Modulating inhibitory response control through potentiation of GluN2D subunit-containing NMDA receptors

Journal Pre-proof Modulating inhibitory response control through potentiation of GluN2D subunitcontaining NMDA receptors Patrick M. Callahan, Alvin V. Terry, Jr., Frederick R. Nelson, Robert A. Volkmann, A.B. Vinod, Mohd Zannudin, Frank S. Menniti PII:

S0028-3908(20)30060-5

DOI:

https://doi.org/10.1016/j.neuropharm.2020.107994

Reference:

NP 107994

To appear in:

Neuropharmacology

Received Date: 10 August 2019 Revised Date:

16 January 2020

Accepted Date: 5 February 2020

Please cite this article as: Callahan, P.M., Terry Jr., , A.V., Nelson, F.R., Volkmann, R.A., Vinod, A.B., Zannudin, M., Menniti, F.S., Modulating inhibitory response control through potentiation of GluN2D subunit-containing NMDA receptors, Neuropharmacology (2020), doi: https://doi.org/10.1016/ j.neuropharm.2020.107994. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Author Credit Statement Patrick M. Callahan -- Data curation; Formal analysis; Investigation; Methodology; Validation; Visualization; Roles/Writing - original draft; Writing - review & editing Alvin V. Terry, Jr. -- Conceptualization; Data curation; Formal analysis; Investigation; Methodology ; Project administration; Supervision;Validation; Visualization; Roles/Writing original draft; Writing - review & editing Frederick R. Nelson -- Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision; Validation; Visualization; Roles/Writing - original draft Robert A. Volkmann -- Conceptualization; Formal analysis; Funding acquisition; Project administration; Supervision; Visualization; Roles/Writing - original draft AB Vinod -- Data curation; Formal analysis; Investigation; Methodology Mohd Zannudin -- Data curation; Formal analysis; Investigation; Methodology; Project administration; Supervision Frank S. Menniti -- Conceptualization; Formal analysis; Funding acquisition; Project administration; Supervision; Visualization; Roles/Writing - original draft; Writing - review & editing.

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Modulating inhibitory response control through potentiation of GluN2D subunitcontaining NMDA receptors Patrick M. Callahana,b*, Alvin V. Terry, Jr.a,b, Frederick R. Nelsonc, Robert A. Volkmannc, AB Vinodd, Mohd Zannudind, and Frank. S. Mennitie*

a

Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta GA, 30912, USA

b

Small Animal Behavior Core, Augusta University, Augusta GA, 30912, USA

c

BioPharmaWorks, LLC, Groton, CT 06340, USA

d

Jubilant Biosys Ltd, Yeshwantpur, Bangalore-560022, Karnataka, India

e

The George & Anne Ryan Institute for Neuroscience, University of Rhode Island, Kingston, RI 02881, USA

* Corresponding Author: Frank S. Menniti, Ph.D., University of Rhode Island, 130, Flagg Rd., Kingston, RI 02881, USA, email: [email protected], tel: 860-271-9706

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Highlights •

PTC-174 is a novel positive allosteric modulator of GluN2D subunit-containing NMDA receptors

•

PTC-174 reduced spontaneous locomotor activity in a novel open field and hyperactivity produced by the stimulants amphetamine or MK-801 in adult rats

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PTC-174 improved inhibitory response control without degrading choice accuracy in a 5choice serial reaction time task in adult rats

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These behavioral effects of PTC-174 may be accounted for by potentiation of GluN2D receptors expressed in the neurons of the indirect/hypredirect basal ganglia pathways

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Abstract NMDA receptors containing GluN2D subunits are expressed in the subthalamic nucleus and external globus pallidus, key nuclei of the indirect and hyperdirect pathways of the basal ganglia. This circuitry integrates cortical input with dopaminergic signaling to select advantageous behaviors among available choices. In the experiments described here, we characterized the effects of PTC-174, a novel positive allosteric modulator (PAM) of GluN2D subunit-containing NMDA receptors, on response control regulated by this circuitry. The indirect pathway suppresses less advantageous behavioral choices, a manifestation of which is suppression of locomotor activity in rats. Systemic administration of PTC-174 produced a dose-dependent reduction in activity in rats placed in a novel open field or administered the stimulants MK-801 or amphetamine. The hyperdirect pathway controls release of decisions from the basal ganglia to the cortex to optimize choice processing. Such response control was modeled in rats as premature responding in the 5-choice serial reaction time (5-CSRT) task. PTC-174 produced a dose-dependent reduction in premature responding in this task. These data suggest that potentiation of GluN2D receptor activity by PTC-174 facilitates the complex basal ganglia information processing that underlies response control. The behavioral effects occurred at estimated free PTC-174 brain concentrations predicted to induce 10-50% increases in GluN2D activity. The present findings suggest the potential of GluN2D PAMs to modulate basal ganglia function and to treat neurological disorders related to dysfunctional response control.

Keywords: NMDA receptors, GluN2D positive allosteric modulators, inhibitory response control, executive function, basal ganglia

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1. Introduction NMDA receptors mediate the slow component of glutamate ionotropic signaling as well as the activity dependent modulation of glutamate synaptic strength that is a molecular basis for learning and memory (Hansen et al., 2017). In the adult brain, NMDA receptors are tetramers composed primarily of 2 GluN1 subunits and 2 GluN2 subunits (Karakas and Furukawa, 2014; Lee et al., 2014). There are 4 GluN2 subunits, GluN2A-D, that are differentially deployed at both the circuit and synapse level to fine tune NMDA receptor signaling (Cull-Candy and Leszkiewicz, 2004; Paoletti et al., 2013; Glasgow et al., 2015). Pharmacological modulators have been essential in elucidating the diverse functions of NMDA receptors and such agents also have considerable therapeutic potential (Ogden and Traynelis, 2011; Monaghan et al., 2012). For example, CP-101,606, a GluN2B-selective negative allosteric modulator (NAM), has demonstrated clinical antidepressant efficacy as well as efficacy for reducing L-DOPA induced dyskinesias and neuropathic pain (Sang et al., 2003; Nutt et al., 2008; Preskorn et al., 2008). Furthermore, human genetic studies are revealing that variations in NMDA receptor subunit structure and/or expression are associated with neurodevelopmental and neuropsychiatric disease (XiangWei et al., 2018). These findings support a strategy of targeting NMDA receptor subtypes to develop new therapeutics. Since such drugs very likely will be administered systemically, their therapeutic benefits and side effect liabilities will result from the integration of their effects at sensitive NMDA receptors throughout the CNS.

