Negative allosteric modulator of the metabotropic glutamate receptor 7 ADX71743 increases wakefulness, reduces sleep, and alters stress-induced changes in the brain neurotransmitter levels

Abstract

Objective

Converging evidence suggests that metabotropic glutamate receptor 7 (mGlu₇) contributes to sleep–wake regulation and stress reactivity. We used the selective negative allosteric modulator (NAM) ADX71743 to examine the role of mGlu₇ in sleep–wake control and in basal and stress-evoked neurotransmitter release in freely moving rats.

Method

Rats were implanted with electrodes and telemetry devices to record electroencephalogram (EEG), neck muscle electromyogram (EMG), motor activity (MA), and body temperature (BT) at baseline and for 9 h after subcutaneous (sc) administration of ADX71743 (50, 100, or 150 mg/kg) or vehicle (50% cyclodextrin in water), given 2 h after lights on. In a separate cohort, rats were implanted with microdialysis probes in the prefrontal cortex (PFC) and ventral hippocampus (vHipp). Samples were collected at baseline, followed by sc ADX71743 (100 mg/kg) or vehicle and 10 min of light handling, 60 min later. Extracellular glutamate (Glu), GABA, glycine (Gly), serotonin (5-HT), norepinephrine (NE), and dopamine (DA) were quantified over the 180 min sampling period.

Results

ADX71743 increased wakefulness by up to 100% and reduced rapid eye-movement (REM) sleep by up to 100% and non-REM (NREM) sleep by up to 75% (all P < .05), with comparable effects across doses. ADX71743 also delayed NREM and REM sleep onset by nearly 2-fold (P < .05) for 2 h after dosing, without altering MA or BT. Basal neurotransmitter concentrations were unchanged. However, ADX71743 attenuated stress-related changes in GABA and 5-HT, maintaining higher levels in the vHipp than in vehicle-treated controls. Stress-evoked NE and DA increases tended to be greater after ADX71743 than after vehicle. Neither treatment nor stress markedly affected Glu or Gly in the PFC or vHipp.

Conclusions

These findings support an important role for mGlu₇ in regulating sleep and wakefulness. The ADX71743-associated maintenance of higher vHipp 5-HT and GABA levels after stress may contribute to reduced stress- and anxiety-like reactivity produced by mGlu₇ inhibition.

Introduction

Metabotropic glutamate receptor 7 (mGlu₇), a G protein-coupled receptor (GPCR), belongs to the family comprising 8 subtypes (mGlu₁-mGlu₈), which are subdivided into 3 groups based on their amino acid sequence, G-protein coupling, and pharmacological profile.¹ mGlu₇ is a part of group III, the largest group of the family, which also includes mGlu₄, mGlu₆, and mGlu₈. mGlu₇, like other members of this group, is negatively coupled with adenylyl cyclase signal transduction pathway through G_i/G_o proteins and is primarily expressed presynaptically both on neurons and the glia. As mGlu₇ is detected on both asymmetrical and symmetrical synapses, it can play a role as both an autoreceptor and a heteroreceptor, regulating release of not only glutamate but also GABA and other neurotransmitters.² Among 8 mGlu subtypes, mGlu₇ has the widest distribution in the brain and shows the highest degree of evolutionary conservation across species.^3,4 A rapidly growing body of evidence points at the importance of mGlu₇ across a broad range of brain functions, including stress and emotional reactivity, fear learning and memory, aggressivity, social and maternal behaviors, and motor function.^5–8

There is also an expanding experimental support to mGlu₇ playing an important role in arousal and sleep–wake regulation, although several studies have delivered contradictory results. For example, mGlu₇ knockout mice were found to exhibit increases in wake, decreases in time spent in non-rapid eye movement (NREM) sleep during both light and dark phases, and reductions in rapid eye movement (REM) sleep bout duration during the light phase.⁹ Also, a study by Cavas et al.¹⁰ described a bell-shaped activity profile of a potent and systemically-active mGlu₇ allosteric agonist AMN082 (5-20 mg/kg) in Wistar rats, where the sleep-promoting effects at 5 and 10 mg/kg were followed by increased waking and reduced REM sleep at 20 mg/kg.¹⁰ On the other hand, a study by Ahnaou et al.¹¹ reported an enhancement in total active waking and reduced sleep in Wistar rats in response to 2.5 mg/kg AMN082, including reductions in deep NREM sleep, REM sleep, and delays in the onset of REM sleep. Adding to the uncertainty is the finding that the wake-promoting effects of AMN082 were both replicated in wildtype mice and in mGlu₇ knockout mice,¹¹ suggesting the AMN082-mediated changes in sleep–wake regulation do not involve mGlu₇ and may be mediated by other targets. Thus, pharmacological tools that exhibit high selectivity at mGlu₇ are needed to improve our understanding of the role of mGlu₇ in sleep and waking.

Here, we investigate the effect of a potent and highly selective mGlu₇ negative allosteric modulator (NAM) ADX71743 (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one on sleep and waking in rats.¹² ADX71743 was discovered through chemical lead optimization of a hit compound identified from a high-throughput screening campaign of the corporate chemical library using a Ca²⁺ mobilization assay.¹² The NAM properties of ADX71743 were confirmed using Schild plot analysis and reversibility tests in vitro, while activity at the native mGlu₇ was shown using ex vivo electrophysiological measures on the mouse hippocampal slices¹² and on human-derived brain tissue.¹³ ADX71743 is highly selective, with no significant activity observed at other mGlu subtypes, as well as lack of activity at 27 additional targets tested functionally using CEREP.¹²  In vivo, ADX71743 (50-150 mg/kg) exhibited anxiolytic-like properties in the marble burying and elevated plus maze tests in rodents and antipsychotic-like profile in the amphetamine-induced locomotor hyperactivity test in rats.¹² Also, ADX71743 (50-150 mg/kg) reduced visceral hypersensitivity in the Wistar Kyoto rat model, increasing visceral sensitivity threshold and reducing the total number of pain behaviors.¹⁴ Recently, ADX71743 was found to disrupt fear memory reconsolidation in rats when administered systemically or microinfused into the lateral amygdala.¹³ Importantly, in ex vivo experiments, at thalamus to lateral amygdala synapses, ADX71743 exhibited a stabilizing effect on glutamatergic synaptic neurotransmission, as it augmented transmission under low stimulus conditions, while attenuating it under high-stimulation conditions.¹³ We hypothesized that negative modulation of the mGlu₇ with ADX71743 would reduce its inhibitory control of the glutamate release, thereby disinhibiting the glutamate synapse and cause increases in brain arousal and, therefore, wake at the expense of sleep.

