# Effects of nitrous oxide and ketamine on electrophysiological and molecular responses in the prefrontal cortex of mice: A comparative study

Stanislav Rozov, Roosa Saarreharju, Stanislav Khirug, Markus Storvik, Claudio Rivera, Tomi Rantamäki

► To cite this version:

Stanislav Rozov, Roosa Saarreharju, Stanislav Khirug, Markus Storvik, Claudio Rivera, et al. Effects of nitrous oxide and ketamine on electrophysiological and molecular responses in the prefrontal cortex of mice: A comparative study. European Journal of Pharmacology, 2024, 968, pp.176426. 10.1016/j.ejphar.2024.176426. hal-04612017

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<figure><img src="image_1.png" /></figure>
# Effects of nitrous oxide and ketamine on electrophysiological and molecular responses in the prefrontal cortex of mice: A comparative study

Stanislav Rozov<sup>a,b,*</sup>, Roosa Saarreharju<sup>a,b</sup>, Stanislav Khirug<sup>c</sup>, Markus Storvik<sup>d</sup>, Claudio Rivera<sup>c,e</sup>, Tomi Rantamäki<sup>a,b</sup>

<figure><img src="image_1.png" /></figure>

## ARTICLE INFO

### Keywords:

Dual specificity phosphatases (DUSPs)

Ketamine

Mitogen-activated protein kinases (MAPKs)

Nitrous oxide

Medial prefrontal cortex

Single-unit activity

## ABSTRACT

Nitrous oxide (N₂O; laughing gas) has recently reported to produce rapid antidepressant effects, but little is known about the underlying mechanisms. We performed transcriptomics, in situ hybridization, and electro-physiological studies to examine the potential shared signatures induced by 1 h inhalation of 50% N₂O and a single subanesthetic dose of ketamine (10 mg/kg, i.p.) in the medial prefrontal cortex (mPFC) in adult mice. Both treatments similarly affected the transcription of several negative regulators of mitogen-activated protein kinases (MAPKs), namely, dual specificity phosphatases (DUSPs). The effects were primarily located in the pyramidal cells. Notably, the overall effects of N₂O on mRNA expression were much more prominent and widespread compared to ketamine. Ketamine caused an elevation of the spiking frequency of putative pyramidal neurons and increased gamma activity (30–100 Hz) of cortical local field potentials. However, N₂O produced no such effects. Spiking amplitudes and spike-to-local field potential phase locking of putative pyramidal neurons and interneurons in this brain area showed no uniform changes across treatments. Our findings suggest that N₂O and subanesthetic-dose ketamine target MAPK pathway in the mPFC but produce varying acute electrophysiological responses.

## 1. Introduction

N-methyl-D-aspartate receptor (NMDAR) antagonist nitrous oxide (N₂O; laughing gas) has been used for several decades as an adjunct anesthetic, analgesic, and anxiolytic. Recently, N₂O has shown promise as a rapid-acting antidepressant (Nagele et al., 2015, 2021), similar to that of ketamine. Notably, the antidepressant effects of subanesthetic-dose ketamine are proposed to be associated with stimu-latory effects on neural ensembles in cortical microcircuits that may occur through the blockade of NMDARs in inhibitory interneurons and subsequent disinhibition of glutamatergic synapses and/or inhibition of pre-synaptic NMDARs on excitatory synapses of pyramidal cortical neurons (Abdallah et al., 2018; Ali et al., 2020; Homayoun and Mog-haddam, 2007; Miller et al., 2016; Widman and McMahon, 2018; Zanos

and Gould, 2018). Increased excitatory drive may then lead to critical molecular changes such as activation of TrkB neurotrophin receptor and mitogen-activated protein kinase (MAPK) signaling (Kohtala et al., 2021). However, ketamine has been also proposed to inhibit the extra-synaptic NMDARs on pyramidal neurons (Abdallah et al., 2018; Miller et al., 2016; Zanos and Gould, 2018) and NMDAR-mediated sponta-neous neurotransmission (Autry et al., 2011). NMDAR inhibition-independent mechanism has also been suggested (via hydroxynorketamine metabolites (Zanos et al., 2016). Intriguingly, a recent study indicates that ketamine directly binds to TrkB receptor and thereby facilitates its responsiveness to TrkB (Casarotto et al., 2021), however, see (Alitalo et al., 2023). Knowledge of the mechanisms un-derlying the antidepressant effects of N₂O remains more limited. How-ever, our recent animal studies indicate that N₂O and ketamine trigger

<footnote><sup>a</sup> Laboratory of Neurotherapeutics, Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>b</sup> SleepWell Research Program, Faculty of Medicine, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>c</sup> Neuroscience Center, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>d</sup> University of Eastern Finland, Kuopio, 70029, Finland</footnote>

<footnote><sup>e</sup> Aix Marseille Univ, INSERM, INMED, Marseille, 13007, France</footnote>

<footnote>* Corresponding author. Laboratory of Neurotherapeutics, Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, 00014, Finland.</footnote>

<footnote>E-mail address: stanislav.rozov@helsinki.fi (S. Rozov).</footnote>
# Effects of nitrous oxide and ketamine on electrophysiological and molecular responses in the prefrontal cortex of mice: A comparative study

Stanislav Rozov<sup>a,b,*</sup>, Roosa Saarreharju<sup>a,b</sup>, Stanislav Khirug<sup>c</sup>, Markus Storvik<sup>d</sup>, Claudio Rivera<sup>c,e</sup>, Tomi Rantamäki<sup>a,b</sup>

<figure><img src="image_2.png" /></figure>

Check for
updates

## ARTICLE INFO

### Keywords:

Dual specificity phosphatases (DUSPs)

Ketamine

Mitogen-activated protein kinases (MAPKs)

Nitrous oxide

Medial prefrontal cortex

Single-unit activity

## ABSTRACT

Nitrous oxide (N₂O; laughing gas) has recently reported to produce rapid antidepressant effects, but little is known about the underlying mechanisms. We performed transcriptomics, in situ hybridization, and electro-physiological studies to examine the potential shared signatures induced by 1 h inhalation of 50% N₂O and a single subanesthetic dose of ketamine (10 mg/kg, i.p.) in the medial prefrontal cortex (mPFC) in adult mice. Both treatments similarly affected the transcription of several negative regulators of mitogen-activated protein kinases (MAPKs), namely, dual specificity phosphatases (DUSPs). The effects were primarily located in the pyramidal cells. Notably, the overall effects of N₂O on mRNA expression were much more prominent and widespread compared to ketamine. Ketamine caused an elevation of the spiking frequency of putative pyramidal neurons and increased gamma activity (30–100 Hz) of cortical local field potentials. However, N₂O produced no such effects. Spiking amplitudes and spike-to-local field potential phase locking of putative pyramidal neurons and interneurons in this brain area showed no uniform changes across treatments. Our findings suggest that N₂O and subanesthetic-dose ketamine target MAPK pathway in the mPFC but produce varying acute electrophysiological responses.

## 1. Introduction

N-methyl-D-aspartate receptor (NMDAR) antagonist nitrous oxide (N₂O; laughing gas) has been used for several decades as an adjunct anesthetic, analgesic, and anxiolytic. Recently, N₂O has shown promise as a rapid-acting antidepressant (Nagele et al., 2015, 2021), similar to that of ketamine. Notably, the antidepressant effects of subanesthetic-dose ketamine are proposed to be associated with stimu-latory effects on neural ensembles in cortical microcircuits that may occur through the blockade of NMDARs in inhibitory interneurons and subsequent disinhibition of glutamatergic synapses and/or inhibition of pre-synaptic NMDARs on excitatory synapses of pyramidal cortical neurons (Abdallah et al., 2018; Ali et al., 2020; Homayoun and Mog-haddam, 2007; Miller et al., 2016; Widman and McMahon, 2018; Zanos

and Gould, 2018). Increased excitatory drive may then lead to critical molecular changes such as activation of TrkB neurotrophin receptor and mitogen-activated protein kinase (MAPK) signaling (Kohtala et al., 2021). However, ketamine has been also proposed to inhibit the extra-synaptic NMDARs on pyramidal neurons (Abdallah et al., 2018; Miller et al., 2016; Zanos and Gould, 2018) and NMDAR-mediated sponta-neous neurotransmission (Autry et al., 2011). NMDAR inhibition-independent mechanism has also been suggested (via hydroxynorketamine metabolites (Zanos et al., 2016). Intriguingly, a recent study indicates that ketamine directly binds to TrkB receptor and thereby facilitates its responsiveness to TrkB (Casarotto et al., 2021), however, see (Alitalo et al., 2023). Knowledge of the mechanisms un-derlying the antidepressant effects of N₂O remains more limited. How-ever, our recent animal studies indicate that N₂O and ketamine trigger

<footnote><sup>a</sup> Laboratory of Neurotherapeutics, Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>b</sup> SleepWell Research Program, Faculty of Medicine, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>c</sup> Neuroscience Center, University of Helsinki, Helsinki, 00014, Finland</footnote>

<footnote><sup>d</sup> University of Eastern Finland, Kuopio, 70029, Finland</footnote>

<footnote><sup>e</sup> Aix Marseille Univ, INSERM, INMED, Marseille, 13007, France</footnote>

<footnote>* Corresponding author. Laboratory of Neurotherapeutics, Drug Research Program, Division of Pharmacology and Pharmacotherapy, Faculty of Pharmacy, University of Helsinki, Helsinki, 00014, Finland.</footnote>

<footnote>E-mail address: stanislav.rozov@helsinki.fi (S. Rozov).</footnote>
similar time-dependent effects on TrkB and MAPK signaling in the cor-
tex. Specifically, both treatments acutely increase MAPK phosphoryla-
tion, which is followed by a brain state characterized by slow-wave
electroencephalographic (EEG) activity, during which MAPK phos-
phylation decreases, whereas TrkB receptor signaling increases
(Kohtala et al., 2019). Indeed, comparative studies with both ketamine
and N₂O may thus reveal shared mechanisms underlying for both ke-
tamine and N₂O.

The objective of this study was to investigate electrophysiological and molecular responses of the prefrontal cortex of adult mice to clinically relevant doses of N₂O and compare the results to those acquired after treatment with a subanesthetic dose of ketamine. Single unit activity (SUA) and phase coupling analyses showed non-uniform responses to treatment in putative pyramidal neurons and interneurons. Gene ontology and rank-rank hypergeometric overlap (RRHO) analyses of the whole-transcriptome sequencing data revealed that both treatments acutely affected the MAPK pathway, in particular the expression of dual-specificity phosphatases (DUSPs). Taken together, these findings demonstrate the presence of multiple molecular targets for N₂O, some of which are also common to ketamine in terms of both direction of regulation and localization, which support our recent findings (Kohtala et al., 2019). The heterogeneity of electrophysiological responses to both treatments suggests that the relationships within and between local circuits are more complex than proposed by the disinhibition hypothesis.