Because of this, subtle differences in

pharmacology, even within a specific class, can have significant consequences with regard to both efficacy and therapeutic index. Thus, an iterative deductive/inductive process will be required to successfully map the new NMDA receptor modulators to therapeutic indications. In a companion article, we report the preliminary pharmacological characterization of PTC-174, an exemplar of a new class of NMDA receptor modulator (Yi et al., 2019). PTC-174 is a highly efficacious GluN2C/D positive allosteric modulator (PAM)1 with two functional modes of action. At maximal concentration, PTC-174 increases the activity of recombinant 1

Abbreviations: 5-CSRT- 5-choice serial reaction time, eGP- external globus pallidus, ITIinter-trial-interval, PAM- positive allosteric modulator, STN- subthalamic nucleus, vSDvariable stimulus duration

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GluN1/2C and GluN1/2D receptors more than 10-fold at saturating glutamate and glycine concentrations. The compound also increases by 2 to 4-fold the apparent affinity of these receptors for the co-agonists. In contrast, PTC-174 weakly potentiates recombinant GluN1/2B receptors and partially inhibits recombinant GluN1/2A receptors. In native systems, GluN2C and GluN2D are deployed primarily, if not exclusively, as triheteromeric GluN1/2A/2C (Bhattacharya et al., 2018) and GluN1/2B/2D receptors (see below). PTC-174 produces robust potentiation of recombinant GluN1/2B/2D receptors but is minimally effective at GluN1/2A/2C receptors.

Consistent with its activity at recombinant receptors, PTC-174 increased the

amplitude and/or slowed the decay of NMDA receptor responses in subthalamic nucleus (STN) neurons and hippocampal interneurons that express GluN2D, but had no effect on NMDA receptor responses of hippocampal pyramidal neurons, which express GluN2A and GluN2B. Because PTC-174 has relatively weak activity at GluN1/2A/2C receptors, the compound may be functionally selective for GluN2D containing receptors in vivo. Whereas GluN2D is broadly expressed early in development, this distribution becomes limited in the adult CNS (Monyer et al., 1994; Standaert et al., 1994; Yamasaki et al., 2014). Notably, GluN2D is expressed at several loci within the basal ganglia circuit, including the subthalamic nucleus (STN) and external globus pallidus (eGP) (Standaert et al., 1994), as well as by dopaminergic neurons of the substantia nigra (Jones and Gibb, 2005; Brothwell et al., 2008), and striatal cholinergic interneurons (Zhang et al., 2014; Zhang and Chergui, 2015). In addition, GluN2D is expressed in neurons of lamina 1 in spinal cord (Hildebrand et al., 2014), cerebellar golgi cells (Misra et al., 2000; Brickley et al., 2003), neurons in the hypothalamic supraoptic nucleus (Hagino et al., 2010), and GABAergic interneurons in the hippocampus (Thompson et al., 2002; von Engelhardt et al., 2015; Perszyk et al., 2016). Given the notable expression of GluN2D in the basal ganglia, we focused our first in vivo assessment of PTC-174 on behavioral models that probe the activity of this circuitry (Robbins, 2002; Freeze et al., 2013; Nelson and Kreitzer, 2014).

We observed that PTC-174 reduced locomotor activity in a novel environment and

reversed amphetamine-, and MK-801-induced hyperactivity. PTC-174 also reduced premature responses and timeout responses in the 5-choice serial reaction time (5-CSRT) task in rats. This pattern of results is consistent with an effect of PTC-174 to increase NMDA receptor signaling in

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indirect/hyperdirect pathways of the basal ganglia circuitry to enhance inhibitory response control. 2. Materials and Methods 2.1 Subjects For the behavioral studies, experimentally naive, 2 month old (locomotor activity studies) or 4 month old (5-CSRT study) male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc, Indianapolis, IN) were housed in pairs in polycarbonate cages (45 X 30 X 18 cm) with corncob bedding in a vivarium of constant temperature (21-23ºC) and humidity (40-50%). Lighting was maintained on a 12-hr light-dark cycle (7:00 a.m.-7:00 p.m.) with free access to water and food during the first week (see subsequent food restriction procedures below). All behavioral testing was performed during the light portion (9 a.m.-5 p.m.) of the light/dark cycle (Monday thru Friday). Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health) and the Institutional Animal Care and Use Committee at Augusta University approved all experimental protocols. The pharmacokinetic studies used 6-10 week old male Sprague Dawley rats purchased from Vivo Biotech Ltd (Hyderabad, India).

Animals were group housed in an AAALAC

accredited laboratory animal facility under standard laboratory conditions at temperature 22 ± 2°C, relative humidity 30-70%, under a 12 h light/dark cycle. Food (Altromin Spezialfutter GmbH & Co. KG, Lage, Germany) and water were provided ad libitum. Animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals (U.S. National Institutes of Health). All animal experimentation was approved by the Institutional Animal Ethics Committee (IAEC) under supervision of Committee for the Purpose of Control and Supervision of Experimentation on Animals (CPCSEA) of India. 2.2 Pharmacokinetic Studies Groups of three rats per time-point were given a subcutaneous (sc) dose of PTC-174 at 10 mg/kg at a concentration of 10 mg/ml and injected at 10 ml/kg. At intervals, rats were placed under isoflurane anesthesia and blood samples (1 ml) were collected by cardiac puncture at 0.25,

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0.50, 1, 2, 4, 8 or 24 hr following drug administration. Whole blood was collected in 0.7-ml heparinized Microtainer tubes and placed on ice. Subsequently, the whole brain was extracted, rinsed of excess blood with ice-cold saline, weighed and frozen on dry ice. Plasma was prepared by centrifugation and all pharmacokinetic samples were stored at -70°C until analysis. At the time of analysis, brain tissues were homogenized in 4 volumes (w/v) of 0.9% saline. Brain homogenate or plasma samples (50 µl) were added to 200 µl acetonitrile containing an internal standard in 96-well polypropylene plates. The acetonitrile mixtures were vortexed and then centrifuged at 1800g for 10 to 15 min. Aliquots of the supernatant were transferred to a 96-well plate and diluted with equal volume of water before analysis by high-performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS). PTC-174 and added internal standard were separated on a reverse-phase Atlantis dC18 XDB-C8 analytical column (4.6 mm x 15 cm, 3 µm particle size) using a discontinuous elution gradient of acetonitrile and 0.2% triflouroacetate in water. Analytes were quantified by mass spectrometric (Perkin-Elmer Sciex API-4000) under conditions of positive ionization using a heated nebulizer probe. 2.3 Locomotor Activity Procedure Rat open field activity monitors (43.2 X 43.2 cm, Med Associates St. Albans, VT) were used for locomotor activity experiments. The following behavioral parameters were recorded during the 2 hr test session:

horizontal activity (distance travelled (cm), vertical counts

(photobeam breaks) and number of stereotypical movements (repeated photobeam breaks). Rats (N=8/treatment group) were administered PTC-174 (15 or 30 mg/kg) or its vehicle (10% DMA, 90% PEG 200) in a dose volume of 3 ml/kg subcutaneously (sc) 30 min prior to placement in the open field, after which activity was monitored for 60 min. Animals were then briefly removed from the activity monitors and administered by intraperitoneal (ip) injection amphetamine (0.5 mg/kg), MK-801 (0.15 mg/kg) or saline (1ml/kg) and returned to the activity chambers for an additional 60 min. Note that PTC-174 was assumed to be maintained at relatively stable levels over the course of these activity measurements based on the pharmacokinetic exposure studies. 2.4 5-Choice Serial Reaction Time (5-CSRT) Procedure

