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. Author manuscript; available in PMC: 2022 Jun 8.
Published in final edited form as: Pharmacol Biochem Behav. 2021 Oct 25;211:173292. doi: 10.1016/j.pbb.2021.173292

Modulation of mGlu5 improves sensorimotor gating deficits in rats neonatally treated with quinpirole through changes in dopamine D2 signaling

Russell W Brown 1, Christopher G Varnum 1, Liza J Wills 1, Loren D Peeters 1, Justin T Gass 1
PMCID: PMC9176413  NIHMSID: NIHMS1799703  PMID: 34710401

Abstract

This study analyzed whether the positive allosteric modulator of metabotropic glutamate receptor type 5 (mGlu5) 3-Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) would alleviate deficits in prepulse inhibition (PPI) and affect dopamine (DA) D2 signaling in the dorsal striatum and prefrontal cortex (PFC) in the neonatal quinpirole (NQ) model of schizophrenia (SZ). Male and female Sprague-Dawley rats were neonatally treated with either saline (NS) or quinpirole HCL (1 mg/kg; NQ). a DAD2 receptor agonist from postnatal days (P) 1–21. Rats were raised to P44 and behaviorally tested on PPI from P44-P48. Before each trial, rats were subcutaneous (sc) administered saline or CDPPB (10 mg/kg or 30 mg/kg). On P50, rats were given a spontaneous locomotor activity test after CDPPB or saline administration. On P51, the dorsal striatum and PFC were evaluated for both arrestin-2 (βA-2) and phospho-AKT protein levels. NQ-treated rats demonstrated a significant deficit in PPI, which was alleviated to control levels by the 30 mg/kg dose of CDPPB. There were no significant effects of CDPPB on locomotor activity. NQ treatment increased βA-2 and decreased phospho-AKT in both the dorsal striatum and PFC. The 30 mg/kg dose of CDPPB significantly reversed changes in βA-2 in the dorsal striatum and PFC and phospho-AKT in the PFC equivalent to controls. Both doses of CDPPB produced a decrease of phospho-AKT in the PFC compared to controls. This study revealed that a mGlu5 positive allosteric modulator was effective to alleviate PPI deficits and striatal DAD2 signaling in the NQ model of SZ.

Keywords: Dopamine D2 receptor, Metabotropic glutamate receptor type 5 (mGlu5), prepulse inhibition, adolescence, Beta arrestin-2, Phospho-AKT

1. Introduction

There has been recent interest in targeting metabotropic glutamate receptors (mGlus) for the treatment of psychosis (Nicoletti et al, 2019). Type 5 metabotropic glutamatergic receptors (mGlu5) have been recognized as potential therapeutic targets for numerous disorders including depression, anxiety, chronic pain, Fragile X, and most relevant to this study, schizophrenia (SZ; Cleva & Olive, 2013). However, the functional knowledge of mGlus relative to psychosis and disorders that present with psychosis is sparse. Interestingly, Fuxe and colleagues (Borroto-Escuela et al 2016) discovered that the DAD2 receptor, which is known to have increased sensitivity in SZ (see Seeman, 2013 for review), forms a mutually inhibitory triple heteromer with the adenosine A(2A) and mGlu5 receptor such that A(2A) or mGlu5 stimulation decreases DAD2 receptor responding (Beggiato et al, 2016; Borroto-Escuela et al, 2016). It is also well-established that N-methyl D-asparate (NMDA) receptor hypofunction is important in the dysregulation of cellular communication that is known to exist in SZ. Interestingly, the mGlu5 colocalizes with the NMDA receptor, and activation of the mGlu5 increases NMDA receptor activity (Conn et al 2005). In addition, CDPPB has been shown to enhance extinction learning (Gass et al 2009, 2014a; 2014b) and facilitate cognition through its action on NMDA receptors which play a critical role in cognition (Geoffroy et al 2021). These cognitive effects, in addition to its indirect effects on DAD2 signaling, was the rationale for analyzing the effects of CDPPB on sensorimotor gating. Therefore, the mGlu5 receptor may be an especially attractive target relative to the treatment of psychosis, because activating this receptor can lead to normalizing DAD2 and NMDA receptor signaling in SZ.

