant effects of ketamine: mechanisms underlying fast-acting novel antidepressants. Front. Pharmacol. 4, 161. Celada, P., Llado-Pelfort, L., Santana, N., Kargieman, L., Troyano-Rodriguez, E., Riga, M.S., Artigas, F., 2013. Disruption of thalamocortical activity in schizophrenia models: relevance to antipsychotic drug action. Int. J. Neuropsychopharmacol. 16, 2145–2163. Chin, C.-L., Upadhyay, J., Marek, G.J., Baker, S.J., Zhang, M., Mezler, M., Fox, G.B., Day, M., 2011. Awake rat pharmacological magnetic resonance imaging as a translational pharmacodynamic biomarker: metabotropic glutamate 2/3 agonist modulation of

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