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One week prior to 5-CSRT training and throughout testing rats were food restricted to approximately 85% of their age-dependent, free-feeding weights based upon Harlan Laboratories growth rate curves. Animals were trained in eight automated 5-CSRT operant chambers (Med controlled by MedPC software (Med Associates), as described previously (Terry Jr et al., 2014). Briefly, each operant chamber was equipped with 5 apertures containing a photocell beam to detect nose pokes and a lamp (2.8 W) that could be illuminated for varying durations. On the wall opposite to the nose poke apertures, food pellets (45 mg chow pellet, BioServ, Frenchtown, NJ, USA) were delivered to a magazine equipped with a light that turned on to indicate that a pellet had been dispensed. The house-light remained on for the entire session unless an error or omission occurred. Training sessions began with the delivery of a food pellet and retrieval triggered the first trial. After a 5 sec inter-trial-interval (ITI), a stimulus light within one of the five apertures was illuminated for a fixed duration (see below) and a single nose-poke into this opening during the signal illumination period or during the 5 sec limited hold period delivered a reward (correct response); a nose-poke into a non-illuminated aperture (incorrect response) resulted in a 5 sec time-out period and no food reward. Failure to respond within the 5 sec limited hold period (omission) also resulted in a time-out. Training began with the stimulus duration set at 10 sec, a limited hold period of 5 sec and ITI of 5 sec. The stimulus duration was gradually reduced (e.g., 5, 2.5, 2.0, 1.5, and 1.25 sec) while maintaining stable performance until a final duration of 1.0 sec was achieved. Sessions ended when 30 minutes had lapsed or 100 trials had been completed. Sixteen animals were trained 5 days per week until they reached stable performance criteria of 70-75% accuracy, <20% omissions and completion of all 100 trials for 5 consecutive days. Upon meeting the performance criteria animals were separated into 2 groups (N=8 per group). Group1 subjects were assessed on the variable stimulus duration (vSD; 0.5, 1.0 and 2.0 sec) test measure and Group2 subjects were assessed on the variable inter-trialinterval (vITI; 1.0, 5.0 and 10.0 sec) test measure. Test sessions were performed one to two times per week with maintenance training interspersed. A within subject’s design was used such that all animals received all treatments in a pseudorandom presentation. Performance parameters measured were: % correct ((# correct /(# correct + # incorrect))x100), premature responses (total # of nose-pokes into any aperture after trial initiation but before onset of the stimulus light), timeout responses (total # of nose pokes into any aperture during a timeout period), perseverative

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responses (total # of nose pokes occurring after the correct response had been made but before reward collection), omissions and total trials completed. Animals were administered PTC-174 (7.5, 15 and 30 mg/kg) or vehicle (10% DMA, 90% PEG200) subcutaneously (sc) in a volume of 3 ml/kg 60 min prior to test. 2.5 Drugs PTC-174

(8-(6-(tert-butyl)pyridin-3-yl)-6-oxo-3,4-dihydro-2H,6H-pyrimido[2,1-

b][1,3]thiazine-7-carbonitrile)

is

disclosed

in

patent

application

PCT/US2017/068135

(compound 174) and was synthesized by Chinglu Pharmaceutical Research (Newington, CT, USA). PTC-174 was administered in 10% DMA and 90% PEG 200 (vehicle) for the behavioral tests, whereas PTC-174 was administered in 5% DMSO, 5 % Cremophore and 90 % saline for the pharmacokinetic characterization. MK-801 and amphetamine were purchased from SigmaAldrich (St Louis MO) and prepared in physiological saline (0.9% NaCl). All test doses refer to the weight of the salt. 2.6 Data Analysis Data was analyzed using StatView 5.0. For the locomotor studies, data for the first 60 min, prior to administration of stimulants, and second 60 min, after administration of stimulants, were analyzed separately. To assess overall effects of PTC-174 dose (0, 15, or 30 mg/kg) on locomotor activity, distance travelled (cm), vertical counts and stereotypy counts were totaled over the 60 min test periods. These data were analyzed by one-way analysis of variance (ANOVA). In addition, distance travelled, vertical counts and stereotopy counts were summed in 5 min bins and analyzed by two-way (PTC-174 dose by time bin) repeated measures ANOVA. The Student Newman Keuls test was used when main effects were significant.

Results are expressed as means (± S.E.M.).

Differences between means from experimental groups were considered significant at the p<0.05 level. For the 5-CSRT task, data for the two cohorts tested under the vSD and vITI conditions were analyzed separately.

The primary measures of interest were Choice accuracy and

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Premature responses. Data were also collected and analyzed for numbers of Timeout responses, Magazine head entries, Perseverative responses, Trial omissions and Trials completed, as well as latencies to Correct or Incorrect responses and to retrieve Reward. To assess overall treatment effects, data on each measure were summed over the different stimulus durations (0.5, 1.0, and 2.0 sec) or intertrial intervals (2.5, 5.0 or 10.0 sec) and analyzed by one-way repeated measures ANOVA. For choice accuracy and premature responses, two-way repeated measures ANOVA were also performed for treatment (PTC-174 doses, 0, 7.5, 15, 30 mg/kg) by stimulus duration or treatment by intertrial interval. Post-hoc assessments were made using the Student Newman Keuls test when main effects were significant. Results are expressed as means (± S.E.M.). Differences between means from experimental groups were considered significant at the p<0.05 level.

3. Results 3.1 Pharmacokinetics of PTC-174 In vitro ADME studies suggested that PTC-174 has excellent cell permeability and no propensity for transporter-mediated brain efflux, which predicted the compound would gain access to the CNS following systemic administration. In vivo studies in rats were undertaken to confirm this prediction and to determine the exposure time course after systemic administration. Animals were administered PTC-174 at 10 mg/kg, sc in a vehicle of 0.5% DMSO, 5% cremophore and 95% saline and sacrificed 30 min later for determination of plasma and brain drug exposure. Free brain and plasma levels were calculated by correction for protein binding (plasma free fraction: 9.9%; brain free fraction: 3.1%). At 30 min, the free brain and free plasma levels were approximately 120 and 100 nM, respectively, for a free brain/free plasma concentration ratio of 1.2 (Table 1). To assess the time-course of drug exposure, animals were administered PTC-174 at 10 mg/kg and sacrificed at different times up to 8 hrs afterwards to obtain plasma samples. PTC-174 plasma exposures were highest at 15 min, the first time point examined, and then were maintained at approximately similar levels through 4 hrs (Fig. 1). Based on corrections for protein binding and brain/plasma ratio, we estimate that free brain

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levels of PTC-174 were maintained at 150-200 nM over 4 hrs. Extrapolating from potencies at recombinant GluN2D receptors (Yi et al., 2019), we estimate approximately 20-30% potentiation of GluN2D mediated responses in brain after systemic administration in the 10 mg/kg dose range, assuming agonist saturation. In other single time point studies, plasma exposures were dose proportional up to 30 mg/kg, thus, the behavioral effects of PTC-174 were examined at doses up to 30 mg/kg. 3.2 PTC-174 effects on locomotor activity We investigated the effects of PTC-174 (15 and 30 mg/kg) on locomotor activities in a novel environment in 2 cohorts of rats.