Our laboratory established that neonatal treatment of the DAD2-like receptor agonist quinpirole (NQ) to rats induces an increase in DAD2 receptor sensitivity throughout the animal’s lifetime (Kostrzewa, 1995; Brown et al 2012) which has validity to SZ and a number of clinical conditions that often present with psychosis (Uchida et al, 2011; Grant et al 2014). In recent work, we have shown that the adenosine A(2A) agonist CGS 21680 was effective to alleviate PPI deficits in adolescent rats given NQ treatment (Brown et al 2020) and decrease the associative rewarding aspects of nicotine, although it was not as effective in female controls (Gill et al 2020). In addition, CGS 21680 reversed increases in cyclic-AMP response element binding protein (CREB) produced by NQ-treatment in the dorsal striatum. CREB protein in dopamine terminal areas of the brain has been shown to be related to both PPI performance and underlying neural plasticity mechanisms (Berger et al 2011). Consistent with past work (Janusz et al 1992), CGS 21680 resulted in locomotor depression, even when a relatively low dose (0.09 mg/kg) was used.

From a therapeutic perspective, traditional agonists that bind at the orthosteric site on a receptor are known to result in a number of side effects. Work has shown that positive allosteric modulation of the mGlu5 receptor is favored over traditional agonists as outlined in a review by Cleva and Olive (2013). Certainly, the hypoactive response produced by CGS 21680 in our past work would be consistent with this concern (Gill et al 2020). Regarding mGlu5 as a therapeutic target, stimulating the excitatory glutamatergic system includes increasing susceptibility to seizures and possible excitotoxic reactions to the drug. Positive allosteric modulation provides several advantages as opposed to direct mGlu5 agonists, including binding to a receptor site that is distinct from the orthosteric glutamate binding site, and increased receptor functioning in the presence of its endogenous ligand glutamate. In addition, positive allosteric modulation has high discrimination towards the mGlu5 receptor and does not result in rapid receptor desensitization (Cleva & Olive, 2013).

This study is focused on analyzing the effects of the mGlu5 positive allosteric modulator 3 Cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide (CDPPB) on auditory sensorimotor gating as measured by prepulse inhibition (PPI) in rats neonatally treated with the DAD2 agonist quinpirole. Sensorimotor gating deficits as measured by PPI is one of the most common behavioral hallmarks tested in individuals diagnosed with psychosis. Studies have shown that PPI deficits are related to increased DAD2 signaling in the brain (During et al 2014) and PPI has been used to screen antipsychotics in animal models (Powell et al 2009). In addition, based on the relationship between mGlu5 and DAD2 signaling. this study will analyze the effects of CDPPB on two proteins involved in G-protein independent signaling of the DAD2 receptor, beta arrestin-2 (βA-2) and phospho-AKT in both striatum and PFC. We have recently shown that NQ treatment results in increased βA-2 and decreased phospho-AKT in the dorsal striatum (Gill et al 2021), consistent with increased signaling at this receptor (Beaulieu et al 2007; 2008). Both proteins have been shown to be important in sensorimotor gating (Liu et al 2017; Aquino-Miranda et al 2019; Gill et al 2020; Mielnik et al 2021). This is the first study to analyze the effectiveness of a positive allosteric modulator of mGlu5 towards sensorimotor gating in a validated rodent model of psychosis.

2. Methods

2.1. Subjects

This study used a total of 68 Sprague-Dawley rats, 35 males and 33 females. All animals were the offspring of a total of 8 male-female pairs that were ordered from Envigo, Inc. (Indianapolis, IN) at 55–60 days of age. Only one male and one female were used per drug conditioned per litter to control for within litter variance. Similar to past work, the day of birth was referred to as postnatal day (P0). All the animals used in this study were housed and raised in an AAALAC-accredited climate-controlled vivarium on a cycle of 12-h on/off light dark and water and food was available ad libitum. All tests and procedures were approved by the East Tennessee State University Committee on Animal Care which is congruent the National Institutes of Health Guide for the Animal Care.

2.2. Drugs

Quinpirole HCl was ordered from Sigma-Aldrich, Inc. (St. Louis, MO, USA) and dissolved in a 0.9% NaCl at a dosage of 1mg/kg. CDPPB was ordered from EAG Laboratories (San Diego, CA, USA). CDPPB was dissolved in 10% Tween-80 from Sigma-Aldrich, Inc. mixed with 0.9% NaCl at two experimental dosages: 10 mg/kg and 30 mg/kg. Quinpirole was intraperitoneal (ip) administered following previous work (Brown et al 2012), and CDPPB was given through subcutaneous (sc) administration following past work by Gass et al. (2016).

