Novel object recognition test as an alternative approach to assessing the pharmacological profile of sigma-1 receptor ligands

Background Although the terms “agonist” and “antagonist” have been used to classify sigma-1 receptor (σ1R) ligands, an unambiguous definition of the functional activity is often hard. In order to determine the pharmacological profile of σ1R ligands, the most common method is to assess their potency to alleviate opioid analgesia. It has been well established that σ1R agonists reduce opioid analgesic activity, while σ1R antagonists have been demonstrated to enhance opioid analgesia in different pain models. Methods In the present study, we evaluated the pharmacological profile of selected σ1R ligands using a novel object recognition (NOR) test, to see if any differences in cognitive functions between σ1R agonists and antagonists could be observed. We used the highly selective PRE-084 and S1RA as reference σ1R agonist and antagonist, respectively. Furthermore, compound KSK100 selected from our ligand library was also included in this study. KSK100 was previously characterized as a dual-targeting histamine H3/σ1R antagonist with antinociceptive and antiallodynic activity in vivo. Donepezil (acetylcholinesterase inhibitor and σ1R agonist) was used as a positive control drug. Results Both tested σ1R agonists (donepezil and PRE-084) improved learning in the NOR test, which was not observed with the σ1R antagonists S1RA and KSK100. Conclusions The nonlinear dose–response effect of PRE-084 in this assay does not justify its use for routine assessment of the functional activity of σ1R ligands. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s43440-023-00516-x.


Introduction
In describing sigma (σ) receptors, the challenge is not that so little is known about these enigmatic proteins [1], but rather that much of what we thought we knew has turned out to be wrong.Sigma receptors, initially described as a subtype of opioid receptors, are now considered unique receptors.Pharmacological studies have distinguished two types of σ receptors, termed σ 1 and σ 2 [2,3].The σ 1 R is an unusual and poorly understood target, although it is thought to act as an intracellular chaperone [4] and is implicated in learning and memory, depression, anxiety, and schizophrenia [2,5].Sigma-1 antagonists are also effective in neuropathic pain [5] and, whilst the mechanism of action is not well understood, several studies suggest σ 1 receptors regulate ion channel function (including NMDA/GluN receptors and K + /Ca 2+ channels) [2].Although the terms "agonist" and "antagonist" have been used to classify σ 1 receptor ligands, an unambiguous definition of the functional activity (agonist versus antagonist) is often hard, especially since σ 1 R ligands are often not selective.Unfortunately, a lack of canonical functional assays for sigma receptors makes classifying the pharmacology of sigma-targeting ligands challenging.
To date, some predictive approaches have been used to identify an agonist/antagonist profile of σ 1 R ligands, and the most common relate to their pain-related behavioral pharmacological evaluation.It has been well established that σ 1 R agonists (e.g.: pentazocine) reduce opioid analgesic activity, while σ 1 R antagonists (e.g.: S1RA, Fig. 1) have been demonstrated to enhance opioid analgesia in different pain models [6][7][8].Ligands that induce the same phenotype as pentazocine are commonly accepted as σ 1 R agonists, while compounds that exhibit the same effects as S1RA are considered as antagonists.Importantly, σ 1 R antagonists also exhibit antinociceptive activity independently of opioids, e.g. in capsaicin and formalin models [2,3].In order to unambiguously determine the functional profile of σ 1 R ligands, in vitro studies were also performed, including a well-established phenytoin-based functional assay [9].Previous studies have shown that phenytoin, a low-potent allosteric modulator for the σ 1 R, differentially modulates the affinity of σ 1 R ligands depending on their agonist versus antagonist functionality.Phenytoin potentiates the receptor binding affinity of σ 1 R agonists, however, it produces no effects or slightly reduced receptor binding affinity for σ 1 R antagonists [9].In fact, the results of phenytoin-based functional assay correlate with the in vivo activity of tested compounds, but the weak point is that the differences in binding affinities with the phenytoin are very small compared to the values obtained for compounds without an allosteric modulator [9].
On the other hand, several studies have demonstrated the efficacy of σ 1 R agonists in some neuropsychiatric and neurodegenerative disorders [10].For example, PRE-084 is a high affinity, selective σ 1 agonist (Fig. 1) that produces Fig. 1 Structure and biological activity of compounds used in this work [3,12,13] an anti-amnesic effect in vivo [11].Furthermore, donepezil (Fig. 1) has been reported to not only inhibit the acetylcholinesterase (AChE) but also activate σ 1 receptors, which is postulated to contribute to its overall neuroprotective and anti-amnesic effects [12].
Noteworthy, some of the pharmacological effects of old-generation σ 1 R ligands were mediated by a naloxonesensitive opioid receptor site, some by the phencyclidine (PCP) site within the NMDA receptor, and some by other, less well-defined mechanisms [1].Thus, one of the key requirements for progress in this field is the development of new compounds having improved affinity and selectivity for the haloperidol-sensitive σ 1 R binding site.
Considering the above, in the present study we decided to evaluate the pharmacological profile of selected σ 1 R ligands using a novel object recognition (NOR) test, to see if any differences in cognitive functions between σ 1 R agonists and antagonists could be observed.We used the highly selective PRE-084 and S1RA as reference σ 1 R agonist and antagonist, respectively.Furthermore, compound KSK100 selected from our ligand library (Fig. 1) was also included in this study.KSK100 was previously characterized as a dual-targeting histamine H 3 /σ 1 R antagonist with antinociceptive and antiallodynic activity in vivo [13].Donepezil (AchE inhibitor and σ 1 R agonist) was used as a positive control drug in the NOR test.