Subsequently, cohorts were administered either

amphetamine or MK-801 and the effects of PTC-174 were assessed on locomotor activity stimulated by these agents. 3.2.1 Locomotor activity in a novel open field Since prior to administration of stimulants, both cohorts of rats were treated identically, we combined the activity data collected during this epoch for the vehicle and PTC-174 dose groups. When placed in the novel open field, animals initially ambulated at a high rate that attenuated over the 60 min data collection period (Fig 2, bottom panels). PTC-174 treatment significantly altered the total horizontal distance travelled during the 60 min recording period [F(2,61)=8.16, p<0.001] (Fig. 2A). Post-hoc analyses indicated that both the 15 and 30 mg/kg test doses (N=16/dose) reduced activity relative to vehicle treated animals. Two way analysis of PTC-174 dose vs distance travelled binned at 5 min intervals (Fig. 2D) indicated a significant effect of time bin [F(11,22)=234.86, p<0.0001], consistent with activity attenuating over time. However, there was no PTC-174 dose by time interaction [F(22,671)=1.17, p>0.05]. PTC-174 treatment also affected the total vertical counts during the 60 min recording period [F(2,61)=16.64, p<0.0001] (Fig 2B). Post-hoc analysis indicated that PTC-174 at 15 mg/kg and 30 mg/kg significantly attenuated vertical counts relative to vehicle treated animals. Two-way analysis of PTC-174 dose vs vertical counts binned at 5 min intervals (Fig. 2E) also revealed a significant effect of time bin [F(11,22)=50.55, p<0.0001], accounted for by higher vertical

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counts early in the session, but there was no PTC-174 dose by time interaction [F(22,671)=1.09, p>0.05]. PTC-174 treatment also significantly affected total stereotypy counts [F(2,61)=6.87, p<0.01]. Post-hoc analysis indicated the 30 mg/kg dose reduced stereotypy counts relative to vehicle treated animals (Fig. 2C). Two-way analysis of PTC-174 dose on stereotopy counts binned at 5 min intervals revealed significant effect of time bin [F(11,22)=123.23, p<0.0001] and a significant dose by time interaction was observed [F(22,671)=1.81, p<0.01]. This appeared to due to a greater effect of PTC-174 to reduce stereoptopy counts at earlier time points compared to later time points (Fig 2F). 3.2.2 Amphetamine-stimulated locomotor activity After 60 min in the open field animals had become quiescent. Animals were briefly removed from the open field and those initially administered PTC-174 vehicle were then administered saline (N=8) or amphetamine (0.5 mg/kg, ip; N=8). Animals that had initially received 15 or 30 mg/kg PTC-174 received amphetamine (N=8/dose group). Animals were returned to the open field and data was collected for an additional 60 min (Fig 3, Table 2). Total horizontal distance travelled over the entire 60 min recording period was significantly altered by treatment [F(3,28)= 7.11, p<0.001] (Fig 3A). Post-hoc analyses indicated that in animals previously administered vehicle, amphetamine administration significantly (p<0.05) increased overall total horizontal distance travelled compared to animals that were again administered vehicle (Fig. 3A). Furthermore, prior administration of 15 or 30 mg/kg PTC-174 significantly reduced total horizontal distance traveled relative to that in animals that received amphetamine only (Fig 3A). Statistical analysis of the effects of treatment on distance travelled binned in 5 min intervals (Fig. 3A, bottom panel) indicated a significant effect of time [F(11,33)=5.75, p<0.0001], but no treatment by time interaction effect [F(33,308)=1.11, p>0.05]. Post-hoc analysis indicated that both doses of PTC-174 reduced the effect of amphetamine in several time bins (Fig. 3C).

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Similar patterns of results were observed for total vertical counts and stereotypy counts (Table 2). In general, amphetamine treatment increased both measures and treatment with either dose of PTC-174 attenuated these increases. 3.2.3 MK-801-stimulated locomotor activity The same paradigm described above was used to assess the effect of PTC-174 on MK-801induced locomotor activity. Animals initially administered PTC-174 vehicle were then administered saline (N=8) or MK-801 (0.15 mg/kg, i.p., N=8) and animals that had initially received 15 or 30 mg/kg PTC-174 received MK-801 (N=8/dose group). Total horizontal distance travelled was significantly altered by treatment [F(3,28)= 5.09, p<0.005]. Post-hoc analyses indicated that administration of MK-801, in comparison to saline, significantly increased (p<0.05) overall total horizontal distance travelled (Fig. 3B). PTC-174 significantly attenuated the MK-801-induced increase at both the 15 and 30 mg/kg test doses (Fig. 3B). Statistical analysis of distance travelled binned in 5 min intervals (Fig. 3D) also indicated a significant effect of time [F(11,33)=0.868, p>0.05] but no treatment by time interaction [F(33,308)=1.16, p>0.05]. Post-hoc analysis revealed that both doses of PTC-174 reduced the effect of MK-801 at several time intervals (Fig. 3D). Similar patterns of results were observed for total vertical counts and stereotypy counts. In general, MK-801 treatment increased both measures and treatment with either dose of PTC-174 attenuated these increases (Table 2).

3.3 PTC-174 effects on performance in the 5-CSRT task The effects of PTC-174 on performance in the 5-CSRT task were assessed in two cohorts of rats. In one cohort, performance was assessed under the condition of variable stimulus duration (vSD) to tax attention. In a second cohort, performance was assessed under condition of variable intertrial interval (ITI) to interrogate impulse control. The effects of PTC-174 were tested using a within subjects experimental design, with each animal receiving vehicle, 7.5, 15, and 30 mg/kg PTC-174 in random order.

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3.3.1 Variable stimulus duration (vSD) Rats were trained at a stimulus duration of 1.0 sec and under this condition had a choice accuracy of ~80% correct (open bar, Fig 4C). In vSD test sessions, stimulus duration was randomly varied at 0.5, 1.0, or 2.0 sec. As expected, in vehicle treated animals, choice accuracy decreased (from ~80% to ~60%) for trials with stimulus duration shorter than the training duration (0.5 sec, compare open bars in Fig 4B vs 4C). Administration of PTC-174 (7.5-30 mg/kg, sc) did not significantly alter % correct choices in comparison to vehicle treated animals. This was the case when % correct choices were collapsed over all stimulus durations (Fig 4A) or analyzed separately at each stimulus duration (Fig 4B-D). In the vSD paradigm, premature responses were low and did not systematically vary following vehicle administration (compare open bars in Fig 5B-D). Nonetheless, PTC-174 treatment significantly affected the total number of premature responses committed over the entire test (i.e., combining premature responses committed at each stimulus duration, [F(3,7)=9.54, p<0.001]).

