Sensorimotor gating is an autonomic process of filtering irrelevant sensory information from salient ones in a stimulus-laden environment which is followed by execution of attention-dependent cognitive processes in order to respond to the salient stimuli (Braff and Geyer, 1990; Braff and Light, 2004; Powell et al., 2012). Prepulse inhibition (PPI) of acoustic startle reflex has been identified as an operational measure of sensorimotor gating since it is one of the forms of startle plasticity that resembles the mechanism of sensorimotor gating. PPI of acoustic startle reflex is defined as attenuation of behavioral response to a strong sensory stimulus (pulse) when preceded by a weaker stimulus (prepulse) by 30–500 ms (Graham, 1975; Hoffman and Ison, 1980; Ison and Hoffman, 1983). The observed reduction in response to the pulse stimulus occurs due to activation of inhibitory mechanisms in the central nervous system (CNS) which screens out incoming sensory stimuli until processing of the prepulse stimulus is completed. Hence, PPI serves as a mechanism to avoid interference between distinguishable stimuli (Kumari and Sharma, 2002). The inability to avoid this interference results in significant inundation or overflow of incoming sensory information leading to impairments in attention-dependent cognitive functions (McGhie and Chapman, 1961; Braff, 1993; Braff and Light, 2004). Deficits in PPI have been reported in a wide spectrum of neuropsychiatric disorders that are characterized by cognitive deficits including schizophrenia (Braff and Geyer, 1990; Geyer et al., 2001; Greenwood et al., 2007; Swerdlow et al., 2008; Swerdlow and Light, 2018), bipolar disorder (Perry et al., 2001), manic depressive disorder (Ludewig et al., 2002), Tourette syndrome (Swerdlow et al., 2001b) and autism spectrum disorders such as autism (Perry et al., 2007) and Fragile X syndrome (Frankland et al., 2004).

Previous investigations from our laboratory have reported a role for purinergic P2X4 receptors (P2X4Rs) in modulation of sensorimotor gating (Bortolato et al., 2013; Wyatt et al., 2013; Khoja et al., 2016). P2X4Rs belong to the 7-member P2X superfamily of cation permeable ligand gated ion channels (LGICs) gated by extracellular adenosine-5′-triphosphate (ATP) (North, 2002; North and Verkhratsky, 2006; Khakh and North, 2012). P2X4Rs are abundantly expressed on neurons and glial cells in the CNS (Abbracchio et al., 2009; Khakh and North, 2012). Ivermectin (IVM), a positive modulator of P2X4Rs, disrupted PPI regulation in male C57BL/6J mice and this effect was attenuated in mice deficient in the p2rx4 gene [i.e., P2X4R knockout (KO) mice] (Bortolato et al., 2013) suggesting that IVM-mediated PPI deficits is P2X4R-dependent. Moreover, male P2X4R KO mice exhibited PPI deficits in comparison to their wildtype (WT) littermates (Wyatt et al., 2013). A possible mechanistic explanation for this behavioral outcome could be the reported increased expression of dopamine (DA) D1 and D2 receptors in the ventral striatum of P2X4R KO mice (Khoja et al., 2016). To further reinforce a role for DA neurotransmission in P2X4R-mediated PPI disruption, DA receptor antagonists significantly ameliorated PPI deficits in P2X4R KO mice (Khoja et al., 2016).

The propensity of IVM to reduce PPI function in C57BL/6J mice is reminiscent of non-selective DA receptor agonists including amphetamine, methamphetamine, cocaine, apomorphine, all of which have been shown to disrupt PPI function in rodents (Ralph et al., 1999; Ralph-Williams et al., 2002, 2003; Doherty et al., 2008). The ability of IVM to mimic pharmacological effects of DA receptor agonists is not surprising considering that we have previously shown that IVM can modulate DA-dependent behaviors and signaling pathways. For instance, IVM potentiated the effects of DA on motor behavior (Khoja et al., 2016). IVM significantly modulated phosphorylation of dopamine and cyclic AMP regulated phosphoprotein of 32 kDa (DARPP-32) (Khoja et al., 2016), which is a critical downstream target regulated by DA receptor agonists and antagonists (Svenningsson et al., 2000, 2003; Nairn et al., 2004; Borgkvist and Fisone, 2007; Bonito-Oliva et al., 2011). The findings from our laboratory and the numerous studies that have described a role for DA in PPI regulation forms the basis for our hypothesis that IVM is causing PPI disruption via activation of DA neurotransmission.

