Sprague-Dawley female and male rats, 10.5 weeks old, weighing between 201 and 374 g (Charles River, Raleigh, North Carolina, United States) were employed in the study. Each animal was identified using a subcutaneously implanted electronic identification chip and randomly assigned to groups. Two to three animals were housed in solid-bottom cages with nonaromatic bedding. The housing was equipped with an automatic watering valve as specified in the USDA Animal Welfare Act (9 Code of Federal Regulations [CFR], Parts 1, 2 and 3) and as described in the Guide for the Care and Use of Laboratory Animals (National Research Council, Current edition). Each cage was clearly labeled with study, group, animal number, and sex. Target temperatures of 68°F–79°F with a target relative humidity of 30–70% were maintained. A 12-h light/12-h dark cycle was maintained. Block Lab Diet (Certified Rodent Diet #5002, PMI Nutrition International, Inc.) was provided ad libitum. Tap water was available ad libitum to each animal via an automatic watering system; the drinking water used was monitored for specified contaminants at periodic intervals. All procedures were conducted in accordance with the Institutional Animal Care and Use Committee and the Eighth Edition of the Guide for Care and Use of Laboratory Animals (National Research Council 2017).

As described in Figure 1 and Table 1, 90 male and 90 female Sprague-Dawley rats were subdivided into 12 groups of 15 rats each. This study was conducted in compliance with Good Laboratory Practice regulations at Charles River Laboratory (Raleigh, North Carolina, United States). Group 1 animals received vehicle control solution via oral gavage (0 mg/kg/day; n = 15 males). Groups 2, 3, and 4 animals received REL-1017 via oral gavage (31.25, 62.5, and 110 mg/kg/day, respectively; n = 15 males/group, total number of animals = 45 males). Group 5 animals received methadone racemate via oral gavage (31.25 mg/kg/day; n = 15 males). Group 6 animals were administered MK-801 subcutaneously (5 mg/kg/day; n = 15 males). Group 7 animals received the vehicle control via oral gavage (0 mg/kg/day; n = 15 females). Group 8, 9, and 10 animals received REL-1017 via oral gavage (20, 40, and 80 mg/kg/day, respectively; n = 15 females/group, total number of animals = 45 females). Group 11 animals were administered methadone racemate via oral gavage (20 mg/kg/day; n = 15 females). Group 12 animals were administered MK-801 subcutaneously (2 mg/kg/day; n = 15 females). Groups 1–5 (males) and 7–11 (females) animals were administered experimental articles (REL-1017 or methadone) or vehicle control solutions once a day for 4 days. These doses were selected based on previously conducted rat studies with REL-1017 that determined tolerability and showed that females had substantially higher systemic exposures at a given dose than males. The positive control, MK-801, was administered once on Day 1 via subcutaneous injection at a dose level of 5 (males) or 2 (females) mg/kg/day and at a dose volume of 1 ml/kg. The subcutaneous injections were administered between the skin and the underlying layers of tissue in the scapular region of the animal. Individual doses were based on the most recent body weights. Clinical evaluation was performed daily on all animals. Five animals/sex/group were euthanized on Day 1 (8 h post first dose), on Day 3 (24 h after the second dose), and on Day 5 (24 h after the fourth dose). The presence of neuronal vacuoles (Olney’s effect) was assessed by light microscopy in H&E stained brain 2 μm thick sections, with emphasis on the cingulate gyrus. Neuronal necrosis was determined by Fluoro Jade B staining. As an additional indication of drug-induced brain toxicity, microgliosis in the brain cortex was also assessed (see Figure 1 and Table 1).

All animals were evaluated daily throughout the study for clinical changes. Observations included evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, respiratory, and circulatory effects, autonomic effects such as salivation, nervous system effects including tremors, convulsions, reactivity to handling, and unusual behavior. Rat body weights were measured and recorded before the study (Day 1) and on the day of necropsy (Days 1, 3, and 5).