Post-hoc analysis indicated that both the 15 and 30 mg/kg PTC-174 doses

significantly reduced premature responses relative to vehicle treated animals (Fig 5A). Two-way analysis of variance of premature responses associated with individual stimulus durations indicated no PTC-174 dose by stimulus duration interaction (Fig 5B-D). PTC-174 treatment had no statistically significant main effects on most of the other measures when data was collapsed over stimulus durations (Table 3). The exception was a small but statistically significant increase in Correct latency at the PTC-174 15 mg/kg dose relative to vehicle control. Nonetheless, there were notable trends. There was a trend for a PTC-174 dosedependent decrease in overall number of Timeout Responses. PTC-174 also tended to increase the number of Trial Omissions and decrease Trial Completions.

Latencies to Correct or

Incorrect responses, and the latency to Reward were lengthened by PTC-174 treatment. PTC174 had no systematic effects on the number of perseverative errors or magazine head entries. 3.3.2 Variable inter-trial-interval (vITI) Rats were trained with an ITI of 5.0 sec and under this condition had a choice accuracy of ~80% correct (open bar, Fig 6C). In vITI test sessions, ITI was randomly varied between 2.5, 5.0, or

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10.0 sec. Choice accuracy was not appreciably changed when ITI was varied (compared open bars in Figs 6B-D). PTC-174 had no effect on choice accuracy when data was collapsed over all trials (Fig 6A) or in trials with any particular ITI (Figs 6B-D). As expected, premature responses increased with increasing ITI duration (compare open bars across ITI durations in Fig. 7B-D). PTC-174 treatment significantly affected the total number of premature responses when data was collapsed over all ITI durations [F(3,7)=12.23, p<0.0001]. Post-hoc analysis indicated that both the 15 and 30 mg/kg doses of PTC-174 statistically significantly reduced premature responses relative to vehicle control treated animals (Fig 7A). Two-way analysis of PTC-174 dose vs ITI revealed a significant main effect of ITI [F(2,21)=65.75, p<0.0001], attributable the increase in premature responses as ITI increased (Fig 7B-D).

There was also a PTC-174 dose by ITI interaction [F(6,63)=7.02, p<0.0001]. The

interaction appears to be due to significant effects of PTC-174 at the 5 and 10 sec ITI durations (Fig 7C & 7D) but not at the 2.5 sec ITI duration (Fig 7B). The lack of statistical effects at the short ITI may have been due to the very low rates of premature responses under this condition. There was also a statistically significant [F(3,7)=7.67, p<0.001] effect of PTC-174 dose on overall number of Timeout Responses collapsed across the different ITIs (Table 4). This effect appeared PTC-174 dose dependent and the reduction in Timeout Responses was statistically significance at the 30 mg/kg dose in post-hoc analysis. PTC-174 treatment had a number of other effects that reached statistical significance (Table 3). There was a statistically significant main effect of PTC-174 dose [F(3,7)=3.24, p<0.05] on Trial omissions, with post-hoc analysis showing a significant increase at the 15 mg/kg dose. Significant effects of PTC-174 dose were also observed for total magazine head entries [F(3,7)=7.07, p<0.001]. This was accounted for in post-hoc analysis by a decrease in head entries at the 30 mg/kg dose. There were also main effects of PTC-174 dose on both Correct latency and Reward latency, accounted for by increases in latencies at the PTC-174 30 mg/kg dose. The 30 mg/kg dose of PTC-174 also decreased the total number of completed trials. There was no trend for a PTC-174 dose effect on incorrect latency and perseverative errors (p>0.05).

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4. Discussion We have discovered a new class of functional GluN2D PAMs exemplified by PTC-174 (Yi et al., 2019). GluN2D is notably expressed in the adult brain within the basal ganglia, among other circuitry. Thus, we focused our first assessments of systemic administration of PTC-174 on behaviors regulated by basal ganglia information processing. We found that PTC-174 caused a dose dependent reduction in locomotor activity when rats were introduced into a novel open field and when locomotor activity was stimulated by administration of amphetamine or MK-801. In the 5-CSRT task, PTC-174 dose dependently reduced premature responding and time out responding, but had no effect on choice accuracy. We conducted preliminary pharmacokinetic/exposure studies with PTC-174 to estimate the level of putative GluN2D receptor potentiation associated with the behavioral effects we observed. PTC-174 readily crosses the blood brain barrier and exposure is proportional at the doses used, allowing for extrapolation of free brain concentrations from plasma concentrations.

Such

extrapolation yielded an estimate of PTC-174 free brain concentrations of approximately 100 to 500 nM over the dose range employed.

Based on the concentration response data for

recombinant receptors expressed in oocytes (Yi et al., 2019), these concentrations would result in approximately 10 to 50 % increases in activity of GluN2D receptors.

Thus, while high

concentrations of PTC-174 are capable of potentiating GluN2D activities as much as 1000%, it appears that relatively low concentrations and correspondingly low levels of potentiation are sufficient for meaningful behavioral modulation. This is similar to what has been observed for AMPA receptor potentiation (Shaffer et al., 2013; Ranganathan et al., 2017), suggesting this may be a common theme for ionotropic glutamate receptor PAMs. Important experiments to test this idea further will be to measure the effects of PTC-174 in vivo on activity at synapses where GluN2D-subunit containing NMDA receptors are expressed. That the behavioral effects of PTC-174 result from potentiation of GluN2D receptors is consistent with other data in the literature. The effects of PTC-174 on stimulated locomotor activity are similar to those reported for CIQ, an exemplar of another structurally and functionally distinct class of GluN2C/D PAMs (Mullasseril et al., 2010; Santangelo Freel et al., 2013).