Another goal of this study was to explore the role of mGlu₇ in stress reactivity, in particular, the mechanisms and anatomical sites mediating amelioration of the effects of stress achieved by mGlu₇ inhibition. In a recent study, pharmacological inhibition of mGlu₇ was found to promote a chronic stress-resilience phenotype in mice.¹⁵ Specifically, a novel, selective orthosteric-like antagonist of mGlu₇, HAP044 (7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one), administered chronically, resulted in highly significant reductions in the effect of chronic psychosocial stress on hypothalamic–pituitary–adrenal axis dysfunctions and stress-mediated exacerbation in anxiety-like reactivity.¹⁵ To achieve this goal, we used the in vivo microdialysis technique to measure the basal and mild stress-induced changes in extracellular concentrations of glutamate (Glu), γ-aminobutyric acid (GABA), glycine (Gly), serotonin (5-hydroxytryptamine; 5-HT), norepinephrine (NE), and dopamine (DA) in freely moving rats. The extracellular concentrations of each neurotransmitter were measured in the prefrontal cortex (PFC) and the ventral hippocampus (vHipp) in light of their central role in reactivity to stress, including stress susceptibility and resilience, as well as high expression of mGlu₇ in these anatomical regions.

Materials and methods

Animals

Thirty-six, adult, male Sprague-Dawley rats (250-300 g), purchased from Charles River Laboratories (Stilwell, KS, United States), were used in the sleep-wake EEG study, among which 20 were used for EEG recording, and 16 were used as satellites for plasma sampling (see below). Animals were group-housed (2/cage) and maintained on a 12 h light/dark schedule (lights on from 6:00 to 18:00). Eleven, adult, male, Sprague-Dawley rats (300-387 g), purchased from Harlan (Horst, The Netherlands), were used in the in vivo microdialysis experiment. Animals were group-housed (5/cage) and maintained on a 12-h light/dark schedule (lights on from 07:00 to 19:00). All animals, maintained under constant temperature (22 ± 2 °C) and humidity (55% ± 15%) conditions, received food and water ad libitum and were acclimated at least 10 days before experimentation. The experimental procedures were approved by the Institutional Animal Care and Use Committees of Melior Discovery, Inc. and University of Groningen and performed in accordance with the guidelines of the National Institutes of Health.

Drugs

ADX71743 (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one was synthesized at Addex Therapeutics (Geneva, Switzerland). The compound was suspended in water containing 50% cyclodextrin (CD), which was also used as vehicle.

E‌EG/EMG activity and telemetry recording

The sleep–wake EEG and electromyogram (EMG) activity was recorded using standard methodology.^16,17 A body temperature (BT)/activity transmitter (TA10TA-F40, Data Sciences International, N. St. Paul, MN, United States) was implanted into the peritoneal cavity to measure motor activity (MA) and BT. The animals were allowed to recover from surgery for at least 1 week prior to recording. In the experiment, animals were placed into individual containers (30 × 30 × 30 cm with a wire-mesh grid top) placed inside sound attenuation cabinets (#ENV-018 V, Med Associates, Inc., St. Albans, VT, United States) equipped with a ventilation fan and a ceiling light (14 lux, on from 06:00 to 18:00). All animals were habituated to the containers overnight prior to actual recording. The EEG and EMG signals were fed via a cable attached to a commutator (Plastics-One, Roanoke, VA. United States) to an amplifier (10 000× gain for EEG, 1000× gain for EMG; model 1700, A-M Systems; Carlsborg, WA, United States), band pass filtered (0.3-500 Hz for EEG, 10-1000 Hz for EMG), and then digitized at 512 samples per second using ICELUS acquisition/sleep scoring software (Mark Opp, University of Michigan, Ann Arbor MI, United States) operating under National Instruments (Austin, TX, United States) data acquisition software (Labview 5.1) and hardware (PCI-MIO-16E-4). In each experiment, 12 h of EEG recording was acquired for each animal. The first 12 h of EEG/EMG data were scored for sleep–wake regulation for each animal, and the full 12 h was subjected to fast-Fourier transform (FFT) analysis to evaluate changes in EEG power over time. Animals were placed into the recording chambers the evening prior to recording, which began automatically 3 h after lights on (at 09:00) and continued for 12 h. Data from four animals were excluded from analysis due to bad signals that could not be accurately scored into sleep/wake stages.

Dosing protocol used in the sleep–wake study

Three experiments were conducted with 20 rats tested in each experiment and 1-week intervals (drug washout period) between experiments, as described previously.¹⁷ The sample size was decided based on the 3R approach and data from previous experiments.¹⁷ Animals were pseudo-randomly assigned to receive a subcutaneous (sc) injection of ADX71743 (50, 100, 150 mg/kg) or vehicle (CD) administered at 2 mL/kg volume. Animals did not receive the same dose of ADX71743 twice, although some animals received vehicle twice. The treatment was administered at 11:00, 2 h after the start of recording, and 5 h after lights on, when the animals were entering their “quiet” period.