## 2. Materials and methods

2.1. *Animals*

Eight to ten weeks-old, male C57BL/6J OlaHsd mice (Envigo,
Netherlands) were used. Animals were group-housed in sound-proof
cages (Scantainer, Scanbur, Sweden) under controlled conditions (22
± 1 °C, 12:12 h light-dark cycle, 6 a.m. lights on) in the animal facility of
the University of Helsinki with free access to food and water, unless
specified otherwise. Treatments were performed during the light phase
(Zeitgeber Time, ZT 3–7). Animals were single-housed for the time of
pharmacological treatment or during electrophysiological recordings to
avoid damage of a headmount. The experiments were performed ac-
cording to the guidelines of the Society for Neuroscience and were
approved by the County Administrative Board of Southern Finland (Li-
cense: ESAVI/5844/2019).

2.2. *Study design and drug treatments*

Ketamine-HCl (Ketaminol®, Intervet International B.V., Vnr. nr. 511485) was diluted in saline and injected intraperitoneally (i.p.) at a dose of 10 mg/kg (Moda-Sava et al., 2019) (injection volume ~10 ml/kg, n = 5-6). Controls received saline (n = 5-6). A 1:1 mixture of N₂O and O₂ (Woikoski, Finland) was administered for 40-60 min, as described in previous clinical (Nagele et al., 2015) and animal (Kohtala et al., 2019) studies (n = 5-6). Because the RNAseq experiment included one control group for both ketamine and N₂O treatments, saline (i.p.) was administered to both the control and N₂O groups to control for the effects of injection. To control for the potential effects of hyper-oxygenation in the 50% N₂O treatment group, an additional experiment was performed using a 50% N₂/50% O₂ mixture. For the biochemical analyses, the animals were euthanized by cervical dislocation 2 h after ketamine/saline injections or 1 h after 1-h N₂O treatment. The whole brain or bilateral mPFC (including prelimbic and infralimbic cortices) tissue was rapidly dissected on a cooled dish (Kohtala et al., 2016) and stored at -80 °C until further processing. The number of animals used (per group): RNAseq: 5 (ketamine), 4 (N₂O); qPCR: n = 5; FISHCR: n = 4-5; electrophysiological experiments: n = 6 (ketamine), n = 7 (N₂O).

2.3. RNA sequencing

mRNA extraction was performed with the NucleoSpin RNA Plus (Macherey-Nagel, Germany) kit in accordance with the manufacturer's instructions. Before sequencing, RNA integrity was tested with a Bio-analyzer (Agilent Technologies, CA, USA), and samples with RIN >9.0 were used in analysis. Samples were sequenced with the Illumina NextSeq sequencer (Illumina, San Diego, CA, USA) at the Biomedicum Functional Genomics Unit (FuGU) in High output runs using the NEB-Next® Ultra™ II Directional RNA Library Prep Kit for Illumina 3. The sequencing was performed as single-end 75 sequencing (SE75 or 1x75bp; read length 75 bp).

Data processing steps included FastQC quality analysis, analysis
summarization with MultiQC (Ewels et al., 2016), light quality trimming
with Trimmomatic (Bolger et al., 2014), alignment of the sample reads
against the mouse GRCm38.p6 (GCA_000001635.8) reference with
spliced transcript alignment to a reference genome (STAR) (Dobin et al.,
2013), mapping quality assessment with Qualimap (García-Alcalde
et al., 2012) and read quantification (featureCounts (Liao et al., 2014),).

2.4. *Quantitative RT-PCR*

mRNA extraction was done with the NucleoSpin RNA Plus (Macherey-Nagel, Germany) kit according to the manufacturer's instructions. RNA concentration and purity were assessed with the Nanodrop 2000 Spectrophotometer (Millipore, USA). Samples with an RNA concentration >100 ng/µl, 260/280 nm ratio >1.9, and 260/230 nm ratio >1.9 were processed for cDNA synthesis (one sample excluded) using the Maxima First Strand cDNA Synthesis Kit and dsDNase mix (K1672, Thermo Scientific, USA). The primers used to amplify specific cDNA regions of the transcripts are listed in Table S1. Primers for *Dusp1*, *Dusp5*, *Dusp6*, and *Nr4a1* were designed using the PrimerBLAST service (NCBI, USA) and tested for specificity and selectivity. Amplification of cDNA and corresponding control samples was done with Maxima SYBR Green/ROX qPCR Master Mix (2X) (K0221, Thermo Scientific, USA) on the Lightcycler® 480 System (Roche, Switzerland). Relative quantification of templates was performed as described in (Pfaffl, 2001), with cDNA data being normalized to the joint *Gapdh* and *beta-actin* transcript levels. Amplification efficiency of PCR reactions was estimated using a dilution series: *Gapdh*: 1.93; *Actb*:1.88, *Fos*: 1.84, *Nr4a1*: 1.91, *Arc*: 1.98, *Dusp1*:1.99, *Dusp5*: 2.04, *Dusp6*: 1.97.

2.5. *Fluorescent in situ hybridization chain reaction (FISHCR)*

Sets of probes for *Fos* (accession # NM_010234.3), *Arc* (NM_018790.3), *Dusp1* (NM_013642.3), *Dusp6* (NM_026268.3), *Nr4a1* (NM_010444.3), *Slc17a7* (NM_182,993), and *Gad2* (NM_008078.2) as well as buffers for hybridization, washing, and amplification steps were produced by and purchased from Molecular Instruments (USA). Twenty micrometer-thick coronal sections of mPFC were prepared at -15 °C with the Leica CM1860 UV (Leica, Germany) and stored at -80 °C until further processing. Prior to FISHCR, sections were fixed in cold 4% paraformaldehyde for 15 min and processed according to the supplier's instructions (Choi et al., 2018) with the following modifications: concentrations of probes in hybridization solutions were 1 nM (*Slc17a7* and *Gad2*) and 16 nM (*Fos*, *Arc*, *Dusp1*, *Dusp6I*, and *Nr4a1*); *Fos*, *Arc*, *Dusp1*, *Dusp6*, and *Nr4a1* probes were designed to link to the B4 amplifier and were conjugated with dye B4-488, and *Slc17a7* and *Gad2* probes were conjugated with B2-647 and B3-546, respectively, to perform the triple hybridization reaction; and the incubation times for hybridization and amplification steps were 20 h. Sections were mounted in Fluoromount-G media (Thermo Fisher Scientific, USA). Data acquisition was performed with a Leica DMi8 system equipped with a HC PL APO 40x/1.25 mot-CORR objective, White Light Laser Stellaris 8, HyD S/HyD X detectors, and LASX software. Image background correction (BaSiC; Peng et al., 2017), stitching, thresholding, and co-localization analysis (BIOP
similar time-dependent effects on TrkB and MAPK signaling in the cor-
tex. Specifically, both treatments acutely increase MAPK phosphoryla-
tion, which is followed by a brain state characterized by slow-wave
electroencephalographic (EEG) activity, during which MAPK phos-
phylation decreases, whereas TrkB receptor signaling increases
(Kohtala et al., 2019). Indeed, comparative studies with both ketamine
and N₂O may thus reveal shared mechanisms underlying for both ke-
tamine and N₂O.

The objective of this study was to investigate electrophysiological and molecular responses of the prefrontal cortex of adult mice to clinically relevant doses of N₂O and compare the results to those acquired after treatment with a subanesthetic dose of ketamine. Single unit activity (SUA) and phase coupling analyses showed non-uniform responses to treatment in putative pyramidal neurons and interneurons. Gene ontology and rank-rank hypergeometric overlap (RRHO) analyses of the whole-transcriptome sequencing data revealed that both treatments acutely affected the MAPK pathway, in particular the expression of dual-specificity phosphatases (DUSPs). Taken together, these findings demonstrate the presence of multiple molecular targets for N₂O, some of which are also common to ketamine in terms of both direction of regulation and localization, which support our recent findings (Kohtala et al., 2019). The heterogeneity of electrophysiological responses to both treatments suggests that the relationships within and between local circuits are more complex than proposed by the disinhibition hypothesis.

## 2. Materials and methods

2.1. *Animals*

Eight to ten weeks-old, male C57BL/6J OlaHsd mice (Envigo,
Netherlands) were used. Animals were group-housed in sound-proof
cages (Scantainer, Scanbur, Sweden) under controlled conditions (22
± 1 °C, 12:12 h light-dark cycle, 6 a.m. lights on) in the animal facility of
the University of Helsinki with free access to food and water, unless
specified otherwise. Treatments were performed during the light phase
(Zeitgeber Time, ZT 3–7). Animals were single-housed for the time of
pharmacological treatment or during electrophysiological recordings to
avoid damage of a headmount. The experiments were performed ac-
cording to the guidelines of the Society for Neuroscience and were
approved by the County Administrative Board of Southern Finland (Li-
cense: ESAVI/5844/2019).

## 2.2. Study design and drug treatments

Ketamine-HCl (Ketaminol®, Intervet International B.V., Vnr. nr. 511485) was diluted in saline and injected intraperitoneally (i.p.) at a dose of 10 mg/kg (Moda-Sava et al., 2019) (injection volume ~10 ml/kg, n = 5-6). Controls received saline (n = 5-6). A 1:1 mixture of N₂O and O₂ (Woikoski, Finland) was administered for 40-60 min, as described in previous clinical (Nagele et al., 2015) and animal (Kohtala et al., 2019) studies (n = 5-6). Because the RNAseq experiment included one control group for both ketamine and N₂O treatments, saline (i.p.) was administered to both the control and N₂O groups to control for the effects of injection. To control for the potential effects of hyper-oxygenation in the 50% N₂O treatment group, an additional experiment was performed using a 50% N₂/50% O₂ mixture. For the biochemical analyses, the animals were euthanized by cervical dislocation 2 h after ketamine/saline injections or 1 h after 1-h N₂O treatment. The whole brain or bilateral mPFC (including prelimbic and infralimbic cortices) tissue was rapidly dissected on a cooled dish (Kohtala et al., 2016) and stored at -80 °C until further processing. The number of animals used (per group): RNAseq: 5 (ketamine), 4 (N₂O); qPCR: n = 5; FISHCR: n = 4-5; electrophysiological experiments: n = 6 (ketamine), n = 7 (N₂O).

2.3. RNA sequencing

mRNA extraction was performed with the NucleoSpin RNA Plus (Macherey-Nagel, Germany) kit in accordance with the manufacturer's instructions. Before sequencing, RNA integrity was tested with a Bio-analyzer (Agilent Technologies, CA, USA), and samples with RIN >9.0 were used in analysis. Samples were sequenced with the Illumina NextSeq sequencer (Illumina, San Diego, CA, USA) at the Biomedicum Functional Genomics Unit (FuGU) in High output runs using the NEB-Next® Ultra™ II Directional RNA Library Prep Kit for Illumina 3. The sequencing was performed as single-end 75 sequencing (SE75 or 1x75bp; read length 75 bp).