Novel object recognition test
The novel object recognition test is now among the most commonly used cognitive behavioral tests in rodents.A rat is presented with two similar objects during the first session, and then one of the two objects is replaced by a new object during a second session (Fig. 2).The amount of time taken to explore the new object provides an index of recognition memory [15].
Rats were transferred to the experimental room for acclimation about 1 h before the test began.Animals were placed in an open field arena made of a dull gray plastic (66 × 56 × 30 cm) and tested in a dimly lit (25 lx).After each measurement, rats were return to a home cage and apparatus was cleaned and dried.
The procedure consisted of habituation to the arena (without any objects) for 5 min, 24 h before the test, and a test session comprised of two 3-min trials separated by an intertrial interval of 24 h (ITI).During the first trial (familiarization, T1) two identical objects (A1 and A2) were presented in opposite corners, approximately 10 cm from the walls of the open field.In the second trial (recognition, T2) one of the objects was replaced by a novel one (A = familiar and B = novel).The objects used were the plastic bottles filled with sand and the glass beakers filled with gravel and.The objects were heavy enough not to be displaced by the animals and had a comparable height (~ 12 cm).The sequence of presentations and the location of the objects were randomly assigned to each rat and counterbalanced for each experimental group.The exploration was defined as a looking, licking, sniffing, or touching the object while sniffing, but not leaning against, standing, or sitting on the object.Any rat spending less than 5 s exploring the two objects within a 3 min trial was eliminated from the study.The exploration time and the distance traveled were measured Fig. 2 Experimental setup for the NOR task.The test takes place over 3 days.The first day is habituation (T0), in which a rat can explore the open field for 5 min.Day 2 is training (T1), in which the rat is allowed to explore the arena with 2 identical objects placed along the diagonal.Testing (T2) takes place 24 h after T1.Rats can explore the arena with one of the familiar objects and one novel object, placed along the diagonal using the Any-maze® video tracking system.Based on the exploration time (E) of two objects during T2, the discrimination index (DI) was calculated according to the formula: DI = (EB -EA)/(EA + EB).