E‌EG/EMG activity scoring and analysis

Sleep–wake scoring was performed according to standard methods^18,19. The EEG and EMG records were analyzed in 6 sec epochs, each of which was classified into 1 of 3 sleep–wake states: wake, NREM sleep, and REM sleep by an investigator blinded to treatment. Waking activity was defined as consisting of relatively low-amplitude EEG activity with low power in the low frequency bands from 0.3 to 5 Hz (delta), accompanied by moderate to high level EMG activity. “Theta-waking”, in which EEG power was relatively focused in the 5.5-9 Hz (theta) range and significant EMG activity was present, was scored as wake. NREM sleep was defined as generally high-amplitude EEG activity with greater power in the delta frequency band, accompanied by minimal EMG activity. REM sleep was characterized by moderate and constant amplitude EEG concentrated in the theta range that was similar to waking theta but with no EMG activity and was preceded by NREM sleep.²⁰ In practice, approximately 45 min of data prior to dosing were scored manually, and the remainder of the data were scored using a semi-automated system based on analysis of EEG frequency and amplitude characteristics and EMG activity. After all epochs were scored, the percentage of time spent in each sleep–wake state was calculated for 30 min intervals before and after dosing. Cumulative values of time spent in wake, NREM sleep, and REM sleep for successive 4 h post-dosing periods (4 h AUCs; maximum 240 min) were also calculated. In addition, cumulative wake surplus (CWS) produced by treatment was also calculated by subtracting the mean cumulative wake time (CWT) value at each time point in the vehicle-treated group from the corresponding CWT value in the compound-treated group. Thus, CWS represents the excess wake activity produced by the drug as a function of time compared to vehicle-treated animals. Latencies to onset of NREM and REM sleep, defined as the time from dosing to detection of 2 consecutive minutes (20 epochs) of NREM sleep and 1 consecutive min (10 epochs) of REM sleep, respectively, were also calculated.

Analysis of motor activity and core temperature

Motor activity and BT were recorded using the Artquest integrated hardware/software system (Data Sciences International, N. St. Paul, MN, United States). The computer saved average activity and temperature values every 2 mins. MA included any movement causing the transmitter to be displaced relative to the receiver, such as locomotor activity and rearing. Average MA and BT values were evaluated in a similar manner as the sleep–wake measures. BT was analyzed by calculating the mean BT for the 2 h prior to dosing, and for 4 h periods post dosing.¹⁹ EMG signals were analyzed by rectifying (making all positive) and integrating them over the same time periods as the sleep–wake measures. Also, EMG activity was normalized by dividing values by the corresponding percent time awake and multiplying by 100.

Analysis of EEG frequency changes

Changes in EEG power were evaluated using FFT analysis, in which the power (amplitude) of the signals was calculated as a function of frequency. Electroencephalogram records were first analyzed to identify periods of electrical noise associated with head movement or transient connection problems known to produce large, readily identifiable voltage transients. These periods, normally short (a few seconds), were flagged as artifacts and excluded from analysis. Quantitative EEG analysis was carried out by calculating the average FFT power of the surface EEG records from 0 to 20 Hz at 0.5 Hz intervals (bins) in epochs of 6 sec duration. The EEG data were not segregated by wake, NREM sleep, and REM sleep states before analysis. FFT records were initially computed for 5-min blocks aligned to the time of dosing. These values were then averaged over 30 min and 4 h intervals from the time of dosing to 10 h post-dosing. Also, data from the first 2 h pre-dosing were averaged together as a reference point (the “Pre-dosing” period). Data were next pooled by frequencies and by time intervals. Several frequency intervals were considered: frequency intervals of delta (0-4.5 Hz, ie, 0 up to but not including 4.5 Hz, characteristic of NREM sleep), theta (4.5-10 Hz, characteristic of REM sleep), and were designated in the present study as the beta frequency range (10-20 Hz). Data were plotted as both raw power levels and relative to the 2 h pre-dosing level by subtracting the pre-dosing mean value for each animal from all post-dosing levels.

Bioanalysis of Plasma Concentrations of ADX71743

In the rat sleep–wake EEG study, we used a group of 16 satellite rats to determine plasma concentrations of ADX71743 (50, 100, or 150 mg/kg sc) 1, 3, and 12 h post-dosing. For this purpose, animals (n = 4/dose) received test compounds at 10:00 am. At 11:00 am and at 1:00 pm, blood (0.3-0.5 cc) was collected by tail vein puncture. For the 12 h time point (10:00 pm), blood was collected by intracardiac puncture during isoflurane anesthesia. At each time point, blood samples were placed into tubes containing di-potassium EDTA, placed on ice for 5-10 min, and spun via centrifuge (Sorvall RT+ centrifuge (ThermoScientific) at 1000× g for 10 min at 4 °C. Plasma (~100 μL) was then extracted, frozen immediately on dry ice, and stored at -60 °C. The bioanalysis of plasma samples was performed as described previously.¹² After quantification of the total plasma concentration of ADX71743, we calculated the cerebrospinal fluid (CSF) concentration of the compound based on CSF/plasma ratio of 5.3% as reported previously.¹²

In vivo microdialysis

Experiment was performed as described previously.^21,22 Rats were anesthetized before microdialysis probes were implanted in the PFC and vHipp. For this purpose, each animal was placed into a stereotaxic frame (Kopf Instruments, Tujunga, CA, United States). A microdialysis probe (polyacrylonitrile membrane; Brainlink, Groningen, The Netherlands) with a 4 mm exposed surface was implanted into the PFC (Coordinates for the Tip of the Probe: AP = +3.4 Mm (to Bregma), Lateral -0.8 Mm (to Midline), Ventral -5.0 Mm (to Dura), the incisor bar was set at -3.3 mm²³ and the vHipp (Coordinates for the Tip of the Probe: AP = -5.3 mm to Bregma, Lateral +4.8 Mm to Midline, Ventral -8.0 Mm (to Dura), the Incisor Bar Was Set at -3.3 Mm²³ and were subsequently attached to the skull with two stainless steel screws and dental cement. Experiments were performed 24-48 h after probe implantation.