Data processing steps included FastQC quality analysis, analysis
summarization with MultiQC (Ewels et al., 2016), light quality trimming
with Trimmomatic (Bolger et al., 2014), alignment of the sample reads
against the mouse GRCm38.p6 (GCA_000001635.8) reference with
spliced transcript alignment to a reference genome (STAR) (Dobin et al.,
2013), mapping quality assessment with Qualimap (García-Alcalde
et al., 2012) and read quantification (featureCounts (Liao et al., 2014),).

2.4. *Quantitative RT-PCR*

mRNA extraction was done with the NucleoSpin RNA Plus (Macherey-Nagel, Germany) kit according to the manufacturer's instructions. RNA concentration and purity were assessed with the Nanodrop 2000 Spectrophotometer (Millipore, USA). Samples with an RNA concentration >100 ng/µl, 260/280 nm ratio >1.9, and 260/230 nm ratio >1.9 were processed for cDNA synthesis (one sample excluded) using the Maxima First Strand cDNA Synthesis Kit and dsDNase mix (K1672, Thermo Scientific, USA). The primers used to amplify specific cDNA regions of the transcripts are listed in Table S1. Primers for *Dusp1*, *Dusp5*, *Dusp6*, and *Nr4a1* were designed using the PrimerBLAST service (NCBI, USA) and tested for specificity and selectivity. Amplification of cDNA and corresponding control samples was done with Maxima SYBR Green/ROX qPCR Master Mix (2X) (K0221, Thermo Scientific, USA) on the Lightcycler® 480 System (Roche, Switzerland). Relative quantification of templates was performed as described in (Pfaffl, 2001), with cDNA data being normalized to the joint *Gapdh* and *beta-actin* transcript levels. Amplification efficiency of PCR reactions was estimated using a dilution series: *Gapdh*: 1.93; *Actb*:1.88, *Fos*: 1.84, *Nr4a1*: 1.91, *Arc*: 1.98, *Dusp1*:1.99, *Dusp5*: 2.04, *Dusp6*: 1.97.

2.5. Fluorescent in situ hybridization chain reaction (FISHCR)

Sets of probes for *Fos* (accession # NM_010234.3), *Arc* (NM_018790.3), *Dusp1* (NM_013642.3), *Dusp6* (NM_026268.3), *Nr4a1* (NM_010444.3), *Slc17a7* (NM_182,993), and *Gad2* (NM_008078.2) as well as buffers for hybridization, washing, and amplification steps were produced by and purchased from Molecular Instruments (USA). Twenty micrometer-thick coronal sections of mPFC were prepared at -15 °C with the Leica CM1860 UV (Leica, Germany) and stored at -80 °C until further processing. Prior to FISHCR, sections were fixed in cold 4% paraformaldehyde for 15 min and processed according to the supplier's instructions (Choi et al., 2018) with the following modifications: concentrations of probes in hybridization solutions were 1 nM (*Slc17a7* and *Gad2*) and 16 nM (*Fos*, *Arc*, *Dusp1*, *Dusp6I*, and *Nr4a1*); *Fos*, *Arc*, *Dusp1*, *Dusp6*, and *Nr4a1* probes were designed to link to the B4 amplifier and were conjugated with dye B4-488, and *Slc17a7* and *Gad2* probes were conjugated with B2-647 and B3-546, respectively, to perform the triple hybridization reaction; and the incubation times for hybridization and amplification steps were 20 h. Sections were mounted in Fluoromount-G media (Thermo Fisher Scientific, USA). Data acquisition was performed with a Leica DMi8 system equipped with a HC PL APO 40x/1.25 mot-CORR objective, White Light Laser Stellaris 8, HyD S/HyD X detectors, and LASX software. Image background correction (BaSiC; Peng et al., 2017), stitching, thresholding, and co-localization analysis (BIOP
JaCoP; Bolte and Cordelières, 2006) were performed using Fiji software v. 1.53 (Schneider et al., 2012).

## 2.6. Electrophysiological recording and spike sorting

Mice were anesthetized using isoflurane (5% induction, 2% maintenance), accompanied by the administration of analgesic (carprofen, s. c. 5 mg/kg) and local analgesic (lidocaine), and craniotomy for electrophysiological recording and montage of a metal frame for head fixation was performed. To facilitate the administration of injectable compounds during recording sessions, the subjects were additionally implanted with a biocompatible catheter (s.c.) connected to the injection port. Animals were allowed to recover for 7 days, followed by 4 days of habituation to Mobile HomeCage® (Neurotar, Helsinki, Finland). Recordings were performed in the anterior cingulate cortex (AP: 2.0, ML: 0.75, angle 45°) on head-restrained conscious subjects with an A4x8-5mm-100-200-177-A32 probe (Neuronexus, USA) connected to an A32 preamplifier and SmartBox Pro amplifier (Neuronexus, USA). A face mask was used for the delivery of gaseous substances. Data acquisition was performed at a sampling rate of 20–30 kHz. At 40 min after control treatment, subjects received either ketamine or N₂O, and recording continued for another 40 min. After the experiment, probe locations were verified on brain slices under a fluorescent microscope.

Power spectra were normalized using the frequency-wise division of power spectral density values of treatment phases per power spectral density values of baseline recordings. We used non-paired t-tests to compare normalized values between the control and treatment groups. To correct for multiple comparisons, we set a threshold for the vectors of t-values to retain only those values that exceeded *p* = 0.01, excluding the remainder from further analysis. Integrated t-values were computed for any contiguous array (cluster) of suprathreshold values. We tested the significance of these values against the distribution of integrated t-values of clusters acquired through the permutation-based generation of sets of matrices of t-values, followed by setting a threshold as described above. We considered clusters with *p* < 0.05 as significant. Spike sorting was performed on band-pass filtered (300–5000 Hz, Butterworth filter, order 3), whitened, median-subtracted data using SpykingCircus software (Yger et al., 2018). Results of spike sorting were manually verified using Phy 2 software (Cyrille Rossant) to identify putative pyramidal neuron- and interneuron-like spikes on the basis of the duration of the after-hyperpolarization phase and autocorrelogram analysis of inter-spike intervals.

## 2.7. Statistics

Differential expression statistics of RNA sequencing data was performed using negative binomial linear model followed by Wald's test, followed by Benjamini-Hochberg correction for multiple comparisons implemented in DESeq2 R library (Love et al., 2014). To identify the molecular processes primarily affected by ketamine or N₂O alone, gene expression data (Wald's test, *p*adj < 0.05) were subjected to gene ontology (GO; Mi et al., 2019) analysis, and pathways that exceeded the significance threshold (Fisher's exact test, *p*adj < 0.05) were considered as significantly enriched. To identify the common molecular signatures of ketamine and N₂O effects, signed log10(P) values from the previous step were used in rank-rank hypergeometric overlap (RRHO) analysis as described earlier (step size = 100) (Plaisier et al., 2010). A *posteriori* power analysis with alpha = 0.1 and beta = 0.25 (Hart et al., 2013) was used as a selection criterion for a commonly regulated subset of genes identified by RRHO (signed log10(P) values higher than maximum estimated co-occurrence rate), to be included in the GO search as described above. Genes that were associated with several pathways and had a >1.5 times difference vs. control (Wald's test, *p*adj < 0.05) were selected for the qPCR validation analysis.

Statistical analysis of qPCR data was performed using R software (version 3.5.2, R core team, 2018). Differences among experimental

groups in qPCR analysis were determined using either a two-tailed, non-paired T-test or one-way analysis of variance (ANOVA), followed by Tukey *post-hoc* test with Bonferroni correction.

Spike-to-LFP coupling, firing rate assessment, and statistical analyses (omnibus test to assess the spike-to-LFP phase lock of single neurons, Watson-Williams test to assess the significance of treatment-induced spike-to-LFP phase lock change of multiple SUA, paired T-test to assess the significance of treatment-induced firing rate change) were performed in Matlab (Natick, USA) with the CircStat toolbox (Berens, 2009).

# 3. Results

## 3.1. Molecular effects of subanesthetic-dose ketamine and N₂O

To examine changes in transcriptome induced by 50% N₂O (60 min inhalation) or ketamine (10 mg/kg, i.p.), we conducted transcriptome-wide analysis of samples derived from mPFC 2 h after treatment onset (Fig. 1A). Gene-wise comparison of groups treated with air or 50% N₂O (15,257 transcripts) revealed approximately 740 differentially expressed genes (Wald's test, *p*adj < 0.05; Fig. 1A–Table S2). In contrast, out of 16,968 transcripts analyzed in groups treated with saline or ketamine, only 15 were found to be differentially expressed: *Dusp5*, *Dusp6*, *Erf*, *Etv6*, *Fam84b*, *Ier5l*, *Junb*, *Kdm6b*, *Klf10*, *Lrtm2*, *Nr4a1*, *Sox 8*, *Spry 4*, *Tiparp*, and *Wisp 1* (Wald's test, *p*adj < 0.05; Fig. 1B–Table S2).

Next, we used RRHO analysis to further analyze the concordance/ discordance in mRNA changes in the mPFC induced by N₂O and ketamine. Non-corrected results of Wald's tests from the previous step were subjected to RRHO analysis, which revealed ~1600 genes with the highest estimated probability of co-occurrence between these treatments (Fig. 1C, *p* = -log₁₀128). A subset of these genes, chosen based on *a posteriori* power analysis (alpha = 0.1, beta = 0.25, 125 genes), were further processed for GO search. This analysis showed 18 biological processes affected by N₂O and only 2 processes by ketamine (Table 1). Moreover, several genes that were significantly influenced by both treatments are known members of the MAPK family as well as a subset of immediate early genes (Table 1).

A substantial portion of the pathways and gene transcripts affected by N₂O are associated with synaptic function and neuronal activity (Table 1). These include synaptic vesicle trafficking factors (*Apha1*, *Cplx1*, *Stxbp2*, *Syt13*, *Snap25*, *Unc13a*, *Picalm*, and *Dnm3*), G-protein-coupled receptors (*Adgrl1*, *Chrm4*, *Gpr3*, *Htr1a*, *Htr2a*, *Htr6*, *Lpar1*, *Mc4r*, and *Ptger4*), and GABA<sub>A</sub> receptor subunits (*Gabra2* and *Gabra3*). Furthermore, N₂O downregulated the expression of postsynaptic scaffold factors such as *Nlgn2*, *Nlgn3*, and *Shank3* and affected the expression of several immediate early genes including *Arc*, *Egr1*, *Egr2*, *Egr4*, *Fos*, *Fosb*, *Junb*, *Klf4*, *Maff*, and *Nr4a1*. Notably, four of the 10 known dual-specificity serine-threonine phosphatases (DUSPs), which are key negative regulators of the three main pathways of the MAPK signaling cascade—JNK (*Dusp1*, *Dusp4*), p38-MAPK (*Dusp1*) and MAPK (*Dusp5*, *Dusp6*, *Dusp4*)—were regulated by N₂O.