Similar to PTC-174, CIQ attenuated hyperactivity produced by MK-801 and by

17

methamphetamine (Suryavanshi et al., 2014). The effects of PTC-174 and CIQ are interesting to consider with respect to phenotypes resulting from genetic deletion of GluN2D in mice. Similar to the PAMs, deleting the GluN2D gene blocked the effect of either acute or repeated intermittent administration of the NMDA receptor channel blocker phencyclidine (PCP) to increase locomotor activity (Hagino et al., 2010). While the similarity of effects of the PAMs and gene deletion may seem incongruous, it may be rationalized if GluN1/2B/2D triheteromeric receptors, which are likely the predominant GluN2D receptors in brain, are replaced by higher conductance GluN1/2B diheteromeric receptors. Thus, the PAMs or the exclusive deployment of GluN1/2B may yield a net increase in NMDA receptor activity that may then result in similar behavioral effects. The behavioral effects of PTC-174 observed in the present studies suggest a first working hypothesis that potentiation of GluN2D subunit containing NMDA receptors biases basal ganglia information processing towards the indirect pathway and facilitates the complex basal ganglia information processing related to response control via the hyperdirect pathway. The basal ganglia are interconnected subcortical nuclei that integrate widespread cortical input with dopaminergic signaling to sort the most advantageous behaviors among the multiple available choices (Alexander and Crutcher, 1990; Stephenson-Jones et al., 2011). This computation is accomplished within three information streams, the direct, indirect, and hyperdirect pathways. GluN2D is conspicuously expressed in the STN and eGP, which are key nuclei of the indirect pathway. Pharmacological treatments such as dopamine D2 receptor inhibitors or pathological conditions such as in Parkinson’s disease that bias toward indirect pathway activity suppress behavior (Nelson and Kreitzer, 2014). In rodents, a simple manifestation of the indirect pathway subroutine is suppression of locomotor activity (Freeze et al., 2013). We observed that PTC-174 attenuated locomotor activity in rats exposed to a novel open field or administered the locomotor stimulants amphetamine or MK-801, consistent with an effect of the compound to potentiate indirect pathway activity. The hyperdirect pathway, an excitatory glutamatergic input from frontal cortical regions into the STN, exerts control over the release of choice decisions processed through the direct and indirect pathways (Nambu et al., 2002; Aron and Poldrack, 2006; Frank, 2006). By delaying release, the hyperdirect pathway promotes more thorough assessment of behavioral choices and, consequently, better outcomes. Given the particularly

18

prominent expression of GluN2D in STN, we also investigated the effect of PTC-174 in a task designed to examine the effects of pharmacological agents on response control, the 5-CSRT task (Robbins, 2002). The principal measure of response control on which we focused was premature responses. As predicted, premature responses increased with increasing ITIs, validating the sensitivity of our assay to assess response control (Terry Jr et al., 2014). We found that PTC-174 produced a robust, dose-related reduction in premature responding. There was also a reduction in timeout responding, another measure that may also reflect response control. In contrast, there were no effects of PTC-174 on choice accuracy, indicating the compound did not degrade attention. These results are consistent with an effect of PTC-174 to potentiate hyperdirect pathway activity to improve response control. The working hypothesis presented above is founded in the notable expression of GluN2D in STN and eGP. Nonetheless, this working hypothesis is likely an over-simplification for several reasons. GluN2D is expressed in other loci within the basal ganglia circuitry as well as outside this circuitry, including interneurons in hippocampal and neocortical circuits. For example, frontal cortical information processing plays a significant role in response control (Kim and Lee, 2011) and it will be of interest to investigate whether GluN2D receptors deployed within this circuitry contributes to the effects of PTC-174 on response control-related behaviors. Such studies may require discreet administration of GluN2D PAMs into different brain regions as opposed to systemic administration as was used here. Along the same line, caution is warranted in attributing effects of a complex pharmacological agent such as PTC-174 on a complex behavioral construct such as response control based on data in a single model, the 5-CSRT task. For example, Cope et al (2016) have presented evidence that rats bridge the ITI in the 5-CSRT task using a temporal strategy and that pharmacological agents may reduce premature responding by altering temporal perception as opposed to response control. Furthermore, the present studies do not inform as to whether motor slowing caused by PTC-174 may have influenced premature responding and time out responses. Motor slowing was clearly evidenced in the locomotor activity assays. However, motor slowing may also have contributed to a number of the subtle effects observed in the 5-CSRT task. These included slightly more trial omissions and less trial completions, as well as slight increase in response latencies. It will be of interest to investigate the behavioral effects of PTC-174 in other paradigms that interrogate response control, such as

19

behavioral assays that feature delayed reward (Dalley and Robbins, 2017). Such studies will be critical in assessing the therapeutic potential of GluN2D PAMs. 5. Conclusions PTC-174 was found to enhance measures of inhibitory response control in rats after systemic administration.

We hypothesize that potentiation of GluN2D subunit containing NMDA

receptors in STN and eGP plays a significant role in this behavioral response by biasing information processing towards the indirect and hyperdirect pathways within the basal ganglia circuitry. This hypothesis is consistent with the relatively high expression of GluN2D in these nuclei and with what would be predicted from increasing excitatory drive in these nuclei based on current basal ganglia models.

Although this working hypothesis is likely an over

simplification, it does provide a framework for further studies of the potential of GluN2D modulators to modulate basal ganglia function and to treat disorders related to dysfunction in this circuitry. It will be of particular interest in future studies to compare the effects of PTC-174 and CIQ. While both compounds potentiate GluN2D receptors, they differ in efficacy for GluN2C receptors and also diverge in modes of PAM activity, which may impact the degree to which these compounds potentiate synaptic versus extrasynaptic receptors (Yi et al., 2019). Comparative studies with these two compounds may shed light on the physiology of GluN2C and GluN2D subunit containing receptors and reveal unique therapeutic niches for these new classes of NMDA receptor modulators.

6. Declaration of Conflicting Interests Volkmann, and Menniti are co-inventors on patent applications that claim PTC-174. The intellectual property rights to PTC-174 are assigned to Cadent Pharmaceuticals and Volkmann and Menniti own stock in this company. All other authors have no commercial or financial relationships that could be construed as a potential conflict of interest. 7. Funding

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This work was funded by SBIR grants from the National Institutes of Health (1R43MH09846701 and 5R43MH098467-02) awarded to Chinglu Pharmaceutical Research LLC, Yuelian Xu Principal Investigator. Additional funding and resources to support this work were from Mnemosyne Pharmaceuticals, Inc., Luc Therapeutics, Inc. and Cadent Therapeutics, Inc. 8. Acknowledgements We thank Kollol Pal (Mnemosyne Pharmaceuticals) for grant preparation, Jinming Xiong (Chinglu Pharmaceutical Research) for contributing to compound synthesis.