On the day of the experiment, animals were placed individually into microdialysis cages and the microdialysis probes were connected to microperfusion pumps (Harvard Apparatus; Holliston, MA, United States) via a flexible PEEK tubing (PK005-020; Western Analytical Products Inc., St. Boise, ID, United States) and perfused with artificial CSF containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl₂, and 1.2 mM MgCl₂ in H₂O at a flow rate of 1.5 μL/min. After a minimum of 2 h of prestabilization, microdialysis sample collection started. Samples were collected into mini-vials (4 001 029; Microbiotech/se AB, Stockholm, Sweden) containing 10 μL 20 mM formic acid +0.04% ascorbic acid in ultrapure water using an automated fraction collector (UV 8301501, TSE; Univentor; Zejtun, Malta). Following basal sampling period, animals were randomly allocated for acute sc treatment with either ADX71743 (100 mg/kg; n = 6) or vehicle (CD; n = 5) administered at 2 mL/kg volume. Following treatment, the microdialysates were collected for 180 mins. At the end of the experiment, brains were collected and sectioned by hand to confirm the location of the probe in the targeted anatomical region and to check for possible bleeding around the probe. At the end of this assessment, none were excluded from the analysis. Quantification of neurotrasmitters in microdialysates was performed according to standard methods described previously.^21,22

Handling stress exposure

Sixty minutes after compound administration, animals were exposed to mild handling stress for 10 min.^24–26 Specifically, rats were removed from the microdialysis cage and held in the hand for 10 min using latex gloves. The rats were mildly immobilized for the first couple of minutes, as they struggled to escape. The rats remained calm for the rest of the handling period, with mild immobilization if they tried to escape.

Statistical analysis

The data obtained from the sleep–wake experiment were generated using a two-period cross-over design with repeated measurements within each test period and were analyzed using three-way repeated measures mixed model approach, with treatment factor Treatment, blocking factor Rat, and repeated factor Time.

The responses from each neurotransmitter/brain region were analyzed using a two-way repeated measures mixed model approach. This was followed by pairwise planned comparisons of the treatment back to control at each timepoint and is not adjusted for multiplicity. The response was Log₁₀ transformed prior to analysis to stabilize the variance.

The repeated measures mixed model analysis is using the compound symmetric covariance structure to model the within-subject correlations.²⁷ The analysis was performed with the InVivoStat software.²⁸ The level of statistical significance was set at P < .05 for all tests.

Results

Sleep–wake EEG/EMG/telemetry study

ADX71743 produced time-dependent increases in time awake and CWS that were similar in magnitude and duration across dose groups (Figure 1A-C). Specifically, ADX71743 resulted in 30% and 100% (both P < .01) increases in the total AUC of CWT during 0-1 h and 1-2 h, respectively, while having no effect at later time-points (Figure 1C).

Figure 1.

Effects of acute ADX71743 administration on sleep–wake regulation in rats. Percent time awake (A), cumulative wake surplus (B), cumulative wake (C), percent time in NREM sleep (D), cumulative NREM sleep surplus (E), cumulative NREM sleep (AUC; F), NREM sleep onset latency (min; G), percent time in REM sleep (H), cumulative REM sleep surplus (I), cumulative REM sleep (AUC; J), and REM sleep onset latency (min; K) in Sprague–Dawley rats at baseline and after acute subcutaneous administration of ADX71743 (50, 100, 150 mg/kg) or vehicle depicted in 30 min bins (A, B, D, E, H, I) or cumulative AUC for 0-1 h, 1-2 h, 2-4 h and 4-8 h (C, F, J). Horizontal dashed line on panels B, E, and I represents the vehicle group. Gray shaded area represents the dark phase of the light–dark 12:12 cycle. Each point represents the observed mean (±SEM). ^*P < .05, ^** P < .01, ^***P < .001 compared with corresponding vehicle-treated group.

ADX71743 produced time-dependent reductions in NREM sleep and cumulative NREM sleep surplus that were similar in magnitude and duration across dose groups (Figure 1D, E). Specifically, 75% and 40% (both P < .01) reductions in the total AUC of cumulative NREM sleep were observed during 0-1 h and 1-2 h, respectively, while no effect was seen at later time-points (Figure 1F). Also, ADX71743-treated animals exhibited >100% increases (all P < .01) in NREM sleep onset latencies compared to controls (Figure 1G).

ADX71743 produced time-dependent reduction in REM sleep and cumulative REM sleep surplus in comparison to vehicle treatment (Figure 1H-J). In fact, REM sleep was fully eliminated 0-1 h, while being suppressed by 50% (P < .05) 1-2 h and unchanged at later time-points (Figure 1J). Also, ADX71743-treated animals exhibited >100% increases in REM sleep onset latencies compared to controls (Figure 1K).

ADX71743 modulated the spectral power across several frequency bands (Figure 2). Specifically, ADX71743 attenuated marked (-1.5 log EEG) and transient reductions in delta band (0.5-3.0 Hz) observed in vehicle-treated animals 60-90 min following dosing (Figure 2A). Consequently, delta band activity was significantly higher in ADX71743-treated animals than in controls 90 min post-dosing (Figure 2A). In addition, marked and transient reductions across theta (3.5-6.5 Hz, 7.0-9.0 Hz) and beta (9.5-12 Hz, 12.5-15 Hz, 15.5-20 Hz) bands observed in vehicle-treated animals were prolonged in animals treated with ADX71743 (Figure 2 B-G). As seen with other sleep readouts, these changes were similar in magnitude across dose groups and, except for delta band, were the most pronounced 30 min following dosing (Figure 2).

Figure 2.