To validate the RNAseq findings, we performed qPCR using selected transcripts. The expression levels of *Dusp1*, *Dusp5*, *Dusp6*, and *Nr4a1* were significantly increased by both ketamine and N₂O treatment, whereas only N₂O treatment increased the expression of *Fos* and *Arc* (Fig. 2A). Notably, in these experiments, we compared N₂O-treated group with a non-treated (air) control group, which raises questions about the potential effects of 50% O₂ on target genes. To address this concern, we compared the expression of *Dusp1*, *Dusp5*, and *Dusp6* in the 50% N₂O/50% O₂ group and 50% N₂/50% O₂ treatment and found no differences between the treatments (Fig. 2A–B).

The results of transcriptome analysis prompted us to examine the spatial distribution of *Fos*, *Arc*, *Nr4a1*, *Dusp1*, and *Dusp6* transcripts and study their co-localization with markers of glutamatergic (*Slc17a7*) and GABAergic (*Gad2*) neurons. Triple fluorescent *in situ* hybridization chain reaction (FISHCR) revealed cortical layers 2–3 of mPFC as a primary
JaCoP; Bolte and Cordelières, 2006) were performed using Fiji software v. 1.53 (Schneider et al., 2012).

## 2.6. Electrophysiological recording and spike sorting

Mice were anesthetized using isoflurane (5% induction, 2% maintenance), accompanied by the administration of analgesic (carprofen, s. c. 5 mg/kg) and local analgesic (lidocaine), and craniotomy for electrophysiological recording and montage of a metal frame for head fixation was performed. To facilitate the administration of injectable compounds during recording sessions, the subjects were additionally implanted with a biocompatible catheter (s.c.) connected to the injection port. Animals were allowed to recover for 7 days, followed by 4 days of habituation to Mobile HomeCage® (Neurotar, Helsinki, Finland). Recordings were performed in the anterior cingulate cortex (AP: 2.0, ML: 0.75, angle 45°) on head-restrained conscious subjects with an A4x8-5mm-100-200-177-A32 probe (Neuronexus, USA) connected to an A32 preamplifier and SmartBox Pro amplifier (Neuronexus, USA). A face mask was used for the delivery of gaseous substances. Data acquisition was performed at a sampling rate of 20–30 kHz. At 40 min after control treatment, subjects received either ketamine or N₂O, and recording continued for another 40 min. After the experiment, probe locations were verified on brain slices under a fluorescent microscope.

Power spectra were normalized using the frequency-wise division of power spectral density values of treatment phases per power spectral density values of baseline recordings. We used non-paired t-tests to compare normalized values between the control and treatment groups. To correct for multiple comparisons, we set a threshold for the vectors of t-values to retain only those values that exceeded *p* = 0.01, excluding the remainder from further analysis. Integrated t-values were computed for any contiguous array (cluster) of suprathreshold values. We tested the significance of these values against the distribution of integrated t-values of clusters acquired through the permutation-based generation of sets of matrices of t-values, followed by setting a threshold as described above. We considered clusters with *p* < 0.05 as significant. Spike sorting was performed on band-pass filtered (300–5000 Hz, Butterworth filter, order 3), whitened, median-subtracted data using SpykingCircus software (Yger et al., 2018). Results of spike sorting were manually verified using Phy 2 software (Cyrille Rossant) to identify putative pyramidal neuron- and interneuron-like spikes on the basis of the duration of the after-hyperpolarization phase and autocorrelogram analysis of inter-spike intervals.

## 2.7. Statistics

Differential expression statistics of RNA sequencing data was performed using negative binomial linear model followed by Wald's test, followed by Benjamini-Hochberg correction for multiple comparisons implemented in DESeq2 R library (Love et al., 2014). To identify the molecular processes primarily affected by ketamine or N₂O alone, gene expression data (Wald's test, *p*adj < 0.05) were subjected to gene ontology (GO; Mi et al., 2019) analysis, and pathways that exceeded the significance threshold (Fisher's exact test, *p*adj < 0.05) were considered as significantly enriched. To identify the common molecular signatures of ketamine and N₂O effects, signed log10(P) values from the previous step were used in rank-rank hypergeometric overlap (RRHO) analysis as described earlier (step size = 100) (Plaisier et al., 2010). A posteriori power analysis with alpha = 0.1 and beta = 0.25 (Hart et al., 2013) was used as a selection criterion for a commonly regulated subset of genes identified by RRHO (signed log10(P) values higher than maximum estimated co-occurrence rate), to be included in the GO search as described above. Genes that were associated with several pathways and had a >1.5 times difference vs. control (Wald's test, *p*adj < 0.05) were selected for the qPCR validation analysis.

Statistical analysis of qPCR data was performed using R software (version 3.5.2, R core team, 2018). Differences among experimental

groups in qPCR analysis were determined using either a two-tailed, non-paired T-test or one-way analysis of variance (ANOVA), followed by Tukey post-hoc test with Bonferroni correction.

Spike-to-LFP coupling, firing rate assessment, and statistical analyses (omnibus test to assess the spike-to-LFP phase lock of single neurons, Watson-Williams test to assess the significance of treatment-induced spike-to-LFP phase lock change of multiple SUA, paired T-test to assess the significance of treatment-induced firing rate change) were performed in Matlab (Natick, USA) with the CircStat toolbox (Berens, 2009).

# 3. Results

## 3.1. Molecular effects of subanesthetic-dose ketamine and N₂O

To examine changes in transcriptome induced by 50% N₂O (60 min inhalation) or ketamine (10 mg/kg, i.p.), we conducted transcriptome-wide analysis of samples derived from mPFC 2 h after treatment onset (Fig. 1A). Gene-wise comparison of groups treated with air or 50% N₂O (15,257 transcripts) revealed approximately 740 differentially expressed genes (Wald's test, *p*adj < 0.05; Fig. 1A–Table S2). In contrast, out of 16,968 transcripts analyzed in groups treated with saline or ketamine, only 15 were found to be differentially expressed: *Dusp5*, *Dusp6*, *Erf*, *Etv6*, *Fam84b*, *Ier5l*, *Junb*, *Kdm6b*, *Klf10*, *Lrtm2*, *Nr4a1*, *Sox 8*, *Spry 4*, *Tiparp*, and *Wisp 1* (Wald's test, *p*adj < 0.05; Fig. 1B–Table S2).

Next, we used RRHO analysis to further analyze the concordance/ discordance in mRNA changes in the mPFC induced by N₂O and ketamine. Non-corrected results of Wald's tests from the previous step were subjected to RRHO analysis, which revealed ~1600 genes with the highest estimated probability of co-occurrence between these treatments (Fig. 1C, *p* = -log₁₀128). A subset of these genes, chosen based on *a posteriori* power analysis (alpha = 0.1, beta = 0.25, 125 genes), were further processed for GO search. This analysis showed 18 biological processes affected by N₂O and only 2 processes by ketamine (Table 1). Moreover, several genes that were significantly influenced by both treatments are known members of the MAPK family as well as a subset of immediate early genes (Table 1).

A substantial portion of the pathways and gene transcripts affected by N₂O are associated with synaptic function and neuronal activity (Table 1). These include synaptic vesicle trafficking factors (*Apha1*, *Cplx1*, *Stxbp2*, *Syt13*, *Snap25*, *Unc13a*, *Picalm*, and *Dnm3*), G-protein-coupled receptors (*Adgrl1*, *Chrm4*, *Gpr3*, *Htr1a*, *Htr2a*, *Htr6*, *Lpar1*, *Mc4r*, and *Ptger4*), and GABA<sub>A</sub> receptor subunits (*Gabra2* and *Gabra3*). Furthermore, N₂O downregulated the expression of postsynaptic scaffold factors such as *Nlgn2*, *Nlgn3*, and *Shank3* and affected the expression of several immediate early genes including *Arc*, *Egr1*, *Egr2*, *Egr4*, *Fos*, *Fosb*, *Junb*, *Klf4*, *Maff*, and *Nr4a1*. Notably, four of the 10 known dual-specificity serine-threonine phosphatases (DUSPs), which are key negative regulators of the three main pathways of the MAPK signaling cascade—JNK (*Dusp1*, *Dusp4*), p38-MAPK (*Dusp1*) and MAPK (*Dusp5*, *Dusp6*, *Dusp4*)—were regulated by N₂O.

To validate the RNAseq findings, we performed qPCR using selected transcripts. The expression levels of *Dusp1*, *Dusp5*, *Dusp6*, and *Nr4a1* were significantly increased by both ketamine and N₂O treatment, whereas only N₂O treatment increased the expression of *Fos* and *Arc* (Fig. 2A). Notably, in these experiments, we compared N₂O-treated group with a non-treated (air) control group, which raises questions about the potential effects of 50% O₂ on target genes. To address this concern, we compared the expression of *Dusp1*, *Dusp5*, and *Dusp6* in the 50% N₂O/50% O₂ group and 50% N₂/50% O₂ treatment and found no differences between the treatments (Fig. 2A–B).

The results of transcriptome analysis prompted us to examine the spatial distribution of *Fos*, *Arc*, *Nr4a1*, *Dusp1*, and *Dusp6* transcripts and study their co-localization with markers of glutamatergic (*Slc17a7*) and GABAergic (*Gad2*) neurons. Triple fluorescent *in situ* hybridization chain reaction (FISHCR) revealed cortical layers 2–3 of mPFC as a primary
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** Results of RNAseq analysis of N₂O- and ketamine-treated groups of animals in comparison with control treatment. Heatmap representation of expression levels of the 25 most significantly upregulated and downregulated genes between control and N₂O (A) and control and ketamine (B) groups. Normalized count values are represented as Z scores; sorting based on adjusted p values. (C) Rank-rank hypergeometric overlap (RRHO) map representing shared mRNA changes in adult mouse medial prefrontal cortex (mPFC) in response to control vs. ketamine and control vs. N₂O treatments. The thin dashed line represents the highest estimated probability of co-occurrence between these treatments (p = -log₁₀128). (D) Schematic overview of the experiment: Male C57BL/6J mice were administered with saline (n = 5), ketamine (10 mg/kg, i.p., n = 5), or 50% N₂O (60 min, n = 4). Two hours after the beginning of administration, samples were collected from the mPFC for further analysis.</figcaption></figure>

place of expression of transcripts of interest (Fig. 3 A-E, upper panels). In addition, *Arc* was expressed in the inner cortical layers (presumably 5–6), and expression of *Nr4a1* was relatively evenly spread across the cortex in the case of saline and ketamine administration. Notably, *Fos*, *Arc*, *Nr4a1*, *Dusp1*, and *Dusp6* transcripts were primarily (M = 0.65–0.85) co-localized with *Slc17a7* (marker of excitatory synapses; Fig. 3F and Fig. S1) and only marginally (M = 0.05–0.20) with *Gad2* (marker of GABAergic neurons). This distribution and co-localization was observed regardless of treatment (p > 0.05; non-paired T-test)