Animals were euthanized by euthanasia solution injection, followed by whole-body intravascular perfusion with 0.9% sodium chloride followed by 4% paraformaldehyde. For histochemical analysis, whole brains were sectioned into 2 μm slices. Brain tissue blocks were embedded in paraffin. Sections were obtained using a 2-micron block advance, slide mounted, and stained with hematoxylin and eosin (H&E). Serial sections were also stained with Fluoro Jade B (FJB), a sensitive and specific fluorescence staining for neuronal necrosis (BSENTR-150-FJB, VWR, Suwanee, GA 30024, United States). FJB staining was evaluated employing a fluorescence microscope with a FITC filter. We performed immunohistochemistry (IHC) analysis for ionic calcium-binding adaptor molecule 1 (IBA-1), a protein expressed in macrophages and microglial cells, which is upregulated when these cells are activated in pathological conditions, making this stain a sensitive indicator of microglial activation in the brain. For IHC, slices were incubated in 0.25%/0.5% Triton X-100, 2% bovine serum albumin (BSA) with a primary antibody (1:600) against rat IBA1 (Wako, Code No. 019-19741) for one hour at room temperature. Antigen detection was performed by polymer methods by using labeled polymer prepared by combining amino acid polymer with multiple molecules of alkaline phosphatase and goat anti-mouse Ig reduced to Fab’. Following incubation with DAB solution (10 mg of 3,3′-diaminobenzidine, tetrahydrochloride 50 ml of 0.05 M Tris-HCl buffer, 15 mM NaN₃ pH 7.6, 50 μL of 5% H₂O₂, 34 mg of imidazole in distilled water), counterstaining with hematoxylin to stain the nuclei was performed.

All stained brain tissue slices were independently assessed by Tox Path Services, LLC (Frederick, Maryland) neuropathologists. Quantification of the severity of morphological and IHC parameters was assessed by using the following 1 to 5 grade score. 1—Minimal severity grade. A microscopic change that barely exceeds normal limits or a normal condition. Typically, these are microscopic changes that are sporadic, focal, and of no biologic significance to the function or structure of the tissue. Microscopic changes graded as “1” are not readily apparent in the tissue. Generally, a minimal change affects less than 1% of the total tissue that might be possibly affected. 2—Mild severity grade. A microscopic change that is more readily apparent in the tissue than a minimal (Grade 1) change but is still unlikely to produce any structural or functional impairment. Typically, a grade of mild indicates the microscopic change was present in one or a few foci. Generally, a microscopic change graded as mild affects less than 5% of the total tissue that might be possibly affected. 3—Moderate severity grade. A microscopic change that is readily observed in the section, but still of limited severity and unlikely to have any biologically significant effect on the overall structure or function of the tissue. Moderate changes may be focal, multifocal, or even diffuse. 4—Marked severity grade. A microscopic change that is a prominent, conspicuous, and easily identified feature of the tissue, but is still not present at what would be considered to be the maximum possible effect. A marked change would reasonably be expected to have some effect on the structure and/or function of a tissue, although the corresponding functional change may or may not be apparent. 5—Severe severity grade. A microscopic change that is prominent, cconspicuous, and easily identified feature of the tissue, is present at a maximum severity (affects the majority of the tissue and/or is very pronounced in one or more foci), and/or is present at a severity that would be expected to have a prominent effect on the structure and/or function of a tissue, although the correlating functional change may or may not be apparent.