2.3. Drug treatment and research design.

All animals were weaned at P21 from the female dam and socially housed (2–4 per cage) throughout the course of the experiment. In the statistical analysis of auditory sensorimotor gating measured by PPI, there were three between subjects factors: sex (male, female) neonatal drug treatment (NQ, NS), and adolescent drug treatment (10 mg/kg CDPPB, 30 mg/kg CDPPB, SAL). The within subjects repeated measure was the different levels of prepulse (73, 76, and 82 dB). An analysis of variance (ANOVA) was the primary statistic and a Newman-Keuls post hoc test was used to analyze any statistically significant interactions (p=.05). This study utilized the SPSS statistical program (Version 24 for Mac) for all statistical analyses. The codes for groups in all figures correspond to the neonatal drug treatment (NQ, NS) and adolescent drug treatment condition (10mg/kg = 10 mg CDPPB or 30 mg/kg = 30mg CDPPB, S = 0.9% NaCl).

2.4. Sensorimotor gating apparatus.

The Startle Monitor II apparatus and software was used (Kinder Scientific, Poway, CA) for the sensorimotor gating behavioral test. The animal was placed in a stainless-steel dome (8cm in height) that was attached to a platform (11 cm wide x 15 cm long) mounted to a stainless-steel ellipse which fit onto a metal platform that was wired into a computer interface. All movements were recorded by the software. The stainless-steel ellipse and platform were in a sound-attenuating chamber (28 cm high x 30 cm wide x 36 cm depth). The animal’s responses were recorded and measured in Newtons (N) within a 250-millisecond (ms) window following the stimulus presentation. All auditory stimuli were presented through a speaker located 18 cm above the stainless-steel dome. The chambers were calibrated following the manufacturer’s instructions provided with the equipment and calibration was conducted daily through the software.

2.5. Prepulse inhibition (PPI) methods and rationale for adolescence.

The prepulse inhibition (PPI) behavioral testing methods are based on previous work (Brown et al., 2020). The animals were tested for 5 consecutive days at the same time each day on PPI starting at day P44 and continued through P48. PPI was analyzed in adolescence. We chose to analyze the adolescent developmental period in part because SZ is defined as a neurodevelopmental disorder (Stachowiak et al 2013). In addition, it is well-established that PPI deficits are present in this period in psychosis (Van den Buuse et al., 2003). Finally, the adolescent period is critical for both dopaminergic (Gee et al 2018) and glutamatergic neurotransmitter system development in the brain (Ma et al 2019). Animals were sc administered saline, 10 mg/kg, or 30 mg/kg CDPPB 20 minutes prior to PPI testing. Once placed in the apparatus on each daily session, the animals were given a 5-min habituation period to acclimatize to the background noise (70 dB white noise). After this habituation period, animals were administered a no stimulus, prepulse, or pulse trial. A no stimulus trial was one in which no stimulus was given. A prepulse trial included an auditory stimulus that was 3, 6, or 12 dB above the 70 dB background noise (73, 76 or 82dB) followed by a 120 dB startle pulse 250 ms later. A pulse trial was a 120 dB startle pulse alone. Each animal underwent 60 randomized trials which included 10 no stimulus, 30 prepulse (10 trials of each dB level of prepulse), and 20 pulse trials. PPI was then calculated using the following equation: 100 – [(mean prepulse response / mean pulse response) x 100]. All measurements were recorded through the Kinder Scientific software (Poway, CA) and were averaged for the 5 days of testing.

2.6. Locomotor Activity Test.

Animals were given one day of saline or CDPPB treatment on P49 with no behavioral testing to allow for a day in between PPI and the locomotor activity test. On P50, all animals were administered an open field spontaneous locomotor activity behavioral test. All animals were administered the same drug as during PPI testing: saline, 10 mg/kg CDPPB, or 30 mg/kg CDPPB. Approximately 20 min after drug administration, the animal was placed into a square locomotor arena (91 cm/side). All movements were recorded using the AnyMaze software (Stoelting Co., Wood Dale, IL) with distance (m) as the dependent measure. All animals were tested 10 min.