Effects of acute ADX71743 administration on relative spectral power changes in rats. Frequency bands at 0.5-3 Hz (A), 3.5-6.5 Hz (B), 7.0-9.0 Hz (C), 9.5-12 Hz (D), 12.5-15 Hz (E), 15.5-20 Hz (F), and 0.5-20 Hz (G) in Sprague–Dawley rats at baseline and after receiving acute subcutaneous administration of ADX71743 (50, 100, 150 mg/kg) or vehicle. Gray shaded area represents the dark phase of the light–dark 12:12 cycle. Each point represents the observed mean (±SEM). ^$P = .06, ^*P < .05, ^**P < .01, ^***P < .001 compared with corresponding vehicle-treated group.

ADX71743 resulted in 30% increases (P < .05 at 50 mg/kg) in EMG during 0-1 h and only as a trend during 1-2 h, but not at later time-points (Figure 3A, B). However, there was no effect of treatment on the EMG intensity over time or on cumulative values (Figure 3C, D). ADX71743 had no effect on either MA (Figure 3E, F) or on BT (Figure 3G, H) throughout the recording period.

Figure 3.

Effects of acute ADX71743 administration on EMG, motor activity and body temperature (BT) in rats. EMG activity (A), cumulative EMG activity (B), EMG intensity (C), cumulative EMG intensity (D), motor activity (E), cumulative motor activity (F), BT (°C; G) and cumulative BT (°C; H) in Sprague–Dawley rats at baseline and after acute subcutaneous administration of ADX71743 (50, 100, 150 mg) or vehicle depicted in 30-min bins (A, C, E, G) or total areas under the curve (AUC) for 0-1 h, 1-2 h, 2-4 h and 4-8 h (B, D, F, H). Gray shaded area represents the dark phase of the light–dark 12:12 cycle. Each point represents the observed mean (±SEM). ^*P < .05 compared with corresponding vehicle-treated group.

Plasma concentration of ADX71743 was high, but lacked dose-proportionality as it ranged between 4848 and 6453 ng/mL, and between 847 and 1294 ng/mL, 1 h and 3 h post-treatment, respectively, while being negligible 12 h post-treatment (Table 1). The calculated CSF concentrations (based on CSF/plasma ratio of 5.3%; see¹² were between 257 and 342 ng/mL and 45 and 69 ng/mL, 1 h and 3 h post-treatment, while being negligible 12 h post-treatment (Table 1). The calculated CSF/in vitro EC₅₀ ratios were between 11 and 14 and between 2 and 3, 1 and 3 h after dosing, respectively, while being <1 12 h after dosing (Table 1).

Table 1.

Measured concentration of ADX71743 in plasma and calculated concentrations of ADX71743 in CSF of satellite rats treated with ADX71743. CSF concentrations were calculated based on CSF/plasma of 5.3% (see Text). Adult Sprague–Dawley rats, used as satellites in the sleep–wake EEG study, received ADX71743 (50, 100, 150 mg/kg) and were sampled for plasma 1, 3, and 12 h following treatment (n = 4/dose).

Pretreatment time (h)	Route	Dose (mg/kg)	N	Plasma exposure (ng/mL)	Plasma exposure (nM)	CSF exposure (ng/mL)	CSF exposure (nM)	CSF/EC50 (in vitro)
1	sc	50	4	4848	17 986	257	961	11
	sc	100	4	5885	21 833	312	1167	13
	sc	150	4	6453	23 940	342	1279	14
3	sc	50	4	847	3142	45	168	1.9
	sc	100	4	1183	4389	63	236	2.7
	sc	150	4	1294	4801	69	258	2.9
12	sc	50	4	8.7	32	1.7	6.4	0.1
	sc	100	4	142	527	7.5	28.1	0.3
	sc	150	4	234	868	12	44.9	0.5

In vivo microdialysis

Glutamate

In the PFC, there was no significant effect of Treatment and no significant Treatment x Time interaction on Glu concentrations, whereas effect of Time was significant [F (9, 78) = 4.32; P < .001]. In the vHipp, we did not see any significant effects, including those of Treatment, Time and Treatment × Time interaction (Figure 4B). We detected transient increases following dosing (PFC) or both dosing and stress (vHipp) in one out of five animals (Figure 4A, B). Also, the reduction in concentrations of Glu on both PFC and vHipp detected near the end of sampling was negligible (Figure 4A, B).

Figure 4.

Effects of acute ADX71743 administration on the basal and handling stress-induced concentrations of neurotransmitters in dialysates from the prefrontal cortex (PFC) and ventral hippocampus (vHipp) in freely moving rats. The extracellular levels of glutamate (Glu; A, B), γ-aminobutyric acid (GABA; C, D), and glycine (Gly; E, F) in the PFC (A, C, E) and vHipp (B, D, F) were determined in Sprague–Dawley rats receiving acute, subcutaneous administration of 100 mg/kg ADX71743 or vehicle 60 min after beginning of probe collections (n = 5-6/group). Sixty minutes after treatment, animals received handling stress for 10 min (shaded area). All values are calculated as a percentage of the basal values within the same group (100%). Each point represents the observed mean (± SEM). ⁺P < .05, ⁺⁺P < .01, vs the basal levels in ADX-treated animals. ^#P < .05, ^##P < .01, ^###P < .001 vs the basal levels in vehicle-treated animals. ^*P < .05, ^**P < .01 vs the vehicle-treated group.

GABA

In the PFC, there were no statistical effects on GABA concentrations, including those of Treatment, Time, and Treatment × Time interaction (Figure 4C). In the vHipp, while there was no significant effect of Treatment or Time on GABA concentrations, the Treatment × Time interaction reached statistical significance [F(9, 79) = 4.00; P < .001]. In the vHipp, the extracellular concentration of GABA showed gradual and consistent reductions that were observed only in vehicle-treated controls and not in ADX71743-treated animals (Figure 4C, D). While seen only as a trend in PFC, reductions in GABA concentrations in the vHipp were more pronounced, reaching the maximal effect of -68 ± 14.6% of baseline (32% reduction; P < .001) near the end of sampling (Figure 4D). In contrast, animals treated with ADX71743 showed either a mild and transient increase (121 ± 13.4% of baseline; P < .05) following handling (PFC; Figure 4C) or no change (vHipp; Figure 4D). Consequently, GABA concentrations in ADX71743-treated animals following handling were higher than those in controls significantly in the vHipp (Figure 4D) and as a trend in the PFC (Figure 4C).