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Figure Legends Figure 1. Pharmacokinetic profile of PTC-174. PTC-174 was administered at 10 mg/kg and concentration in plasma was quantified at intervals from 0.25 to 8 h afterwards (data for -.25 to 4 h are shown). Free brain concentrations (y-axis) were calculated from total plasma concentration by correcting for plasma and brain protein binding and the free brain/free plasma ratio (see Table 1). Each point represents a mean (± S.E.M) derived from 3 Sprague Dawley rats. At 300 nM (hash horizontal bar), PTC-174 potentiates the response of recombinant GluN1/2D receptors expressed in oocytes by approximately 35% (Yi et al., 2019). Figure 2. Effects of PTC-174 on open field activity in a novel environment. Rats were administered vehicle (N= 32) or PTC-174 15 and 30 mg/kg (N= 16/dose group) and then 30 min later introduced to a novel open field. Histographs (top) show (A) total distance travelled, (B) total vertical counts (photobeam breaks) and (C) total stereotypy counts (photobeam breaks) over the subsequent 60 min. Line graphs (bottom) show (D) distance travelled, (E) vertical counts and (F) stereotypy counts over the 60 min observation session binned in 5 min intervals. Data are expressed as the mean (± S.E.M.) values for each treatment. For Histographs * = significantly different from vehicle. For line graphs * = significantly different from PTC-174 15 mg/kg, whereas, # = significantly different from PTC-174 30 mg/kg. Statistical significance was considered as p<0.05. Figure 3 Effects of PTC-174 on amphetamine- or MK-801-induced hyperlocomotion. After 60 min in the open field (as described in Figure 2), animals received vehicle, amphetamine (0.5 mg/kg or MK-801 (0.5 mg/kg; N=8/treatment group). Histographs (top) show effects of PTC174 on total distance travelled for the 60 min after amphetamine (A) or MK-801 (B). Line graphs (bottom) show distance travelled over the 60 min test period binned in 5 min intervals. Data are expressed as the mean (± S.E.M.) values for each treatment. For Histographs * = significantly different from vehicle, whereas # = significantly different from either amphetamine or MK-801. For line graphs * = significantly different from vehicle, + = significantly different from PTC-174 15 mg/kg and # = significantly different from PTC-174 30 mg/kg. Statistical significance was considered as p<0.05.

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Figure 4 Effects of PTC-174 on choice accuracy in the 5-CSRT task under the variable stimulus duration (vSD) test condition. Stimulus duration was randomly varied at 0.5, 1 and 2 sec. (A) Choice accuracy when averaged across all stimulus durations. The insets B, C and D show the response accuracy at individual stimulus duration. Note that in vehicle treated animals (open bars), choice accuracy decreased to approximately 60% at the 0.5sec stimulus duration (compare B with C and D). Data are expressed as the mean (± S.E.M.) values for each treatment. * = significantly different (p<0.05) from vehicle. N=8 rats. Figure 5 Effects of PTC-174 on premature responses in the 5-CSRT task under the variable stimulus duration (vSD) test condition. Stimulus duration was randomly varied at 0.5, 1 and 2 sec. (A) Premature responses were averaged across all stimulus durations. The insets B, C and D show the number of premature responses at individual stimulus durations. When averaged across stimulus durations (A), PTC-174 caused a dose-dependent reduction in premature responses compared to vehicle control treated animals that was statistically significant at 15 and 30 mg/kg (*, p <0.05). Data are expressed as the mean (± S.E.M.) values for each treatment. * = significantly different (p<0.05) from vehicle control performance. N=8 rats. Figure 6 Effects of PTC-174 on choice accuracy in the 5-CSRT task under the variable inter-trial interval (vITI) test condition. The inter-trial interval was randomly varied at 2.5, 5 and 10 sec. (A) Choice accuracy when averaged across all ITIs. The insets B, C and D show choice accuracy at individual inter-trial intervals. There were no significantly differences (*, p<0.05) between vehicle control and PTC-174 treated subjects. Data are expressed as the mean (± S.E.M.) values for each treatment. N=8 rats. Figure 7 Effects of PTC-174 on premature responses in the 5-CSRT task under the variable inter-trial interval (vITI) test condition. The inter-trial interval was randomly varied at 2.5, 5 and 10 sec. (A) Premature responding when averaged across all ITIs. The insets B, C and D show premature responses at individual inter-trial intervals. When averaged across ITIs (A), PTC-174 caused a dose-dependent reduction in premature responses compared to vehicle control treated animals that was statistically significant at 15 and 30 mg/kg (*, p< 0.05). Data are expressed as the mean (± S.E.M.) values for each treatment. N=8 rats.

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Table 1. PTC-174 exposure in brain and plasma in rats. ______________________________________________________________________________ Drug Dose

Total Concentration Free Concentration Free Brain/Free Plasma Brain Plasma Brain Plasma Ratio (ng/g) (ng/ml) (nM) (nM) ______________________________________________________________________________ 10 mg/kg

1213 ± 148

317 ± 28

115 ± 14 96 ± 9

1.2

______________________________________________________________________________ PTC-174 was administered subcutaneously (sc) and 30 min later animals were sacrificed. Brain and plasma samples were then collected for determination of PTC-174 total brain and total plasma concentrations. Free brain and plasma levels were calculated by correcting for protein binding (plasma free fraction: 9.9%; brain free fraction: 3.1%). Data represents the mean (± S.E.M) from 3 animals.

1

Table 2. Effects of PTC-174 on locomotor activity stimulated by amphetamine or MK-801 in rats. _______________________________________________________________________________________ Treatment

PTC-174 Distance Vertical Stereotypy (mg/kg) Traveled (cm) Counts Counts _______________________________________________________________________________________ Amphetamine Study Vehicle + Vehicle

2574.5 ± 275.4

282.5 ± 42.3

6707.7 ± 816.4

Vehicle + Amphetamine

5932.1± 892.8*

356.6 ± 60.4

10223.0 ± 1707.2*

4012.9 ± 636.1**

151.0 ± 45.9**

8733.7 ± 1021.9

PTC-174 + Amphetamine

15.0

30.0 2659.7 ± 327.1** 117.5 ± 35.59** 7763.0 ± 543.4 _______________________________________________________________________________________ MK-801 Study Vehicle + Vehicle

1447.9 ± 255.5

101.0 ± 21.1

5623.6 ± 1641.3

Vehicle + MK-801

6790.8 ± 1755.8*

243.1 ± 43.7*

8592.1 ± 1373.8

15.0

3312.4± 772.9 **

185.2 ± 43.0

7315.8 ± 919.6

30.0

2373.6 ± 731.4**

101.5 ± 41.0**

5390.2 ± 793.1

PTC-174 + MK-801

______________________________________________________________________________________ Data represent the mean (± S.E.M.) values summed over the 60 min data recording period for each treatment (n = 8). In both studies, one-way analyses of variance revealed main effects of treatment for both Distance

2

Traveled and Vertical Counts. There was also a main effect of treatment for Stereotopy Counts in the Amphetamine study but not in the MK-801 study. Where main treatment effects were observed, post-hoc analyses were performed and significant distances are indicated as: *Amphetamine + Vehicle or MK-801 + Vehicle significantly different (p<0.05) from respective Vehicle + Vehicle groups. ** PTC-174 treated groups significantly (p<0.05) different from respective Amphetamine + Vehicle or MK-801 + Vehicle groups.