3.2. Electrophysiological effects of subanesthetic-dose ketamine and N₂O

To characterize the acute electrophysiological responses to 50% N₂O and subanesthetic-dose ketamine (10 mg/kg, i.p.), we first examined their effects on local field potentials (LFPs), firing rate, amplitude, and spike-to-LFP coupling of SUA in the anterior cingulate cortex (ACC; Fig. 4C). Ketamine (Fig. 4A) caused an elevation of gamma activity (30–100 Hz; perm. T-test; *p* < 0.05) in this cortical region, whereas N₂O (Fig. 4B) significantly increased theta rhythm (7–9 Hz) and suppressed
<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

**Fig. 1.** Results of RNAseq analysis of N₂O- and ketamine-treated groups of animals in comparison with control treatment. Heatmap representation of expression levels of the 25 most significantly upregulated and downregulated genes between control and N₂O (A) and control and ketamine (B) groups. Normalized count values are represented as Z scores; sorting based on adjusted p values. (C) Rank-rank hypergeometric overlap (RRHO) map representing shared mRNA changes in adult mouse medial prefrontal cortex (mPFC) in response to control vs. ketamine and control vs. N₂O treatments. The thin dashed line represents the highest estimated probability of co-occurrence between these treatments (p = -log₁₀128). (D) Schematic overview of the experiment: Male C57BL/6J mice were administered with saline (n = 5), ketamine (10 mg/kg, i.p., n = 5), or 50% N₂O (60 min, n = 4). Two hours after the beginning of administration, samples were collected from the mPFC for further analysis.

place of expression of transcripts of interest (Fig. 3 A-E, upper panels). In addition, *Arc* was expressed in the inner cortical layers (presumably 5–6), and expression of *Nr4a1* was relatively evenly spread across the cortex in the case of saline and ketamine administration. Notably, *Fos*, *Arc*, *Nr4a1*, *Dusp1*, and *Dusp6* transcripts were primarily (M = 0.65–0.85) co-localized with *Slc17a7* (marker of excitatory synapses; Fig. 3F and Fig. S1) and only marginally (M = 0.05–0.20) with *Gad2* (marker of GABAergic neurons). This distribution and co-localization was observed regardless of treatment (*p* > 0.05; non-paired T-test)

## 3.2. Electrophysiological effects of subanesthetic-dose ketamine and N₂O

To characterize the acute electrophysiological responses to 50% N₂O and subanesthetic-dose ketamine (10 mg/kg, i.p.), we first examined their effects on local field potentials (LFPs), firing rate, amplitude, and spike-to-LFP coupling of SUA in the anterior cingulate cortex (ACC; Fig. 4C). Ketamine (Fig. 4A) caused an elevation of gamma activity (30–100 Hz; perm. T-test; *p* < 0.05) in this cortical region, whereas N₂O (Fig. 4B) significantly increased theta rhythm (7–9 Hz) and suppressed
<caption>**Table 1**</caption>

Results of gene ontology analysis of genes, which expression level in response to treatment with N₂O/O₂ and/or ketamine significantly changed compared to control (Wald's test, *p* < 0.05).

<table><thead><tr><th>Biological Process (PANTHER GO-Slim)</th><th>over/ under</th><th>Fold enrichment</th><th>raw P-value</th><th>FDR</th></tr></thead><tbody><tr><td>N₂O/O₂ (50%/50%, 60 min)</td><td></td><td></td><td></td><td></td></tr><tr><td>epidermal cell differentiation (GO:0009913)</td><td>+</td><td>16.22</td><td>&lt;0.001</td><td>0.007</td></tr><tr><td>circadian regulation of gene expression (GO:0032922)</td><td>+</td><td>12.16</td><td>&lt;0.001</td><td>0.001</td></tr><tr><td>'de novo' protein folding (GO:0006458)</td><td>+</td><td>7.21</td><td>0.004</td><td>0.045</td></tr><tr><td>transforming growth factor beta receptor signaling pathway (GO:0007179)</td><td>+</td><td>6.28</td><td>0.001</td><td>0.014</td></tr><tr><td>cellular response to peptide hormone stimulus (GO:0071375)</td><td>+</td><td>5.16</td><td>0.001</td><td>0.014</td></tr><tr><td>synaptic vesicle cycle (GO:0099504)</td><td>+</td><td>4.42</td><td>&lt;0.001</td><td>0.009</td></tr><tr><td>autophagosome assembly (GO:0000045)</td><td>+</td><td>4.32</td><td>0.004</td><td>0.046</td></tr><tr><td>response to abiotic stimulus (GO:0009628)</td><td>+</td><td>3.96</td><td>&lt;0.001</td><td>0.005</td></tr><tr><td>heart development (GO:0007507)</td><td>+</td><td>3.72</td><td>0.004</td><td>0.047</td></tr><tr><td>central nervous system development (GO:0007417)</td><td>+</td><td>3.37</td><td>0.004</td><td>0.045</td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>3.09</td><td>&lt;0.001</td><td>0.008</td></tr><tr><td>transmembrane receptor protein serine/threonine kinase signaling pathway (GO:0007178)</td><td>+</td><td>3.07</td><td>0.004</td><td>0.046</td></tr><tr><td>neuron projection morphogenesis (GO:0048812)</td><td>+</td><td>2.57</td><td>0.002</td><td>0.027</td></tr><tr><td>G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger (GO:0007187)</td><td>+</td><td>2.48</td><td>0.004</td><td>0.042</td></tr><tr><td>chemical synaptic transmission (GO:0007268)</td><td>+</td><td>2.36</td><td>0.001</td><td>0.017</td></tr><tr><td>positive regulation of cellular protein metabolic process (GO:0032270)</td><td>+</td><td>2.01</td><td>0.004</td><td>0.047</td></tr><tr><td>regulation of transcription, DNA-templated (GO:0006355)</td><td>+</td><td>1.72</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>immune response (GO:0006955)</td><td>-</td><td>0.44</td><td>0.003</td><td>0.039</td></tr><tr><td>Ketamine (10 mg/kg, i.p.)</td><td></td><td></td><td></td><td></td></tr><tr><td>tissue morphogenesis (GO:0048729)</td><td>+</td><td>84,9</td><td>&lt;0.001</td><td>0.0193</td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>32,05</td><td>&lt;0.001</td><td>0.0123</td></tr><tr><td>N₂O/O₂ and ketamine (RRHO analysis)</td><td></td><td></td><td></td><td></td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>10.5</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>regulation of transcription, DNA-templated (GO:0006355)</td><td>+</td><td>2.48</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>apoptotic process (GO:0006915)</td><td>+</td><td>6.72</td><td>&lt;0.001</td><td>0.018</td></tr><tr><td>regulation of cell cycle (GO:0051726)</td><td>+</td><td>5.54</td><td>&lt;0.001</td><td>0.0027</td></tr></tbody></table>

beta activity (15–29 Hz; perm. T-test; *p* < 0.05). Spike sorting identified two major extracellular electrophysiological responses—which are characteristic of putative pyramidal neuron and interneuron cell types—on the basis of spike shapes and autocorrelograms (Fig. 4D and E). Ketamine treatment increased the firing rate of approximately 70% of putative pyramidal SUA (Fig. 4F; *p* = 0.002, paired T-test, Table S3), whereas N₂O treatment increased the firing rate of only 38% of putative pyramidal SUA (Fig. 4G; *p* = 0.76, paired T-test; Table S3). Moreover, approximately 60% of interneuron-like SUA in the ketamine-treated group (Fig. 4H; *p* = 0.22, paired T-test) and 65% in the N₂O-treated group (Fig. 4I; *p* = 0.25, paired T-test) showed higher firing rates. No significant changes in spike amplitude were registered for both putative neuronal types (Fig. 4F–I, lower panels). Spectral analysis of the vicinity of spikes revealed dominant 3 Hz frequency and, in some cases, 7 Hz

oscillations. Subsequent spike-to-LFP phase analysis followed by an omnibus test (histogram of spike-to-LFP phase distribution; Fig. S2E) showed that approximately half of putative pyramidal SUA either coupled to the dominant 3 Hz rhythm irrespective of treatment with ketamine (Fig. S2A *p* = 0.83, Watson-Williams test) or N₂O (Fig. S2B; *p* = 0.76, Watson-Williams test) or developed coupling to that frequency in response to treatment. The other half of the SUA did not demonstrate coupling to any LFP frequencies (Table S3). In turn, virtually all interneuron-like SUA showed strong coupling to the dominant 3 Hz frequency, which was nevertheless not altered by either ketamine (Fig. S2C; *p* = 0.56, Watson-Williams test) or N₂O (Fig. S3D; *p* = 0.56, Watson-Williams test); however, the strength of phase locking was somewhat reduced for both putative pyramidal neurons and interneurons in the case of N₂O treatment (Figs. S2B and D).

### 4. Discussion

N₂O has shown promise as a putative rapid-acting antidepressant (Nagele et al., 2015, 2021) and has several favorable translational properties compared to ketamine. N₂O is administered via inhalation, does not undergo biotransformation (Banks and Hardman, 2005), and shows relatively similar (rapid) pharmacokinetics across mammalian species. Similar to ketamine, N₂O acts as an NMDAR antagonist. However, the precise mechanisms underlying the antidepressant effects of N₂O remain poorly studied.

Our comparative RNA screening analysis of the mouse mPFC, a brain area recognized for its role in the development of depressive states in humans and animal models of depression (Wellman et al., 2020), suggests that the MAPK signaling pathway is a common target of both N₂O and subanesthetic-dose ketamine. In particular, both treatments upregulated the expression of *Dusp*, negative regulators of MAPK signaling. This cascade has been consistently associated with the antidepressant effects of ketamine (Humo et al., 2020), and both MAPK and *Dusp* are also activated by non-pharmacological treatments of depression such as electroconvulsive shock (a model of electroconvulsive therapy; Kodama et al., 2005). Previous transcriptomic studies addressing the effects of ketamine have reported varying results (Bagot et al., 2017; Ficek et al., 2016; Orozco-Solis et al., 2017). However, the MAPK pathway has been highlighted in one of these studies (Ficek et al., 2016).

Recently, we demonstrated N₂O-mediated elevation of *Dusp*1 (Mkp1; Kohtala et al., 2019), however, no consensus has been reached concerning the effects of ketamine on this target, as it has been shown to be either upregulated (Bagot et al., 2017) or downregulated (Ficek et al., 2016) in the prefrontal cortex of rodents. In contrast, MAPK pathway (ERK1/2) inhibitors have been shown to cause depressive-like behavior (Duric et al., 2010) and the phosphorylation of MAPKs has been demonstrated to be altered in depressed patients and in animal models of depression (Meller et al., 2003). Moreover, inhibition of ERK1/2 activity blocks antidepressant-like behavioral effects of antidepressant drugs, including ketamine (Duman et al., 2007).