Glycine

In the PFC and vHipp, there were no changes in concentrations of Gly either after treatment or after handling (Figure 4E, F).

5-HT

In the PFC, while there were no effects of Treatment and no Treatment × Time interaction on the concentration of 5-HT, the effect of Time reached statistical significance [F(9,77) = 2.23; P < .05]. Specifically, in vehicle-treated animals, 5-HT concentrations increased, reaching 136 ± 18.3% and 133 ± 10.1% of baseline (both P < .05) following dosing and handling, respectively (Figure 5A). In ADX71743-treated animals, increases in 5-HT reached 141 ± 10.6% and 158 ± 24.0% of baseline (both P < .05) following dosing and handling, respectively (Figure 5A).

Figure 5.

Effects of acute ADX71743 administration on the basal and handling stress-induced concentrations of neurotransmitters in dialysates from the prefrontal cortex (PFC) and ventral hippocampus (vHipp) in freely moving rats. The extracellular levels of serotonin (5-hydroxytriptamine, 5-HT; A, B), norepinephrine (NE; C, D) and dopamine (DA; E, F) in the PFC (A, C, E) and vHipp (B, D, F) were determined in Sprague–Dawley rats receiving acute, subcutaneous administration of 100 mg/kg ADX71743 or vehicle 60 min after beginning of probe collections (n = 5-6/group). Sixty minutes after treatment, animals received handling stress for 10 min (shaded area). All values are calculated as a percentage of the basal values within the same group (100%). Each point represents the observed mean (± SEM). ⁺P < .05, ⁺⁺P < .01, vs the basal levels in ADX-treated animals. ^#P < .05, ^##P < .01, ^###P < .001 vs the basal levels in vehicle-treated animals. ^*P < .05, ^**P < .01 vs the vehicle-treated group.

In the vHipp, while there was no effect of Treatment on the concentration of 5-HT, the effect of Time was statistically significant [F(9, 77) = 2.92; P < .01] and Treatment × Time interaction was present as a trend [F(9, 77) = 1.75; P = .09]. Specifically, in vehicle-treated animals we observed increases in 5-HT concentrations to 174 ± 67.6% of baseline (P < .01) that were followed by a rapid decline to baseline levels 60 min later (Figure 5B). In ADX71743-treated animals, however, the maximum increase to 167 ± 19.9% of baseline (P < .001) observed 40 min after dosing was maintained until nearly the end of sampling (Figure 5B). Subsequently, the 5-HT concentrations were markedly and significantly higher in ADX71743-treated animals than in controls 40-160 min following dosing (P < .01 most of the timepoints; Figure 5B).

Norepinephrine

In the PFC, there was no significant effect of Treatment and no significant Treatment × Time interaction on the concentration of NE, while the effect of Time was statistically significant [F(9, 79) = 6.54; P < .0001]. Specifically, we observed increases in intracellular concentrations of NE that were virtually identical in ADX71743- and vehicle-treated animals, reaching approximately 140% and >160% of baseline (both P < .001) following dosing and handling, respectively, in both groups (Figure 5C). Thereafter, the concentrations of NE declined rapidly in both groups and by the end of sampling period reached the baseline (Figure 5C).

In the vHipp, while there was no effect of Treatment on the concentration of NE, the effect of Time was statistically significant [F(9, 77) = 21.77; P < .0001], and Treatment × Time interaction was present as a trend [F(9, 79) = 1.73; P = .09]. We observed dosing-induced increases in DA concentrations that were similar in both groups (~150%, both P < .001), while following handling, the maximum increase reached 165 ± 12.7% of baseline in control animals and 203 ± 11.1% (both P < .001) of baseline in ADX71743-treated animals (Figure 5D). Thereafter, the concentration of NE declined rapidly 40 min following handling, with more gradual decline to the baseline levels thereafter (Figure 5D).

Dopamine

In the PFC, there was no significant effect of Treatment and no significant Treatment × Time interaction on the concentration of DA, while the effect of Time was statistically significant [F(9, 68) = 6.65; P < .0001]. There were increases in the concentration of DA as they reached 137 ± 10.5% and 201 ± 33.6% (both P < .001) of baseline in vehicle-treated and ADX71743-treated animals, respectively, 40 min after dosing (Figure 5E). Similarly, handling-induced DA increases reached 203 ± 17.3% and 261 ± 61.9% (both P < .001) of baseline in vehicle-treated and ADX71743-treated animals, respectively (Figure 5E). The concentration of DA declined gradually thereafter, reaching the baseline by the end of sampling (Figure 5E).

In the vHipp, there was no significant effect of Treatment and no significant Treatment x Time interaction on the concentration of DA, while the effect of Time was statistically significant [F(9, 61) = 34.60; P < .0001]. Specifically, there were increases in DA concentration that reached almost 200% (both P < .001) of baseline in both groups 40 min after dosing (Figure 5F). Twenty min after handling stress, however, the maximum increase in DA reached 256 ± 28.6% and 307 ± 29.0% (both P < .001) of baseline in vehicle-treated and ADX71743-treated animals, respectively (Figure 5F). The concentrations gradually declined thereafter but remained elevated to approximately 150% of baseline in both groups at the end of the sampling period (Figure 5F).