1

Table 3. Effects of PTC-174 on 5-CSRT task performance in rats _________________________________________________________________________________________________________ PTC-174 (mg/kg)

Timeout Responses

Magazine Head Entries

Perseverative Trial Correct Incorrect Reward Trials Responses Omissions Latency Latency Latency Completed (sec) (sec) (sec) _________________________________________________________________________________________________________ variable Stimulus Duration (vSD) 0.0

26.2 ± 7.6

271.8 ± 57.8

2.87 ± 1.1

9.62 ± 1.8

0.87 ± 0.03

1.95 ± 0.18

1.97 ± 0.23

98.5 ± 1.22

7.5

21.1 ± 4.8

267.1 ± 60.6

1.75 ± 0.79

13.6 ± 3.2

0.89 ± 0.06

2.12 ± 0.19

1.94 ± 0.22

98.7 ± 0.84

15.0

13.7± 3.8

182.7 ± 18.7

3.12 ± 0.66

13.2± 1.2

1.1 ± 0.10*

2.61 ± 0.42

1.72 ± 0.14

82.5 ± 11.3

30.0 12.8 ± 2.7 245.5 ± 82.2 2.80 ± 1.04 16.6 ± 2.9 1.0 ± 0.06 2.63 ± 0.13 2.67 ± 0.53 90.0 ± 4.05 _________________________________________________________________________________________________________ variable Inter-Trial Interval (vITI) 0.0

24.0 ± 5.1

374.6 ± 62.2 2.7 ± 0.97

16.6 ± 4.6

0.93 ± 0.06

2.50 ± 0.18

1.75 ± 0.23

99.9 ± 0.48

7.5

27.3 ± 6.0

336.8 ± 47.4 1.8 ± 0.54

21.8 ± 1.9

0.98 ± 0.06

2.5 ± 0.18

1.67 ± 0.06

99.9 ± 0.54

15.0

22.3 ± 5.1

312.5 ± 41.7 3.1 ± 0.61

24.8 ± 3.6*

1.0 ± 0.07

2.2 ± 0.13

2.11 ± 0.20

99.9 ± 0.48

30.0 9.5 ± 1.7* 245.0 ± 58.6* 3.8 ± 0.71 22.6 ± 3.3 1.1 ± 0.06* 2.4 ± 0.22 3.00 ± 0.62* 83.5 ± 8.12* __________________________________________________________________________________________________________ Data represent the mean (± S.E.M.) values for each treatment (n = 8) summed over the different Stimulus Durations or Intertrial Intervals. For both studies, one-way repeated measures analyses of variance to identify main effects of treatment. Where main treatment effects were observed, post-hoc analyses were performed and * denotes significant differences between vehicle treated animals (PTC-174 0.0 mg/kg) are PTC-174 dose groups (p<0.05).

Es/mated free brain conc. nM

Fig 1

Approximately 35% poten2a2on at 300 nM

300

200

100

0

Rat (n = 3) 10 mg/kg, s.c. B/P =1.2 0

1

2 Time, h

3

4

6000

*

4000

*

2000 0

2000

Veh

D

1600

Vehicle PTC-174 15 mg/kg PTC-174 30 mg/kg

*

400

#

*#

#

#

#

0

10

300

20 30 40 50 Time Elapsed (min)

60

*

200 100

100

1200 800

*

400

80

Veh

Stereotypy

12000

500

0

15 30 Dose (mg/kg)

C

Vertical Stereotypy Counts

600

B

10000

*

8000 6000 4000 2000 0

15 30 Dose (mg/kg)

Veh

15 30 Dose (mg/kg)

F

E Stereotypy Counts

Horizontal Vertical Counts

8000

A

Vertical Counts

Distance Traveled (cm)

Distance Traveled (cm)

Fig 2

#

60

* # * # # *# *# *

40

*# * #

#

20 0

10

20 30 40 50 Time Elapsed (min)

60

2000 1600

#

*#

1200 800

#

*#

400 0

10

20 30 40 50 Time Elapsed (min)

60

Fig 3 10000

Distance Traveled (cm)

B Amphetamine

8000

*

6000

#

4000

#

2000 0

Veh Veh 15 30 Dose (mg/kg)

Distance Traveled (cm)

C

MK-801

10000

*

8000 6000

#

4000

#

2000 0

Veh Veh 15 30 Dose (mg/kg)

D Vehicle AMP 0.5 mg/kg PTC-174 15 mg/kg + AMP PTC-174 30 mg/kg + AMP

1600 1200

*

800 400

* *

+ #

0

10

#

* * * #

*

#

20 30 40 50 Time Elapsed (min)

60

Distance Traveled (cm)

Distance Traveled (cm)

A

Vehicle MK801 0.15 mg/kg PTC-174 15 mg/kg + MK801 PTC-174 30 mg/kg + MK801

1600 1200

*

800 400 0

*

+ #

10

*

* #

*

+ + + #

20 30 40 50 Time Elapsed (min)

#

#

60

100

Fig 4

100

% Correct

90

A

VEHICLE PTC-174

B

Stimulus Duration 0.5 sec

80 70 60 50 0

90

0.0

7.5

15

30

80

100 90

% Correct

70 60

C

Stimulus Duration 1.0 sec

80 70 60 50

50 0

0

0.0

7.5

15

30

Dose (mg/kg)

0.0

7.5

15

30

100

Dose (mg/kg)

90

% Correct

% Correct

Dose (mg/kg)

D

Stimulus Duration 2.0 sec

80 70 60 50 0

0.0

7.5

15

Dose (mg/kg)

30

VEHICLE PTC-174

A

Premature Responses

20

8

B

16

4 2 0.0

7.5

15

30

8

*

4

*

Premature Responses

Dose (mg/kg)

12

10 8

C

0.0

7.5

15

Dose (mg/kg)

Stimulus Duration 1.0 sec

6 4

*

2 0

0

Stimulus Duration 0.5 sec

6

0

0.0

7.5

* 15

* 30

Dose (mg/kg)

30 Premature Responses

Premature Responses

Fig 5

10

10

D

Stimulus Duration 2.0 sec

8 6 4

*

2 0

0.0

7.5

15

Dose (mg/kg)

30

100

Fig 6

100

A

VEHICLE PTC-174

% Correct

90

Inter-trial Interval 2.5 sec

B

80 70 60 50

90

0

0.0

7.5

15

30

100

80 % Correct

90

70 60

C

Inter-trial Interval 5.0 sec

80 70 60 50 0

50 0

0.0

7.5

15

30

Dose (mg/kg)

0.0

7.5

15

Dose (mg/kg)

100

30

90

% Correct

% Correct

Dose (mg/kg)

D

Inter-trial Interval 10.0 sec

80 70 60 50 0

0.0

7.5

15

Dose (mg/kg)

30

A

VEHICLE PTC-174

Premature Responses

50

1.5

Inter-trial Interval 2.5 sec

B

1.0 0.5 0.0

0.0

40

*

20

*

10

Premature Responses

30

0

7.5

15

30

Dose (mg/kg) 4

C

Inter-trial Interval 5.0 sec

3 2 1

*

0

0.0

7.5

15

30

Dose (mg/kg)

0.0

7.5

15

30

Dose (mg/kg)

Premature Responses

Premature Responses

Fig 7

2.0

50 40

D

Inter-trial Interval 10.0 sec

30

*

20

*

10 0

0.0

7.5

15

Dose (mg/kg)

30