Animals
90 Male Sprague-Dawley rats (Charles River, Germany) weighing ~ 250 g at the arrival were housed in the standard laboratory cages, under standard colony A/C controlled conditions: room temperature 21 ± 2 °C, humidity (40-50%), 12-h light/dark cycle (lights on: 06:00) with ad libitum access to food and water.Rats were acclimatized for at least 7 days before the start of the experimental procedure.During this week animals were handled at least 5 times.Behavioral testing was carried out during the light phase of the light/ dark cycle.Rats were randomly allocated to 9 experimental groups (each n = 10).Three rats from group KSK100 dose 1 and 3 mg/kg and PRE-084 0.3 mg/kg were excluded from the analysis due to exploration time < 5 s in trial T1.Once the experiment was completed rats were killed.All procedures and animals' maintenance were approved by the II Local Ethics Committee for Animal Experiments at the Maj Institute of Pharmacology, Polish Academy of Science, Kraków, Poland (ethical allowance 254/2022, 22-09-2022).All procedures followed the European Guidelines for animal welfare (2010/63/EU).

Drugs
Donepezil hydrocholoride (Sigma Aldrich, US) was administered at the dose of 1 mg/kg, S1RA hydrochloride (Biosynth, UK) was administered at the doses of 15 and 30 mg/ kg, PRE-084 hydrochloride (Angene, UK) was administered at the doses of 0.3 and 1 mg/kg.KSK100 was synthesized at the Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College (identity and purity were assessed by NMR and HPLC techniques, the purity was above 95%) and administered at the dose of 1, 3, and 10 mg/kg.All drugs were dissolved in the mixture of DMSO (5%) and Tween 80 (1%) in the 0.9% NaCl, which also served as a vehicle administered to controls.The doses of tested compounds were chosen based on our previous experiments on analgesic efficacy of sigma-1 receptor ligands [13].Unfortunately, studies addressing the effects of S1RA and PRE-084 on memory in rats are limited.The selection of S1RA doses of 15 and 30 mg/kg is supported by the report of Ruiz-Leyva et al. [16].The authors found that S1RA at neither the dose of 4, 16 nor 64 mg/kg affected Wistar rats' memory in NOR, although at the doses of 16 and 64 mg/kg it demonstrated a central activity manifesting in reduced binge alcohol drinking.In mice, PRE-084 alleviated ischemic memory impairments at the doses of 1 and 3 mg/kg (examined in NOR, Morris water-maze and Y maze [17]) and reduced MK-801-induced memory impairments at the doses 0.3 and 1 mg/kg (examined in Y-maze and passive avoidance test [18,19]), but not 0.1 or 3 mg/kg [20].Maurice reported that PRE-084 at the dose of 0.5 mg/ kg reduced age-related memory deficits in Morris watermaze test in Wistar rats [21].The positive control group was selected based on our earlier preliminary studies and literature reports [22,23] that found donepezil 1 mg/kg effective in NOR (ITI = 24h) in Sprague Dawley rats.All drugs were injected to rats ip 30 min before trial T1.

Statistical analysis
We used G*Power v. 3.1.9.6 for a priori effect and sample size analyses; a sufficient number of animals was estimated at least 7 per group (p = 0.05, Cohen's f = 0.8129).Due to the exclusion criterion (exploration < 5 s) 10 animals per group was assumed.For statistical evaluation, we employed one-way ANOVA followed by Dunnett's post hoc test.All datasets passed the assumption of parametric analysis, here examined by the test of Shapiro-Wilk test of normal data distribution and Levene's test for variances' equality.We used IBM SPSS Statistics v. 25 for statistical examination.

Results
One-way ANOVA demonstrated that the discrimination index differed across tested groups (F 8,78 = 2.292, p = 0.029).Dunnett's post hoc found that animals treated with donepezil 1 mg/kg and PRE-084 0.3 mg/kg presented a significantly increased discrimination index than controls (p < 0.05) (Fig. 3).Total exploration time during T1 was at a similar level for all tested groups (F 8,78 = 1.984; p = ns).However, KSK100 at the highest tested dose of 10 mg/kg reduced the rearing time (F 8,78 = 2.864, p = 0.008) and distance traveled (F 8,78 = 4.596, p < 0.0001) in T1 (i.e. 30 min after ip injection) as compared to control animals (p < 0.01).No significant differences in locomotor activity (rearing and distance) were found 24 h after injection in T2 (Fig. 3).Representative plots of animal head position occupancy and movement trajectories are shown in Fig. 4.