Discussion

Here, for the first time, the role of mGlu₇ in sleep–wake regulation was investigated using a potent, selective, and brain-penetrant mGlu₇ NAM ADX71743, a pharmacological tool compound with well-characterized activity profiles in vitro and in vivo. ADX71743, administered during the light phase, caused rapid wake-promoting effects at the expense of sleep. Specifically, ADX71743-treated animals showed similar increases in percent time awake and CWS, in combination with reductions in both percentage and cumulative surplus measures of NREM and REM sleep. Also, ADX71743-treated animals showed significant, nearly 2-fold delays in the onset of NREM and REM sleep. The magnitude of wake-promoting effects of ADX71743 in rats was similar to those observed with caffeine,²⁹ while being milder than those of amphetamine or cocaine.¹⁷ Wake-promoting effects of ADX71743 were also free from locomotor stimulant effects of dopaminergic agents (see below).

The concentration of ADX71743 (50, 100, 150 mg/kg) measured in the plasma of satellite animals was high, but lacked dose-proportionality, most likely due to suboptimal solubility of the compound. Near-identical in vivo effects of the three doses of ADX71743 on sleep and waking, detected during the first 2 h after treatment, are likely to reflect insufficient separation in exposure. The pharmacodynamic effects of ADX71743 over time were aligned with the estimated CSF to in vitro EC₅₀ ratios that ranged between 11 and 14, 1 h post-dosing, between 2 and 3, 3 h post-dosing, and were <1, 12 h post-dosing.

The wake-promoting effects of the mGlu₇ NAM ADX71743 in rats are well-aligned with the sleep–wake profile of mGlu₇ knockout mice.⁸ As seen here, mGlu₇ knockout mice showed enhanced total time awake at the expense of NREM and REM sleep, accompanied by delays in the onset of REM sleep.⁸ Altogether, we hypothesize that activation of mGlu₇ with an allosteric agonist or a PAM would show sleep-promoting effects. We propose that the constitutive activity of mGlu₇ receptors located in the brain regions modulating sleep/waking reduces glutamate release from synaptic terminals, and therefore is promoting sleep, both NREM and REM, and reducing wake. Support for this hypothesis can be seen in sleep-promoting effects of lower doses (5 and 10 mg/kg) of mGlu₇ allosteric agonist AMN082 in rats.¹⁰ Even though a higher dose of AMN082, 20 mg/kg, showed the opposite effects on sleep and waking, this can be explained by potential internalization of the mGlu₇³⁰ upon activation by AMN082, resulting in functional antagonism, thus mimicking the effects of a NAM such as ADX71743. However, given AMN082’s “rich” pharmacology, these and other effects of AMN082, previously attributed to the allosteric agonist effect on mGlu₇, could be linked to its activity on multiple off-targets for this compound.³¹

We saw signs that ADX71743 modulated low-power EEG in rats, as seen in profiles linked to increased arousal.³² Specifically, in comparison to vehicle treatment, ADX71743 administration attenuated reductions in delta band (0.5-3.0 Hz), while augmenting theta (3.5-9.0 Hz) and beta (9.5-20 Hz) bands. However, we need to keep in mind that, as the EEG data were not segregated by wake/NREM/REM before FFT analysis, we can’t exclude the possibility that the band power changes were driven by state mixing rather than within-state oscillatory modulation. Previous reports on the role of mGlu₇ on power bands provided conflicting evidence. On the one hand, mGlu₇ knockout mice were found not to differ from the wildtype controls in relative spectral power across main frequency bands.⁸ On the other hand, 2.5 mg/kg AMN082, which enhanced active waking, also increased EEG power in the 5-8 Hz range.¹¹ As the effect was identical both in mGlu₇ knockout and wildtype mice, it appears not to be mediated by mGlu₇ receptor. Additional studies using alternative mGlu₇ inhibitors as well as segregating the EEG data before FFT can shed further light on the role of the receptor on EEG power spectra and overall sleep–wake regulation.^8,11,32,33 The wake-promoting effects ADX71743 were not driven by caffeine-, nicotine-, or amphetamine-like psychostimulant properties of the compound, as ADX71743- and vehicle-treated animals did not differ in MA throughout the experiment, as seen previously.¹² Neither did ADX71743 change the BT in rats. A putative role of mGlu₇ in sleep is further supported by the anatomical distribution of the receptor, as the brain regions that show its highest concentration, the locus coeruleus, hippocampus, frontal and cingulate cortices,^34,35 among other functions, are involved in regulation of brain arousal and sleep–wake regulation.^36,37

Here, for the first time, we evaluated the effects of selective mGlu₇ inhibition on basal and mild stress-induced release of key neurotransmitters in the rodent brain using the in vivo microdialysis technique. Acutely, ADX71743 had no effect on basal concentrations of neurotransmitters, but markedly modulated the stress-induced alterations in the extracellular concentration of GABA and 5-HT in the vHipp and, as a trend, in the PFC. Neither treatment nor stress impacted the concentrations of Glu or Gly in the PFC or the vHipp. The lack of activity of ADX71743 on baseline extracellular concentrations of Glu in the PFC and vHipp can be interpreted as a sign of mGlu₇ quiescence, at least in these two anatomical regions, as proposed by others.^38–40 In contrast mGlu₇ allosteric agonist AMN082, administered systemically or locally, increased basal concentrations of Glu, while decreasing basal concentrations of GABA in the NAc.⁴¹ As the effects of AMN082 on glutamate and GABA were blocked by a group III mGlu antagonist MSOP, the effects of AMN082 in their study appear mGlu₇-specific. The effect of mGlu₇ activation on the extracellular levels of Glu appears to be anatomical region dependent. In the rat ex vivo slice preparation, AMN082 resulted in concentration-dependent inhibition of synaptic transmission evoked by 2 Hz stimulation, but not by 0.05 Hz stimulation.⁴⁰ The question of how selective inhibition of mGlu₇ alters stress-induced increases or reductions in extracellular Glu requires additional studies involving other types of stressors and/or other anatomical regions mediating stress effects of Glu, such as the ventral tegmental area or the NAc.