2.7. Brain tissue harvest and homogenization.

On P51, 24 h following the locomotor activity test, brain tissue was harvested and flash frozen. The dorsal striatum and PFC (prelimbic and infralimbic cortices) were dissected away from the rest of the brain. This tissue was then homogenized using a Fisher Scientific (Atlanta, GA, USA) Dismembrator 5000 suspended in a RIPA buffer that contained both protease and phosphatase inhibitors (P0044, P5726, P8340, and phenylmethylsulfonyl fluoride; Sigma Aldrich, Inc., St. Louis, MO). Homogenized samples were analyzed in duplicate in both ELISA assays.

2.7.1. Rationale for brain regions chosen for analysis.

The rationale for analyzing DAD2 signaling in the dorsal striatum was due to its heavy dopaminergic innervation and its important role in sensorimotor gating as measured by PPI (Mohr et al 2007; Sotoyama et al 2007). In addition, DAD2 receptors in both the dorsal and ventral striatum have been shown to be important in sensorimotor gating (Shoemaker et al 2005; Ortega-Alvaro et al, 2011; Takahishi et al, 2006). There are equivocal data on whether the prelimbic and infralimbic cortices of the PFC are involved in PPI, and whether the disruption is related to DAD2 receptor signaling in this brain area (Osterbog et al., 2020). The prelimbic and infralimbic cortices do receive major dopaminergic projections from the ventral tegmental area which play a role in reward processes (Han et al., 2017; Vranjkovic et al., 2018), and PFC DAD2 receptors and their relationship to the glutamatergic system are known to be important in the expression of symptoms related to SZ and psychosis (Domino et al 2004; Borroto-Escuela et al 2016).

2.8. Beta Arrestin-2 (βA-2) ELISA.

The βA-2 ELISA kit was ordered from MyBioSource.com (San Diego, CA, USA CAT No. MBS917674) and the protocol was closely followed. In brief, standards were prepared according to the manufacturer and applied in duplicate to the 96 well plate, followed by 100 μl of each homogenized sample also added in duplicate. The plate was covered and incubated for 2 h at 37°C. The liquid was then removed from the plate, and 100 μl of Biotin-antibody was added to each well. The plate was covered and incubated for 1 h at 37°C. The plate was then aspirated and washed a total of three times using the wash buffer provided, followed by 100 μl of horseradish peroxidase (HRP)-avidin added to each well and incubated for 1 h at 37°C. The plate was then aspirated and washed a total of 5 times followed by application of 90 μl of tetramethylbenzidine (TMB) substrate added to each well, incubated for 20 mins at 37°C and protected from light. At the end of the 20 min period, 50 μl of stop solution was added to each well. The plate was then immediately read at 450 nm on a BioTek Plate reader elx800 (BioTek, Winooski, VT). Concentrations were calculated according to the standard curve and converted to pg/mg weight of the tissue.

2.9. Phospho-AKT ELISA.

The phospho-AKT ELISA kit was also ordered from MyBioSource.com (San Diego, CA, USA; CAT. No. MBS775153) and the protocol was closely followed. The standard and samples were added to the plate in duplicate along with 100 μl of horseradish peroxidase-conjugate reagent and covered. and gently shaken for 60 mins at 37°C. After this incubation, the liquid on the plate was drained, dried, and the plate was washed 5 times using the washing solution provided in the kit. Once dry, 50 μl of chromogen solution A and 50 μl of chromogen B provided with the kit were added to each well., the plate was incubated for 15 mins at 37°C and was covered away from light. After this period, 50 μl of stop solution was added to stop the reaction. The plate was then immediately read at 450 nm using the plate reader. The data is presented as moles per pg of tissue/

3. Results

3.1. Statistical Analyses with sex as a variable.

In all statistical analyses, sex was included as a variable in the initial ANOVA. However, there were no significant main effects or interactions with sex as a variable with the exception of locomotor activity. Therefore, when sex was dropped as a factor, a two- (neonatal drug treatment and adolescent drug treatment) or three-way ANOVA (neonatal drug treatment, adolescent drug treatment, decibel of prepulse) was used in analyses below.