The ability of ketamine to regulate MAPKs is likely associated with its stimulatory effects on neural ensembles in cortical microcircuits (Rantamäki and Kohtala, 2020). These effects are suggested to arise through the blockade of NMDARs in either inhibitory interneurons and subsequent disinhibition of glutamatergic synapses or direct inhibition of pre-synaptic NMDARs on excitatory synapses of pyramidal cortical neurons, which leads to an increase in excitatory synapse drive (Miller et al., 2016). Our *in situ* data show that the expression of *Dusp*1 and *Dusp*6 was primarily co-localized with markers of glutamatergic cells, which may either support the latter hypothesis or indicate that the activation of these genes was not directly linked to the inhibition of NMDARs in interneurons. N₂O-induced activation of *Dusp* may be related to either its ability to block NMDARs (Jevtović-Todorović et al., 1998) or its interaction with serotonergic (Tyssowski et al., 2018) and noradrenergic (Mukaida et al., 2007) receptors and cation channels (Gruss et al., 2004).
<caption>**Table 1**</caption>

Results of gene ontology analysis of genes, which expression level in response to treatment with N₂O/O₂ and/or ketamine significantly changed compared to control (Wald's test, *p* < 0.05).

<table><thead><tr><th>Biological Process (PANTHER GO-Slim)</th><th>over/ under</th><th>Fold enrichment</th><th>raw P-value</th><th>FDR</th></tr></thead><tbody><tr><td>N₂O/O₂ (50%/50%, 60 min)</td><td></td><td></td><td></td><td></td></tr><tr><td>epidermal cell differentiation (GO:0009913)</td><td>+</td><td>16.22</td><td>&lt;0.001</td><td>0.007</td></tr><tr><td>circadian regulation of gene expression (GO:0032922)</td><td>+</td><td>12.16</td><td>&lt;0.001</td><td>0.001</td></tr><tr><td>'de novo' protein folding (GO:0006458)</td><td>+</td><td>7.21</td><td>0.004</td><td>0.045</td></tr><tr><td>transforming growth factor beta receptor signaling pathway (GO:0007179)</td><td>+</td><td>6.28</td><td>0.001</td><td>0.014</td></tr><tr><td>cellular response to peptide hormone stimulus (GO:0071375)</td><td>+</td><td>5.16</td><td>0.001</td><td>0.014</td></tr><tr><td>synaptic vesicle cycle (GO:0099504)</td><td>+</td><td>4.42</td><td>&lt;0.001</td><td>0.009</td></tr><tr><td>autophagosome assembly (GO:0000045)</td><td>+</td><td>4.32</td><td>0.004</td><td>0.046</td></tr><tr><td>response to abiotic stimulus (GO:0009628)</td><td>+</td><td>3.96</td><td>&lt;0.001</td><td>0.005</td></tr><tr><td>heart development (GO:0007507)</td><td>+</td><td>3.72</td><td>0.004</td><td>0.047</td></tr><tr><td>central nervous system development (GO:0007417)</td><td>+</td><td>3.37</td><td>0.004</td><td>0.045</td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>3.09</td><td>&lt;0.001</td><td>0.008</td></tr><tr><td>transmembrane receptor protein serine/threonine kinase signaling pathway (GO:0007178)</td><td>+</td><td>3.07</td><td>0.004</td><td>0.046</td></tr><tr><td>neuron projection morphogenesis (GO:0048812)</td><td>+</td><td>2.57</td><td>0.002</td><td>0.027</td></tr><tr><td>G protein-coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger (GO:0007187)</td><td>+</td><td>2.48</td><td>0.004</td><td>0.042</td></tr><tr><td>chemical synaptic transmission (GO:0007268)</td><td>+</td><td>2.36</td><td>0.001</td><td>0.017</td></tr><tr><td>positive regulation of cellular protein metabolic process (GO:0032270)</td><td>+</td><td>2.01</td><td>0.004</td><td>0.047</td></tr><tr><td>regulation of transcription, DNA-templated (GO:0006355)</td><td>+</td><td>1.72</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>immune response (GO:0006955)</td><td>-</td><td>0.44</td><td>0.003</td><td>0.039</td></tr><tr><td>Ketamine (10 mg/kg, i.p.)</td><td></td><td></td><td></td><td></td></tr><tr><td>tissue morphogenesis (GO:0048729)</td><td>+</td><td>84,9</td><td>&lt;0.001</td><td>0.0193</td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>32,05</td><td>&lt;0.001</td><td>0.0123</td></tr><tr><td>N₂O/O₂ and ketamine (RRHO analysis)</td><td></td><td></td><td></td><td></td></tr><tr><td>MAPK cascade (GO:0000165)</td><td>+</td><td>10.5</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>regulation of transcription, DNA-templated (GO:0006355)</td><td>+</td><td>2.48</td><td>&lt;0.001</td><td>&lt;0.001</td></tr><tr><td>apoptotic process (GO:0006915)</td><td>+</td><td>6.72</td><td>&lt;0.001</td><td>0.018</td></tr><tr><td>regulation of cell cycle (GO:0051726)</td><td>+</td><td>5.54</td><td>&lt;0.001</td><td>0.0027</td></tr></tbody></table>

beta activity (15–29 Hz; perm. T-test; *p* < 0.05). Spike sorting identified two major extracellular electrophysiological responses—which are characteristic of putative pyramidal neuron and interneuron cell types—on the basis of spike shapes and autocorrelograms (Fig. 4D and E). Ketamine treatment increased the firing rate of approximately 70% of putative pyramidal SUA (Fig. 4F; *p* = 0.002, paired T-test, Table S3), whereas N₂O treatment increased the firing rate of only 38% of putative pyramidal SUA (Fig. 4G; *p* = 0.76, paired T-test; Table S3). Moreover, approximately 60% of interneuron-like SUA in the ketamine-treated group (Fig. 4H; *p* = 0.22, paired T-test) and 65% in the N₂O-treated group (Fig. 4I; *p* = 0.25, paired T-test) showed higher firing rates. No significant changes in spike amplitude were registered for both putative neuronal types (Fig. 4F–I, lower panels). Spectral analysis of the vicinity of spikes revealed dominant 3 Hz frequency and, in some cases, 7 Hz

oscillations. Subsequent spike-to-LFP phase analysis followed by an omnibus test (histogram of spike-to-LFP phase distribution; Fig. S2E) showed that approximately half of putative pyramidal SUA either coupled to the dominant 3 Hz rhythm irrespective of treatment with ketamine (Fig. S2A *p* = 0.83, Watson-Williams test) or N₂O (Fig. S2B; *p* = 0.76, Watson-Williams test) or developed coupling to that frequency in response to treatment. The other half of the SUA did not demonstrate coupling to any LFP frequencies (Table S3). In turn, virtually all interneuron-like SUA showed strong coupling to the dominant 3 Hz frequency, which was nevertheless not altered by either ketamine (Fig. S2C; *p* = 0.56, Watson-Williams test) or N₂O (Fig. S3D; *p* = 0.56, Watson-Williams test); however, the strength of phase locking was somewhat reduced for both putative pyramidal neurons and interneurons in the case of N₂O treatment (Figs. S2B and D).

## 4. Discussion

N₂O has shown promise as a putative rapid-acting antidepressant (Nagele et al., 2015, 2021) and has several favorable translational properties compared to ketamine. N₂O is administered via inhalation, does not undergo biotransformation (Banks and Hardman, 2005), and shows relatively similar (rapid) pharmacokinetics across mammalian species. Similar to ketamine, N₂O acts as an NMDAR antagonist. However, the precise mechanisms underlying the antidepressant effects of N₂O remain poorly studied.

Our comparative RNA screening analysis of the mouse mPFC, a brain area recognized for its role in the development of depressive states in humans and animal models of depression (Wellman et al., 2020), suggests that the MAPK signaling pathway is a common target of both N₂O and subanesthetic-dose ketamine. In particular, both treatments upregulated the expression of *Dusp*, negative regulators of MAPK signaling. This cascade has been consistently associated with the antidepressant effects of ketamine (Humo et al., 2020), and both MAPK and *Dusp* are also activated by non-pharmacological treatments of depression such as electroconvulsive shock (a model of electroconvulsive therapy; Kodama et al., 2005). Previous transcriptomic studies addressing the effects of ketamine have reported varying results (Bagot et al., 2017; Ficek et al., 2016; Orozco-Solis et al., 2017). However, the MAPK pathway has been highlighted in one of these studies (Ficek et al., 2016).

Recently, we demonstrated N₂O-mediated elevation of *Dusp*1 (Mkp1; Kohtala et al., 2019), however, no consensus has been reached concerning the effects of ketamine on this target, as it has been shown to be either upregulated (Bagot et al., 2017) or downregulated (Ficek et al., 2016) in the prefrontal cortex of rodents. In contrast, MAPK pathway (ERK1/2) inhibitors have been shown to cause depressive-like behavior (Duric et al., 2010) and the phosphorylation of MAPKs has been demonstrated to be altered in depressed patients and in animal models of depression (Meller et al., 2003). Moreover, inhibition of ERK1/2 activity blocks antidepressant-like behavioral effects of antidepressant drugs, including ketamine (Duman et al., 2007).

The ability of ketamine to regulate MAPKs is likely associated with its stimulatory effects on neural ensembles in cortical microcircuits (Rantamäki and Kohtala, 2020). These effects are suggested to arise through the blockade of NMDARs in either inhibitory interneurons and subsequent disinhibition of glutamatergic synapses or direct inhibition of pre-synaptic NMDARs on excitatory synapses of pyramidal cortical neurons, which leads to an increase in excitatory synapse drive (Miller et al., 2016). Our *in situ* data show that the expression of *Dusp*1 and *Dusp*6 was primarily co-localized with markers of glutamatergic cells, which may either support the latter hypothesis or indicate that the activation of these genes was not directly linked to the inhibition of NMDARs in interneurons. N₂O-induced activation of *Dusp* may be related to either its ability to block NMDARs (Jevtović-Todorović et al., 1998) or its interaction with serotonergic (Tyssowski et al., 2018) and noradrenergic (Mukaida et al., 2007) receptors and cation channels (Gruss et al., 2004).
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** (A) Results of qPCR validation of expression of genes, selected based on RRHO analysis and fulfilled criteria of *α*-posteriori power test (alpha = 0.1, beta = 0.25). Data were analyzed gene-wise with two-tailed non-paired Student's T-test (n = 5). (B) Exposure to 50% O₂ per se does not evoke any changes in Dusp1, 5 and 6. Subjects were treated with either 50% N₂O/50% O₂ or 50% N₂/50% O₂. Only group, treated with N₂O showed significant alterations in Dusp1 and Dusp5 gene expression levels.</figcaption></figure>

The diversity of targets of N₂O suggests complex pharmacological effects of this drug. Indeed, treatment with N₂O affected the transcription of not only several genes associated with the MAPK pathway but also hundreds of other genes, including synaptic vesicle trafficking factors (*Apba1*, *Cplx1*, *Stxbp2*, *Syt13*, *Snap25*, *Unc13a*, *Picalm*, *Dnm3*), G-protein-coupled receptors (*Adgrl1*, *Chrm4*, *Gpr3*, *Htr1a*, *Htr2a*, *Htr6*, *Lpar1*, *Mc4r* and *Ptger4*), ion channels (*Gabra2* and *Gabra3*), post-synaptic scaffold factors (*Nlgn2*, *Nlgn3*, *Shank3*), and immediate early genes (*Arc*, *Egr1*, *Egr2*, *Fos Egr4*, *Fosb*, *Junb*, *Klf4*, *Maff* and *Nr4a1*), which may be regulated by neuronal activity and subsequent activation of

MAPK. Notably, effects of ketamine were much less prominent. However, one of the hits, *Nr4a1* (also known as *Nurr77*), has also previously been shown to be upregulated by ketamine in primary neuronal cultures (Choi et al., 2015).