In particular, to evaluate the severity of the Olney effect, we used the following scoring:

Vacuolation
Severity Grade 1 (minimal)	One or a few foci (or affected cells) present
Severity Grade 2 (mild)	More than a few foci (or affected cells) present
Severity Grade 3 (moderate)	Focally extensive to more diffuse area/groups of cells affected
Severity Grade 4 (marked)	Prominent portion of the tissue and/or cell type affected
Severity Grade 5 (severe)	Greater than 40% of the region/cells affected

To evaluate the severity of the neuronal necrosis, we used the following scoring:

Necrosis, Neuronal
Severity Grade 1 (minimal)	<1% of neurons in a tissue/region affected
Severity Grade 2 (mild)	1–5% of neurons in a tissue/region affected
Severity Grade 3 (moderate)	5–15% of neurons in a tissue/region affected
Severity Grade 4 (marked)	15–40% of neurons in a tissue/region affected
Severity Grade 5 (severe)	>40% of neurons in a tissue/region affected

To evaluate the severity of the microgliosis, we used the following scoring:

Migrogliosis
Severity Grade 1 (minimal)	Microgliosis barely exceeds normal limits; it is typically focal and/or sporadic and of no biological relevance; it is a common change in control animals, especially monkeys
Severity Grade 2 (mild)	Microgliosis more readily apparent than minimal severity grade, typically focal or multifocal; typically a level of severity uncommonly encountered in controls
Severity Grade 3 (moderate)	Microgliosis readily apparent; typically focally extensive or multifocal and typically a level of severity uncommonly encountered in controls
Severity Grade 4 (marked)	Microgliosis pronounced, affecting a prominent portion of a given brain/spinal cord region, typically at a level of severity not encountered in controls
Severity Grade 5 (severe)	Microgliosis very pronounced, affecting a prominent portion of a given brain/spinal cord region, typically at a level of severity not encountered in controls

Statistical analysis was performed with GraphPad Prism 8 software (GraphPad Software, Inc. La Jolla, CA, United States). Data are shown as the mean ± standard deviation (SD) or standard error of the mean (SEM) of the number of measures as indicated. To assess the drug effect at different time-points, the different scores were compared using the two-way analysis of variance (ANOVA), corrected by the Tukey test. p ≤ 0.05 was considered statistically significant; p > 0.05 was considered not statistically significant (ns).

The behavioral effects of MK-801 treatment include a rapid onset of ataxia, which is followed by recumbency (Clineschmidt et al., 1982). In this protocol, clinical evaluation was done daily. In male (5 mg/kg/day) and female (2 mg/kg/day) mice treated with MK-801, we observed clinical findings related to decreased activity (92.5%), impaired limb function (66.7%), ventral and/or lateral recumbency (63.4%), and ataxia (57.5%). None of these clinical parameters were neither statistically nor meaningfully altered in REL-1017 at any dose (low, medium, and high) in either male or females (Table 2a, 2b).

We also assessed whether REL-1017 affected the body weight of treated animals. As shown in Table 3, a slight decreased in body weight was observed in animals receiving the positive control (MK-801) at 5 and 2 mg/kg/day and in females receiving REL-1017.

In 1989, the neuropathological changes consisting of the formation of multiple vacuoles of heterogeneous size occupying the cytoplasmic compartment (vacuolization of neuronal cytoplasm) in the posterior cingulate and retrosplenial neocortices following acute exposure to NMDAR channel blockers were described (Olney et al., 1989). In the current study, five animals/sex/treatment were sacrificed eight hours following administration of vehicle, or REL-1017 at different doses: low (20–31.25 mg/kg/day, female and male, respectively), medium (40–62.5 mg/kg/day female and male, respectively), or high (80–110 kg/mg, female and male, respectively), or racemic methadone at 31.25 mg/kg/day, or MK-801 at 2–5 mg/kg female and male respectively. Rat brains were analyzed for the presence of neuronal vacuoles (Olney’s effect) by light microscopy on H&E stained brain sections. As shown in Figure 2, Olney lesions, indicative of NMDA receptor antagonist neurotoxicity, were present only in the positive control group MK-801 (6/10, 60% animals). We did not observe Olney lesions in the vehicle, REL-1017 at any dose, or racemic methadone (p = ns between REL-1017 at any dose and the vehicle; p < 0.001 between MK-801 and all other experimental groups). We scored Onley lesions according to their severity, (see M&M), and found that MK-801 produced mild/moderate (grade 2–3) vacuolation severity with foci of vacuolated neurons forming more diffuse groups of affected cells.