3.2. Prepulse Inhibition (PPI).

Percent PPI is represented as a function of decibel of the prepulse as shown in Fig 1(A). A three-way ANOVA yielded significant main effects of neonatal drug treatment F (1,63) = 22.56, p < 0.001, adolescent drug treatment, F (2,63) = 11.75, p < 0.001 and a significant neonatal drug treatment x adolescent drug interaction F (2,63) = 16.68, p < 0.001. The QS group demonstrated a significant deficit across all decibels of prepulse compared tp the SS control group. The NQ-30mgCDPPB group was equivalent to SS controls and across all decibels of prepulse and both groups demonstrated significantly improved PPI performance compared to the NQ group. The NQ-10mgCDPPB group demonstrated improved PPI performance at 73dB compared to the NQ group, but was equivalent the NQ group at 76 and 82 dB. Overall, NQ treatment produced a PPI deficit that was alleviated to control levels by a 30 mg/kg dose of CDPPB.

Figure 1 (A).

Figure 1 (A).

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Percent prepulse inhibition (PPI) is presented as a function of decibel of prepulse and group. On 73dB trials, Group NS, NS-10MG CDPPB, NS-30 MG CDPPB, NQ-10MG CDPPB and NQ-30GCDPPB all demonstrated significantly higher levels of PPI than Group NQ (indicated by *, p<.05). At 76 and 82dB trials, Group NS, NS-10MG CDPPB, NS-30MG CDPPB and NQ-30MG CDPPB all demonstrated significantly higher levels of PPI than Group NQ and NQ-10MGCDPPB (indicated by *, p<.05). (B) Startle response (N) is presented as a function of group. There were no significant differences between groups. (C) Force exerted on No Stimulus Trials is presented as a function of group. There were no significant differences between groups.

Acoustic startle is presented in Figure 1(B) and No Stimulus Trials in Figure 1(C) as a function of drug treatment group. A three-way ANOVA revealed no significant main effects or interactions on this dependent variable.

3.3. Spontaneous Locomotor Activity.

Locomotor activity is presented as a function of drug treatment condition in Figure 2. A three-way ANOVA including sex, neonatal drug treatment and adolescent treatment revealed a significant sex main effect F (1,67) = 16.06, p < 0.001. Females were significantly more active than males, which is a common observation in adolescent rats (Krentzel & Meitzen, 2018). However, there were no other statistically significant differences between groups. Therefore, CDPPB did not affect spontaneous locomotor activity.

Figure 2.

Figure 2.

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Locomotor activity is presented as function of group and sex. There was significant sex difference, in that females demonstrated significantly higher levels of spontaneous locomotor activity compared to males (indicated by *, p<.05), but there no other significant differences between groups.

3.4.1. Beta-Arrestin-2 (βA-2) ELISA results: Dorsal Striatum.

Dorsal striatum βA-2 (pg/mg) is presented as a function of drug treatment condition in Figure 3(A). A two-way ANOVA revealed a significant main effect of neonatal drug treatment F (1,40) = 10.98, p < 0.002 and a significant two-way interaction of neonatal drug treatment x adolescent drug treatment F (2,40) = 19.88, p < 0.001. Post hoc analysis revealed that the QS group was significantly greater than all other groups (indicated by **, p < 0.05), and all CDPPB groups were equivalent to SS controls. Therefore, the NQ group resulted in a significant increase of βA-2 protein in the dorsal striatum, which was reversed by both doses of CDPPB.

Figure 3.

Figure 3.

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(A) β arrestin-2 (βA-2) is presented as a function of group for the dorsal striatum. Group NQ demonstrated significantly higher levels of of βA-2 than all other groups (indicated by *, p<.05), and there were no other significant group differences. (B) βA-2 is presented as a function of group for the prefrontal cortex (PFC). Group NQ demonstrated significantly higher levels of of βA-2 than all other groups (indicated by *, p<.05), and there were no other significant group differences.

3.4.2. βA-2 ELISA results: Prefrontal Cortex (PFC).

PFC βA-2 (pg/mg) is presented as a function of drug treatment condition in Figure 3(B). A two-way ANOVA revealed significant main effects of neonatal drug treatment F (1,33) = 4.9, p < 0.03, adolescent drug treatment F (2,33) = 7.9, p < 0.002, and a significant interaction of neonatal drug treatment x adolescent drug treatment F (2,33) = 3.8, p < 0.03. The NQ group was significantly higher than all other groups, and there were no other significant differences between groups. NQ treatment results in a significant increase of βA-2 protein in the PFC, which was significantly reversed by both doses of CDPPB (10 and 30 mg/kg) to control levels.