Previous studies have shown that *Dusps* and *Nr4a1* were upregulated in animal models of depression; however, the results vary depending on the brain region and method used to generate the model (Duric et al., 2010; Elizalde et al., 2010; Gourley et al., 2008; Jeanneteau et al., 2018; Liu et al., 2020). Expression of *Arc* was downregulated in the frontal cortex and CA1 area of the hippocampus both after chronic mild stress
<figure><img src="image_8.png" /><figcaption>**Fig. 2.** (A) Results of qPCR validation of expression of genes, selected based on RRHO analysis and fulfilled criteria of *α*-posteriori power test (alpha = 0.1, beta = 0.25). Data were analyzed gene-wise with two-tailed non-paired Student's T-test (n = 5). (B) Exposure to 50% O₂ per se does not evoke any changes in Dusp1, 5 and 6. Subjects were treated with either 50% N₂O/50% O₂ or 50% N₂/50% O₂. Only group, treated with N₂O showed significant alterations in Dusp1 and Dusp5 gene expression levels.</figcaption></figure>

The diversity of targets of N₂O suggests complex pharmacological effects of this drug. Indeed, treatment with N₂O affected the transcription of not only several genes associated with the MAPK pathway but also hundreds of other genes, including synaptic vesicle trafficking factors (*Apba1*, *Cplx1*, *Stxbp2*, *Syt13*, *Snap25*, *Unc13a*, *Picalm*, *Dnm3*), G-protein-coupled receptors (*Adgrl1*, *Chrm4*, *Gpr3*, *Htr1a*, *Htr2a*, *Htr6*, *Lpar1*, *Mc4r* and *Ptger4*), ion channels (*Gabra2* and *Gabra3*), post-synaptic scaffold factors (*Nlgn2*, *Nlgn3*, *Shank3*), and immediate early genes (*Arc*, *Egr1*, *Egr2*, *Fos Egr4*, *Fosb*, *Junb*, *Klf4*, *Maff* and *Nr4a1*), which may be regulated by neuronal activity and subsequent activation of

MAPK. Notably, effects of ketamine were much less prominent. However, one of the hits, *Nr4a1* (also known as *Nurr77*), has also previously been shown to be upregulated by ketamine in primary neuronal cultures (Choi et al., 2015).

Previous studies have shown that *Dusps* and *Nr4a1* were upregulated in animal models of depression; however, the results vary depending on the brain region and method used to generate the model (Duric et al., 2010; Elizalde et al., 2010; Gourley et al., 2008; Jeanneteau et al., 2018; Liu et al., 2020). Expression of *Arc* was downregulated in the frontal cortex and CA1 area of the hippocampus both after chronic mild stress
<figure><img src="image_4.png" /><figcaption>**Fig. 3.** Co-localization of *Fos*, *Arc*, *Dusp1*, *Dusp6*, and *Nr4a1* transcripts with markers of glutamatergic (Slc17a7) and GABAergic (Gad2) neurons was assessed using fluorescent *in situ* hybridization chain reaction. Fragments of the medial prefrontal cortex (mPFC) labeled with oligonucleotide probe sets against *Fos* (A), *Arc* (B), *Dusp1* (C), *Dusp6* (D), and *Nr4a1* (E) (upper panels; fluorescence is pseudo-colored in grayscale) and their colocalization (cyan) with Slc17a7 (red) and Gad2 (green) markers (lower panels; fluorescence is pseudo-colored in red, green, and cyan). Dashed lines represent approximate borders between cortical layers; arrowheads point to cells with overlapped signals. F) Fraction of Fos-positive cells co-localized with Slc17a7-or Gad2-positive cells in the mPFC, as measured by Mander's coefficient. G) Region of interest used for co-localization analysis (shadowed area) and a part of the mPFC used in the upper panels of A-E (rectangular outline).</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 3.** Co-localization of *Fos*, *Arc*, *Dusp1*, *Dusp6*, and *Nr4a1* transcripts with markers of glutamatergic (Slc17a7) and GABAergic (Gad2) neurons was assessed using fluorescent *in situ* hybridization chain reaction. Fragments of the medial prefrontal cortex (mPFC) labeled with oligonucleotide probe sets against *Fos* (A), *Arc* (B), *Dusp1* (C), *Dusp6* (D), and *Nr4a1* (E) (upper panels; fluorescence is pseudo-colored in grayscale) and their colocalization (cyan) with Slc17a7 (red) and Gad2 (green) markers (lower panels; fluorescence is pseudo-colored in red, green, and cyan). Dashed lines represent approximate borders between cortical layers; arrowheads point to cells with overlapped signals. F) Fraction of Fos-positive cells co-localized with Slc17a7-or Gad2-positive cells in the mPFC, as measured by Mander's coefficient. G) Region of interest used for co-localization analysis (shadowed area) and a part of the mPFC used in the upper panels of A-E (rectangular outline).</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Effects of ketamine (10 mg/kg, i.p.) or 50% N2O/50% O2 on local field potentials and firing rates of putative pyramidal neurons and interneurons of the anterior cingulate cortex (ACC). A) Gamma activity (30–100 Hz) considerably elevated in response to ketamine (spectrogram [upper panel]—ketamine administration is shown with an arrow head; normalized power spectrum [lower panel; black bars represent differences at p < 0.05; gap in the power spectrum at 50 Hz—mains interference removed by a notch filter]). B) N2O significantly increased theta rhythm (7–9 Hz) while suppressing beta activity (15–29 Hz) (spectrogram [upper panel]—black bar represents time of administration; normalized power spectrum [lower panel]). C) Topographical localization of probes in the ACC (upper panel) and its visualization with fluorescent tracer DiI (lower panel). Putative pyramidal neurons (D) and interneurons (E) were identified on the basis of their autocorrelograms (left panels) and spike shapes (right panels). Firing rates (upper panels) and amplitudes (lower panels) of putative pyramidal cells in response to saline and ketamine (F) or 50% N2O/50% O2 (G). Firing rates (upper panels) and amplitudes (lower panels) of putative interneurons in response to saline and ketamine (H) or 50% N2O/50% O2 (I). The number of subjects was 7 (N2O) and 6 (ketamine); total number of units was 44 + 18 (N2O) and 46 + 6 (ketamine).</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** Effects of ketamine (10 mg/kg, i.p.) or 50% N2O/50% O2 on local field potentials and firing rates of putative pyramidal neurons and interneurons of the anterior cingulate cortex (ACC). A) Gamma activity (30–100 Hz) considerably elevated in response to ketamine (spectrogram [upper panel]—ketamine administration is shown with an arrow head; normalized power spectrum [lower panel; black bars represent differences at p < 0.05; gap in the power spectrum at 50 Hz—mains interference removed by a notch filter]). B) N2O significantly increased theta rhythm (7–9 Hz) while suppressing beta activity (15–29 Hz) (spectrogram [upper panel]—black bar represents time of administration; normalized power spectrum [lower panel]). C) Topographical localization of probes in the ACC (upper panel) and its visualization with fluorescent tracer DiI (lower panel). Putative pyramidal neurons (D) and interneurons (E) were identified on the basis of their autocorrelograms (left panels) and spike shapes (right panels). Firing rates (upper panels) and amplitudes (lower panels) of putative pyramidal cells in response to saline and ketamine (F) or 50% N2O/50% O2 (G). Firing rates (upper panels) and amplitudes (lower panels) of putative interneurons in response to saline and ketamine (H) or 50% N2O/50% O2 (I). The number of subjects was 7 (N2O) and 6 (ketamine); total number of units was 44 + 18 (N2O) and 46 + 6 (ketamine).</figcaption></figure>
and in a genetic animal model of depression (Elizalde et al., 2010). Effects of N₂O have been rarely studied in animals (Liu et al., 2020); therefore, assessment of the expression of these targets in a model subjected to N₂O treatment would provide a better understanding of its putative antidepressant properties.

Our study demonstrated an increase in high-frequency (>30 Hz) cortical LFPs in response to ketamine treatment, which is consistent with several reports in humans and rodents (Foster and Liley, 2011; Kohtala et al., 2019). Strong enhancement of the 7 Hz band and suppression of beta activity (15–30 Hz) were induced by N₂O. To bridge these macroscopic EEG observations to SUA recorded, we assessed SUA in relation to LFPs registered at the same location and time. Spike-to-LFP coupling analysis showed that although a 3 Hz oscillation was dominant in the vicinity of spikes in all analyzed SUA, units that demonstrated persistent coupling to this oscillation were neither synchronous nor changed in response to treatment with either compound. Additionally, a small fraction of SUA had a second dominant frequency at 7 Hz, which is likely related to the increase in its power observed in spectrograms. Another noteworthy observation that is yet to be explained is the stronger coupling of putative interneurons to the 3 Hz activity than that of pyramidal cells. Slow oscillations during wakefulness in frontal cortical areas have been attributed to the activity of several circuits. Recent reports (Biskamp et al., 2017) show strong phase-locked spiking activity of orbitofrontal neurons in response to the 4 Hz rhythm of respiration. However, similar to our finding, Kőszeghy et al. (2018) demonstrated modest phase locking of mPFC neurons to this rhythm with no phase preference throughout their population. Another explanation may be synchronization of the cortico-amygdala circuit, which is related to fear response (Karalis et al., 2016), or the cortico-ventral tegmental area-hypothalamic circuit, which may be related to working memory (Fujisawa and Buzsáki, 2011).

Furthermore, we observed a significant increase in firing rate in the majority of recorded putative pyramidal cells in response to ketamine, which was consistent with the observed elevation of gamma activity and is in line with recent findings (Ali et al., 2020; Miller et al., 2016); moreover, in combination with our *in situ* data showing primary localization of immediate early genes Fos and Arc with vesicular glutamate transporter 1, the finding aligns with the inhibition-disinhibition hypothesis (Miller et al., 2016). However, in addition to these cells, a fraction of interneuron-like SUA also showed increased spiking frequency, whereas the firing rate of the rest of them decreased moderately. In contrast, no increase in the firing rate of putative pyramidal cells was observed in the N₂O-treated group. Because of the small number of recorded responses from putative interneurons, which indicates the overall low relative numbers of these cells in the cerebral cortex, it is not possible to generalize observed effects. Nevertheless, our data show heterogeneity in the response of both cell types to ketamine and marked difference in the firing rate response between both treatment groups, suggesting more complex relationships within and between local circuits than a simple on-off response.