The concentrations of GABA in the PFC and vHipp exhibited gradual and sustained reductions of vehicle-treated animals, while remaining unchanged in ADX71743-treated animals throughout the sampling period. Consequently, the extracellular GABA concentrations were 13% (PFC) or 32% (vHipp) higher in ADX71743-treated animals than in controls. We can speculate that the reductions in GABA concentrations are stress-related; however, as a group of non-stressed animals was not included in the study, we can’t exclude the possibility that changes in GABA concentrations reflect post-surgical stabilization, sampling dynamics, or other processes. Reductions in hippocampal GABA levels have been observed in response to 15 mins of forced swimming in water at 25 °C.⁴² Activation of mGlu₇, on the other hand, appears to inhibit GABA release in certain regions. Specifically, systemic or local administration of AMN082 resulted in reduced GABA concentrations in the NAc.⁴¹ As a voltage-dependent Na blocker, TTX could not impact these changes; the effects of AMN082 on GABA appear to involve action potential-independent non-vesicular sources of GABA.⁴¹ Additional studies that combine assessment of stress reactivity with in vivo microdialysis are needed to better understand how mGlu₇ modulates GABA responses to stress. Modulation of stress-induced increases in 5-HT in the vHipp and PFC, to a lesser degree, was another robust effect of ADX71743 discovered in this study. Specifically, dosing-induced increase in 5-HT concentration to 160% of baseline in both groups rapidly declined to the baseline levels in control animals, while remaining elevated virtually until the end of sampling in ADX71743-treated animals. Subsequently, extracellular concentrations of 5-HT in the vHipp were between 100% and 150% higher in ADX71743-treated animals than in controls for up to 160 min following dosing. As discussed above for modulation of GABA responses, as we did not include groups of non-stressed animals in the current study, caution is needed in interpreting these data and additional experiments combining stress-reactivity and in vivo microdialysis are needed to better understand the role of mGlu₇ in modulation of 5-HT responses to stress. Increases in 5-HT concentration of similar magnitudes (160%) were also measured in the vHipp of rats exposed to the elevated plus maze, whereas no change in 5-HT release was seen in animals exposed to white noise in their home cage or those exposed to a maze with only closed arms.⁴³ The authors argued that 5-HT release in response to the elevated plus maze exposure was relevant to anxiety-like conditions rather than behavioral arousal.⁴³

The magnitudes of maximal increases in DA and NE following dosing or handling stress are well aligned with the pattern of DA and NE releases in the PFC following handling reported by others.⁴⁴ While we did not observe statistically-significant difference between the ADX71743-treated and vehicle-treated groups, there was a trend of stress-stimulated concentrations of DA and NE being higher in ADX71743-treated animals than in controls in the vHipp. AMN082, administered systemically or locally in the NAc, failed to alter basal concentrations of DA, while reducing concentrations of GABA and increasing concentrations of Glu.⁴¹ The lack of effects of AMN082 on DA concentration is in conflict with the evidence that activation of group III mGlu by L-AP4 inhibited basal DA release in the NAc⁴⁵ and dorsal striatum.⁴⁶ Alternative pharmacological tool compounds, both potentiators and inhibitors, with high selectivity for mGlu₇, administered in a range of doses, are needed to further investigate the role of the receptor in stress-induced releases of DA and NE.

A limitation of the current study is that we assessed only higher doses of ADX71743 (50-150 mg/kg) and did not include lower doses. As a result, we could not define a dose range producing submaximal or no effects, nor relate any effects to plasma exposure to inform PK/PD evaluation. This information is particularly relevant given reports that lower doses of ADX71743 (2.5, 5, 10 mg/kg), administered via intraperitoneal route, were efficacious in several in vivo assays relevant to schizophrenia in mice and rats.⁴⁷ However, those findings should be interpreted with caution because (1) plasma concentrations associated with efficacy were not measured in each experiment and (2) plasma concentrations extrapolated from the reported pharmacokinetic study are likely too low to support in vivo efficacy. A second limitation is the relatively high CD content used to formulate ADX71743. Although there is no evidence that CD affects sleep–wake activity in rodents, the 50% CD formulation may complicate translational interpretation of the data. Overall, further studies using additional selective, orally bioavailable mGlu₇ NAMs with alternative formulations are needed to advance our understanding of the role of mGlu₇ in sleep–wake regulation.

In conclusion, selective mGlu₇ NAM ADX71743 showed a clear effect on sleep–wake regulation, increasing time in wake, reducing time in NREM and REM sleep, and delaying their onset, without any confounding effects. While ADX71743 did not alter basal concentrations of neurotransmitters, it modulated stress-induced changes in the extracellular concentration of GABA and 5-HT in the vHipp. These effects may contribute to the efficacy of mGlu₇ NAMs in models of anxiety and stress.

Author contributions

Mikhail Kalinichev (Conceptualization [lead], Writing—original draft [lead], Writing—review & editing [lead]), John A. Gruner (Formal analysis [lead], Investigation [lead], Methodology [lead], Visualization [equal], Writing—original draft [supporting]), Gunnar Flik (Conceptualization [equal], Formal analysis [lead], Methodology [lead], Supervision [lead], Visualization [equal], Writing—original draft [supporting]), Mariette Heins (Data curation [supporting], Formal analysis [lead], Investigation [lead]), Hasnaa Haddouk (Formal analysis [equal], Methodology [equal], Validation [equal]), Isabelle Royer-Urios (Formal analysis [equal], Methodology [equal], Validation [equal]), Simon T. Bate (Data curation [lead], Formal analysis [lead], Methodology [equal]), and Sonia Poli (Conceptualization [supporting], Methodology [equal], Supervision [equal]), Robert Lütjens (Supervision [equal], Validation [equal], Writing—review & editing [equal])

Conflicts of interest

M.K., H.H., I.R.-U., S.P., and R.L. were employees of Addex Therapeutics at the time of this research. J.A.G. was the employee of Melior Discovery at the time of this research. S.T.B. was the employee of HLC at the time of this research. G.F. and M.H. were employees of Brain On-Line BV at the time of this research.

Funding

This work was funded by Addex Therapeutics.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.