3.5.1. Phospho-AKT ELISA results: Dorsal Striatum.

Phospho-AKT (mol/mg) in the dorsal striatum is presented as a function of drug treatment condition in Figure 4(A). A two-way ANOVA on phospho-AKT protein levels (pg/mg) revealed a significant main effect of adolescent drug treatment F (2,42) = 3.99, p < 0.027 and a significant neonatal drug treatment x adolescent drug treatment interaction F (2,42) = 6.48, p < 0.004. Post hoc analyses revealed that the NQ group was significantly lower than both the NS and NQ-30mgCDPPB groups, demonstrating that 30 mg/kg CDPPB reversed this decrease in phospho-AKT. This suggests that 30 mg/kg CDPPB, but not the 10 mg/kg dose of CDPPB, reversed the increase in DAD2 signaling in NQ-treated rats 24h after drug treatment in the dorsal striatum.

Figure 4.

Figure 4.

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(A) Phospho-AKT is is presented as a function of group for the dorsal striatum. Group NQ demonstrated significantly lower levels of phosphor-AKT than Groups NS, NS-10MG CDPPB, NS-30 MG CDPPB, and NQ-30MG CDPPB, but was equivalent to NQ-10 MG CDPPB. Group NQ-10MG CDPPB also did not significantly differ from NQ-30 MG CDPPB. (B) Phospho-AKT is is presented as a function of group for the prefrontal cortex. Group NS demonstrated significantly higher levels of phospho-AKT than all other groups (indicated by *p<.05), and there no other significant group differences.

3.5.2. Phospho-AKT ELISA results: PFC.

Phospho-AKT (mol/mg) in the medial PFC is presented as a function of drug treatment condition in Figure 4(B). A two-way ANOVA on phospho-AKT protein levels (pg/mg) in the PFC revealed a significant main effect of adolescent drug treatment F (2,33) = 5.0, p < 0.01 and a significant two-way interaction of neonatal drug treatment x adolescent drug treatment F (2,33) = 6.82, p < 0.004. Group NS demonstrated significantly higher levels of p-AKT protein levels in the PFC compared to all other groups, however, there were no other group differences. CDPPB did not reverse changes produced by NQ treatment in phospho-AKT, but also reduced phospho-AKT relative to NS-treated controls.

4. Discussion

The purpose of this study was to investigate the effects of the mGlu5 positive allosteric modulator CDPPB on auditory sensorimotor gating as measured by PPI and DAD2 signaling in the NQ rodent model of increased DAD2 sensitivity. Results revealed that a 30 mg/kg dose of CDPPB alleviated PPI deficits in NQ-treated rats and reversed increases of DAD2 signaling as measured by βA-2 and phospho-AKT in the dorsal striatum. CDPPB also reversed changes in βA-2 in the PFC, but produced decreases of phospho-AKT in the PFC across both neonatal drug treatment group. Regarding PPI behavioral results, there were no effects of CDPPB on the no stimulus trials or the startle response, and neither dose produced any effects on spontaneous locomotor activity. This is the first demonstration of a positive allosteric modulator of mGlu5 CDPPB improving sensorimotor gating deficits in a developmental rodent model of increased DAD2 sensitivity with relevance to SZ.

Consistent with this finding, past work demonstrated that both 10 and 30 mg/kg CDPPB was effective in alleviating PPI deficits in adult animals administered an acute dose of d-amphetamine (2 mg/kg). Specifically, the 30 mg/kg dose was effective at prepulse trials that were 15 dB above background and the 10 mg/kg dose was effective at 20 dB above background (Kinney et al 2005). D-amphetamine is a dopaminergic agonist, and acute treatment has been used as a rodent model to mimic the increased dopaminergic activity associated with SZ. Here, we demonstrated that the 30 mg/kg dose was effective at all three decibels of prepulse tested: 3, 6, and 12 dB above background, and CDPPB did not produce any deficit in NS-treated controls. The lack of an effect in NS-treated controls coupled with no significant changes in locomotor activity is advantageous to many antipsychotics which often result in extrapyramidal motor side effects (Chow et al 2020).