Our study has some limitations. First, only naïve mice were used and therefore follow-up studies employing animal models of depression are important to carry out. Moreover, while we demonstrate the effects of treatment with N₂O and ketamine on abundance of mRNA of expressed genes we focused our analysis on crude brain homogenates; corresponding proteins were also not analyzed. Recently single-cell transcriptomics revealed the presence of multiple neuronal cell types of both excitatory and inhibitory modalities in the mouse cortex (Zeisel et al., 2018). This might also explain observed variability in electrophysiological responses. However, tools that we used do not allow us to address this heterogeneity since identification of neuronal subtypes was done only according to their electrophysiological characteristics. Recording with higher electrode density and quantity accompanied by optogenetic stimulation/inhibition of selected cell types would provide certainty regarding cell types under investigation, including types of interneurons, and in addition could reveal changes in connectivity of local

cortical circuits in response to these treatments.

Collectively, our study characterized transcriptome-wide changes in mPFC triggered by exposure to N₂O and compared them with those caused by the rapid-acting antidepressant ketamine in terms of both the direction of their regulation and localization, thus further supporting our recent observations (Kohtala et al., 2019). In addition, the heterogeneity of electrophysiological responses to both treatments suggests more complex relationships within and between local circuits than proposed by the disinhibition hypothesis.

### CRediT authorship contribution statement

**Stanislav Rozov:** Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. **Roosa Saarreharju:** Investigation. **Stanislav Khirug:** Methodology, Investigation. **Markus Storvik:** Validation, Formal analysis. **Claudio Rivera:** Writing – review & editing, Funding acquisition. **Tomi Rantamäki:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no conflicts of interest.

### Data availability

Data will be made available on request.

### Acknowledgments

S.R. received funding from the Academy of Finland. T.R. received funding from the Academy of Finland, Business Finland and Sigrid Jusélius Foundation. S.K. and C.R. received funding from the Academy of Finland, Era-Net Neuron III program and ANR project. The funding sources were not involved in writing or decision to submit the work. The authors report no competing interests.

We are grateful to the personnel at the animal facility of the University of Helsinki for helping to conduct animal experiments and take care of the animals. We would like to thank the Light Microscopy Unit, Institute of Biotechnology (supported by HiLIFE and Biocenter Finland) for providing equipment and professional assistance and the Functional Genomics Unit (FUGU), University of Helsinki, for assistance with RNAseq data analysis. We express gratitude to Woikoski OY for the provision of oxygen and nitrous oxide. Additionally, we thank Okko Alitalo, M.Sci., and Dr. Samuel Kohtala for helping to conduct the experiments and improve the manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejphar.2024.176426.

### References

Abdallah, C.G., Sanacora, G., Duman, R.S., Krystal, J.H., 2018. The neurobiology of depression, ketamine and rapid-acting antidepressants: is it glutamate inhibition or activation? Pharmacol. Ther. 190, 148–158. https://doi.org/10.1016/j.pharmthera.2018.05.010.

Ali, F., Gerhard, D.M., Sweasy, K., Pothula, S., Pittenger, C., Duman, R.S., Kwan, A.C., 2020. Ketamine disinhibits dendrites and enhances calcium signals in prefrontal dendritic spines. Nat. Commun. 11, 72. https://doi.org/10.1038/s41467-019-13809-8.

Alitalo, O., González-Hernández, G., Rosenholm, M., Kohtala, P., Matsui, N., Müller, H. K., Theilmann, W., Klein, A., Kärkkäinen, O., Rozov, S., Rantamäki, T., Kohtala, S., 2023. Linking hypothermia and altered metabolism with TrkB activation. ACS Chem. Neurosci. 14, 3212–3225. https://doi.org/10.1021/acschemneuro.3c00350.

Autry, A.E., Adachi, M., Nosyreva, E., Na, E.S., Los, M.F., Cheng, P., Kavalali, E.T., Monteggia, L.M., 2011. NMDA receptor blockade at rest triggers rapid behavioural
and in a genetic animal model of depression (Elizalde et al., 2010). Effects of N₂O have been rarely studied in animals (Liu et al., 2020); therefore, assessment of the expression of these targets in a model subjected to N₂O treatment would provide a better understanding of its putative antidepressant properties.

Our study demonstrated an increase in high-frequency (>30 Hz) cortical LFPs in response to ketamine treatment, which is consistent with several reports in humans and rodents (Foster and Liley, 2011; Kohtala et al., 2019). Strong enhancement of the 7 Hz band and suppression of beta activity (15–30 Hz) were induced by N₂O. To bridge these macroscopic EEG observations to SUA recorded, we assessed SUA in relation to LFPs registered at the same location and time. Spike-to-LFP coupling analysis showed that although a 3 Hz oscillation was dominant in the vicinity of spikes in all analyzed SUA, units that demonstrated persistent coupling to this oscillation were neither synchronous nor changed in response to treatment with either compound. Additionally, a small fraction of SUA had a second dominant frequency at 7 Hz, which is likely related to the increase in its power observed in spectrograms. Another noteworthy observation that is yet to be explained is the stronger coupling of putative interneurons to the 3 Hz activity than that of pyramidal cells. Slow oscillations during wakefulness in frontal cortical areas have been attributed to the activity of several circuits. Recent reports (Biskamp et al., 2017) show strong phase-locked spiking activity of orbitofrontal neurons in response to the 4 Hz rhythm of respiration. However, similar to our finding, Kőszeghy et al. (2018) demonstrated modest phase locking of mPFC neurons to this rhythm with no phase preference throughout their population. Another explanation may be synchronization of the cortico-amygdala circuit, which is related to fear response (Karalis et al., 2016), or the cortico-ventral tegmental area-hypothalamic circuit, which may be related to working memory (Fujisawa and Buzsáki, 2011).

Furthermore, we observed a significant increase in firing rate in the majority of recorded putative pyramidal cells in response to ketamine, which was consistent with the observed elevation of gamma activity and is in line with recent findings (Ali et al., 2020; Miller et al., 2016); moreover, in combination with our *in situ* data showing primary localization of immediate early genes Fos and Arc with vesicular glutamate transporter 1, the finding aligns with the inhibition-disinhibition hypothesis (Miller et al., 2016). However, in addition to these cells, a fraction of interneuron-like SUA also showed increased spiking frequency, whereas the firing rate of the rest of them decreased moderately. In contrast, no increase in the firing rate of putative pyramidal cells was observed in the N₂O-treated group. Because of the small number of recorded responses from putative interneurons, which indicates the overall low relative numbers of these cells in the cerebral cortex, it is not possible to generalize observed effects. Nevertheless, our data show heterogeneity in the response of both cell types to ketamine and marked difference in the firing rate response between both treatment groups, suggesting more complex relationships within and between local circuits than a simple on-off response.

Our study has some limitations. First, only naïve mice were used and therefore follow-up studies employing animal models of depression are important to carry out. Moreover, while we demonstrate the effects of treatment with N₂O and ketamine on abundance of mRNA of expressed genes we focused our analysis on crude brain homogenates; corresponding proteins were also not analyzed. Recently single-cell transcriptomics revealed the presence of multiple neuronal cell types of both excitatory and inhibitory modalities in the mouse cortex (Zeisel et al., 2018). This might also explain observed variability in electrophysiological responses. However, tools that we used do not allow us to address this heterogeneity since identification of neuronal subtypes was done only according to their electrophysiological characteristics. Recording with higher electrode density and quantity accompanied by optogenetic stimulation/inhibition of selected cell types would provide certainty regarding cell types under investigation, including types of interneurons, and in addition could reveal changes in connectivity of local

cortical circuits in response to these treatments.

Collectively, our study characterized transcriptome-wide changes in mPFC triggered by exposure to N₂O and compared them with those caused by the rapid-acting antidepressant ketamine in terms of both the direction of their regulation and localization, thus further supporting our recent observations (Kohtala et al., 2019). In addition, the heterogeneity of electrophysiological responses to both treatments suggests more complex relationships within and between local circuits than proposed by the disinhibition hypothesis.

### CRediT authorship contribution statement

**Stanislav Rozov:** Writing – review & editing, Writing – original draft, Visualization, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. **Roosa Saarreharju:** Investigation. **Stanislav Khirug:** Methodology, Investigation. **Markus Storvik:** Validation, Formal analysis. **Claudio Rivera:** Writing – review & editing, Funding acquisition. **Tomi Rantamäki:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no conflicts of interest.

### Data availability

Data will be made available on request.

### Acknowledgments

S.R. received funding from the Academy of Finland. T.R. received funding from the Academy of Finland, Business Finland and Sigrid Jusélius Foundation. S.K. and C.R. received funding from the Academy of Finland, Era-Net Neuron III program and ANR project. The funding sources were not involved in writing or decision to submit the work. The authors report no competing interests.

We are grateful to the personnel at the animal facility of the University of Helsinki for helping to conduct animal experiments and take care of the animals. We would like to thank the Light Microscopy Unit, Institute of Biotechnology (supported by HiLIFE and Biocenter Finland) for providing equipment and professional assistance and the Functional Genomics Unit (FUGU), University of Helsinki, for assistance with RNAseq data analysis. We express gratitude to Woikoski OY for the provision of oxygen and nitrous oxide. Additionally, we thank Okko Alitalo, M.Sci., and Dr. Samuel Kohtala for helping to conduct the experiments and improve the manuscript.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejphar.2024.176426.

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Ficek, J., Zygmunt, M., Piechota, M., Hoinkis, D., Rodriguez Parkitna, J., Przewlocki, R.,
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its rapid antidepressant action. BMC Genom. 17, 362. https://doi.org/10.1186/
s12864-016-2713-3.

Foster, B.L., Liley, D.T.J., 2011. Nitrous oxide paradoxically modulates slow
electroencephalogram oscillations: implications for anesthesia monitoring. Anesth.
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Fujisawa, S., Buzsáki, G., 2011. A 4 Hz oscillation adaptively synchronizes prefrontal,
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neuron.2011.08.018.

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sequencing alignment data. Bioinformatics 28, 2678–2679. https://doi.org/
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pore-domain K+ channels are a novel target for the anesthetic gases xenon, nitrous
oxide, and cyclopropane. Mol. Pharmacol. 65, 443–452. https://doi.org/10.1124/
mol.65.2.443.

Hart, S.N., Therneau, T.M., Zhang, Y., Poland, G.A., Kocher, J.-P., 2013. Calculating
sample size estimates for RNA sequencing data. J. Comput. Biol. 20, 970–978.
https://doi.org/10.1089/cmb.2012.0283.

Homayoun, H., Moghaddam, B., 2007. NMDA receptor hypofunction produces opposite
effects on prefrontal cortex interneurons and pyramidal neurons. J. Neurosci. 27,
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Humo, M., Ayazgök, B., Becker, L.J., Waltisperger, E., Rantamäki, T., Yalcin, I., 2020.
Ketamine induces rapid and sustained antidepressant-like effects in chronic pain
induced depression: role of MAPK signaling pathway. Prog. Neuro-
Psychopharmacol. Biol. Psychiatry 100, 10989. https://doi.org/10.1016/j.
pnpbp.2020.109898.

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