Discussion
Both tested σ 1 R agonists (donepezil and PRE-084) have been shown to improve learning and memory in the NOR test, which was not observed with the σ 1 R antagonists S1RA and KSK100.However, it should be emphasized that donepezil has a more complex mechanism of action than PRE-084 and was only used as a positive control in the NOR test.Nevertheless, compared to vehicle, administration of σ 1 R agonists significantly enhanced memory, however, the effect of PRE-084 was observed only at a lower dose.This compound was active in the NOR test at 0.3 mg/kg but completely lost activity at 1 mg/kg.Interestingly, PRE-084 has been used in animal models of several central nervous system (CNS) pathologies, particularly related to cognitive functions such as learning and memory, and administered in mice in a wide range of doses (from 0.1 to 64.0 mg/kg) [24].In fact, a biphasic nonlinear dose-response relation has been previously reported for cognitive enhancers such as AChE inhibitors [25,26], nootropics [27] and α7 nicotinic acetylcholine receptor modulators [28].This hormesis has been previously noted also for PRE-084 in mice [18,20] and most likely underlies our finding of the potential procognitive effect of this ligand at the dose of 0.3 mg/kg but not 1 mg/kg in rats.Moreover, it has been proposed that σ 1 R agonists fail to improve cognition at higher doses due to a non-selective σ 2 R activation or NMDA receptor modulation [29].However, with the high selectivity of PRE-084, we do not suspect an interaction with another biological target that could attenuate its pharmacological effect at a higher dose.
Since our study follows the EQIPD guidelines (a system that aims to optimize the robustness and reliability of preclinical biomedical research) and was carefully designed in this particular way, it is not possible to test a wider range of PRE-084 doses and compare the results of two separate experiments.Moreover, the doses, routes, and time of drug administration were selected based on our previous study [13] and literature data [16,17,19,30].In fact, they indicate doses of 0.3 and 1 mg/kg for PRE-084 as potentially effective in such an experiment [17,19,20].
In the present study, neither KSK100, donepezil, SR1A nor PRE-084 affected exploration time in NOR familiarization (T1) phase.However, one of the tested σ 1 R antagonists KSK100 at the dose of 10 mg/kg significantly affected the locomotor activity (rearing and distance) in T1.The high sedative activity is considered an undesirable property, which may lead to an incorrect or ambiguous interpretation of the in vivo results, and may limit the potential clinical application of new drug candidates.Interestingly, in our previous study, KSK100 significantly decreased spontaneous locomotor activity, but at a much higher dose of 30 mg/kg and in a different animal model [13].At doses of 5 and 15 mg/kg, we observed no significant effect on locomotor activity.In addition, KSK100 appears to be a potent H 3 R antagonist, which could be important due to the involvement of histaminergic system(s) in the pathophysiology of psychiatric disorders [31].For instance, the learning and memory-facilitating potential of plentiful H 3 R antagonists have been shown.However, in our study, KSK100 did not significantly influence object recognition.
In conclusion, despite differences in the effects on learning and memory between σ 1 R agonists and antagonists, the nonlinear dose-response effect of the reference compound PRE-084 does not justify the use of the NOR test for routine assessment of functional activity of σ 1 R ligands.It appears that the tests involving opioid-induced analgesia allow for a more precise assessment of their pharmacological profile and determination of whether they are σ 1 R agonists or antagonists.
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Fig. 3
Fig. 3 Effects of KSK100, donepezil, SR1A and PRE084 on object recognition (A), distance traveled (B), exploration time in familiarization (T1) phase (C) and rearing time (D) in the novel object recognition (NOR) test in rats.Drugs were administered ip 30 min before NOR familiarization trial.Data are expressed as mean ± SEM.Statistical analysis: Shapiro-Wilk test for normality, Levene's test for homogeneity of variance and 1-way ANOVA followed by Dunnett's