CDPPB was effective to reverse the effects of NQ-treatment on βA-2 in both the dorsal striatum and PFC. Dopamine mediates its effects via the metabotropic G-protein coupled D1 and D2 families of receptors. β-arrestins are scaffolding proteins involved in the desensitization and internalization of receptors at the plasma membrane. Importantly, they can initiate intracellular signaling cascades along a pathway that is independent of canonical G protein signaling (Smith & Rajagopal, 2016). In this G-protein independent pathway, βA-2 works at the membrane and leads to inhibition of phospho-AKT and activation of GSK-3β, which can also modulate glutamatergic NMDA and AMPA receptors downstream (Li & Gao, 2011). We recently published a study showing that βA-2 was increased in the dorsal striatum in NQ-treated rats, and these animals also demonstrate sensorimotor gating deficits, consistent with several past studies which have shown changes in DAD2 signaling and its relationship to sensorimotor gating (Geyer et al 2000; Gill et al., 2021). Increases in striatal DAD2 expression, but not DAD2 expression in the PFC, has been shown to correlate with deficits in PPI (Kononoff et al 2016; Osterbog et al 2020). Importantly, DAD2 antagonists infused into the striatum rescued PPI deficits in cases where increased DAD2 signaling was present (Manago et al., 2016). Social isolation during the adolescent period has been shown to decrease DAD2 expression in the PFC, as well as result in PPI deficits. However, this decrease was only evident in neurons not contacted by axon terminal containing cannabinoid receptor 1 (CB1; Fitzgerald et al 2013). Regardless, the majority of studies point towards changes in DAD2 signaling in the dorsal striatum versus PFC relative to PPI performance.

There has been little work on the relationship between mGlu5 and phospoho-AKT. We report here that CDPPB reversed changes in phospho-AKT produced by NQ treatment in the dorsal striatum but did not reverse changes in phospho-AKT levels in NQ-treated rats, and reduced phospho-AKT in this same brain region in NS-treated controls. There have only been two studies published on the effects of CDPPB on phospho-AKT levels. Past work has shown that chronic treatment with a low dose (1.5 mg/kg/day) of CDPPB results in activation of phosphorylated-AKT in the cortex, striatum, and hippocampus, which is hypothesized to play a role in neuroprotection produced by CDPPB (Doria et al 2015). Likewise, a 100nM concentration of CDPPB applied to a primary culture of corticostriatal neurons also resulted in a significant increase of p-AKT (Batista et al., 2016). Therefore, it appears there may be a dose-dependent relationship between CDPPB and phospho-AKT, with lower doses resulting in activation and higher doses resulting in no change or inhibition which are brain region-dependent.

It is well-known that NMDA receptor hypofunction exists in SZ (Kahn & Sommer, 2015). The mGluR5 is co-localized and physically linked with NMDA receptors (Kim et al 2020). In addition, it has been shown that increased activation of mGluR5 using a positive allosteric modulator can potentiate NMDA receptor function which can alleviate hypofunction at this receptor (Lindsley et al 2006; Nickols & Conn, 2014). We have not determined whether NMDA hypofunction exists in the NQ rodent model, however, the fact that the DAD2 receptor forms mutually inhibitory heterocomplexes with mGlu5 may lead to an influence in NMDA receptor activity. For example, Liu et al. (2006) showed that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between the DAD2 receptor and the NR2B subunits of the NMDA receptor in the dorsal striatum in vivo. The enhanced D2R-NR2B interaction disrupts the association of Ca (2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site, and inhibits NMDA receptor-mediated currents in medium striatal spiny neurons. Therefore, CDPPB could be working through an influence of the mGlu5 on the NMDA receptor which may ultimately influence DAD2 activity.

4.1. Conclusions.

In conclusion, this study revealed that 30 mg/kg CDPPB was effective to reduce PPI deficits in adolescent rats treated with NQ, an increase in the level of phospho-AKT in the dorsal striatum, and an increase in βA-2 protein in the dorsal striatum. These findings corroborate with previous work on discovering the therapeutic dosage of CDPPB (Parmentier-Batteur et al., 2012). These findings have important implications towards the potential discovery of a new novel drug treatment for SZ. The relationship between mGluR5 and SZ is still full not understood. Future work will be directed towards investigating this relationship and further testing CDPPB as a novel drug treatment.

5. Acknowledgements

This work was supported in part by the East Tennessee State University Honors College and NIH R15DA046926-A1 to RWB.

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