It has been previously shown that a high dose (2–5 mg/kg) of MK-801 induces neuronal necrosis in the posterior cingulate/retrosplenial cortex of adult rats (Fix et al., 1993). We assessed whether administration of the new NMDAR channel blocker REL-1017 at different doses could cause any neuronal death. We first analyzed brain sections stained by H&E at Day 1, 3, and 5 after different doses administrations. As previously shown, we also observed in MK-801 treated animals the presence of necrotic neurons in the posterior cingulate/retrosplenial cortex, appearing as shrunken, angular cells with pyknotic nuclei and bright eosinophilic cytoplasm (Figure 3A, black asterisk).

Importantly, as shown in Figures 3A, B, no signs of neuronal necrosis were found in the brains of animals treated with vehicle, REL-1017 at any dose, and racemic methadone (p = ns). To further confirm and extend these morphological data, we employed Fluoro-Jade B, which is a specific and sensitive fluorescence staining for necrotic neurons. This anionic fluorescein derivative increases the signal to background ratio, thus improving both the contrast and the resolution of the staining of neurons undergoing degeneration. Fluoro-Jade B stained necrotic neurons were detected in the posterior cingulate/retrosplenial cortex of animals treated with MK-801 (white asterisks, Figure 3A). In contrast, no Fluoro-Jade B positive necrotic neurons were detected in any animal from the control, REL-1017, or methadone groups (p = ns between REL-1017 at any doses and vehicle). Quantification of the degree and severity of necrosis found in the posterior cingulate/retrosplenial cortex indicated that all MK-801 treated animals had necrosis (100%, p < 0.0001 versus all other groups on both Day 3 and 5; Figure 3B). The mean severity of the necrosis of each sample was assessed by scoring the degree of necrosis and was moderate to marked (ranging from 5 to 40% of necrotic cells) in the posterior cingulate/retrosplenial cortex of animals treated with MK-801.

Although less severely, NMDAR antagonists have also been shown to induce irreversible neuronal degeneration in other brain regions, including the olfactory bulb (Newcomer et al., 2000). Therefore, we assessed whether administration of the new NMDAR channel blocker REL-1017 at different doses caused any neuronal death in this region. We confirmed the presence of necrosis in the olfactory bulb neurons of 80% of the rats treated with MK-801. The severity of the neuronal necrosis in this region was scored as minimal (81%) or mild (19%) (Figure S1). Similar to vehicle, REL-1017 did not trigger any necrosis of the olfactory bulb neurons (p = ns between REL-1017 at any doses and vehicle).

We confirmed (Figure 4) the sexual dimorphism of neurotoxic MK-801 effects on neuronal necrosis previously demonstrated by others (Auer, 1996; D'Souza et al., 2002; Honack and Loscher, 1993; Hur et al., 1999). MK-801 triggered more severe neuronal necrosis in female rats (Figure 4, p < 0.001 at Day 3). Similarly to the vehicle, there were no lesions in REL-1017 groups of female and male rats (Figure 4).

Necrotic posterior cingulate/retrosplenial cortical neurons, which are present after 3–5 days following single doses of MK-801, have been shown to be associated with an inflammatory response involving microglia (Harry and Kraft, 2008). To further assess the effect of the NMDAR channel blocker REL-1017, we evaluated and quantified the presence of IBA1 positive microglia in the posterior cingulate/retrosplenial cortical region of treated rats. As expected following the increase in neuronal necrosis, we found a statistical increase of IBA positive microglia cells in the posterior cingulate/retrosplenial cortex after 3 and 5 days of MK-801 treatment (Figure 5A). As shown in Figure 5B, mild microgliosis was observed in 40 and 80% of brains treated with MK-801 on Day 3 and 5, respectively (p < 0.001 versus all other groups). Consistent with the absence of neuronal necrosis, no increases in microglia cell number were observed in animals treated with any dose of REL-1017 and racemic methadone (p = ns).