To investigate this hypothesis, we tested the effects of IVM (10 mg/kg, i.p.) on PPI function in the presence of DA receptor antagonists, SCH 23390 (for D1 receptors) and raclopride (for D2 receptors). We also tested the effects of IVM (5 mg/kg, i.p.) in combination with a selective D1 receptor agonist, SKF 82958. Furthermore, to gain mechanistic insights into interaction between IVM and DA receptor antagonists/D1 agonist in PPI regulation, we tested the effects of IVM on phosphorylation of signaling molecules linked to PPI regulation including DARPP-32, Ca²⁺/calmodulin kinase IIα (CaMKIIα) and neuronal nitric oxide synthase (nNOS) in the ventral striatum in the presence of the dopaminergic drugs.

The current study investigated the mechanisms underlying IVM induced PPI disruption in C57BL/6J mice. PPI deficits induced upon P2X4R potentiation by IVM were significantly attenuated to a greater extent via antagonism of D1 than D2 receptors, implicating a more essential role for D1 than D2 receptors in IVM-mediated PPI dysfunction. Additionally, neither of the DA receptor antagonists modified the effects of IVM on startle magnitude suggesting that DA antagonists were able to restore IVM-induced information processing deficits without having any impact on auditory function. The findings from the DA receptor antagonist experiments are in agreement with previous investigations that have reported a more important role for D1 receptors than D2 receptors in PPI regulation in mice (Ralph-Williams et al., 2002, 2003; Ralph and Caine, 2005). For instance, non-selective DA receptor agonists such as apomorphine, cocaine or pergolide have been reported to disrupt PPI function in a D1 receptor-specific manner (Ralph-Williams et al., 2002, 2003; Doherty et al., 2008). Selective D1 agonists such as SKF 82958, SKF 81297 and dihydrexidine have also been shown to disrupt PPI functioning in C57BL/6J and 129S6 mice (Ralph-Williams et al., 2003; Ralph and Caine, 2005) at doses lower than those required to induce similar behavioral effects with rats (Wan et al., 1996; Swerdlow et al., 2000). Moreover, findings from our laboratory reported that D1 receptor antagonism rescued PPI dysfunction in P2X4R KO mice (Khoja et al., 2016). Taken together, the current body of evidence strongly suggests that D1 receptors have a prominent role in PPI regulation in mice.

In addition to the importance of D1 receptors in PPI regulation with mice; the role of D2 receptors cannot be disregarded based on our findings where raclopride significantly blocked IVM-mediated PPI dysfunction. Moreover, indirect DA receptor agonists including amphetamine and cocaine can disrupt PPI function via D2 receptor-dependent manner in mice (Ralph et al., 1999; Ralph-Williams et al., 2002; Doherty et al., 2008). In addition, multiple genetic knockout mouse models for dopamine transporter (DAT) (Ralph et al., 2001), disrupted in schizophrenia complex (DISC) (Lipina et al., 2010) and trace amine receptor-1 (TA1) (Wolinsky et al., 2007) exhibited PPI deficits that were linked to aberrant D2 receptor activity. In agreement with pharmacological and genetic findings, our laboratory has previously demonstrated that PPI deficits in P2X4R KO mice were also reversed by raclopride, in addition to SCH 23390, indicating that D2 receptors can be involved in PPI dysfunction in P2X4R KO mice (Khoja et al., 2016).

The findings from our IVM/P2X4R KO PPI investigations (and others) suggest a plausible D1–D2 receptor interaction in PPI regulation in mice. The argument for this hypothesis is reinforced by previous reports showing that antagonism of either D1 or D2 receptors can negate PPI deficits induced by D1 agonists in mice (Ralph and Caine, 2005). For example, administration of either D2 antagonist eticlopride or D1 antagonist SCH 39166 significantly restored SKF 82958-mediated PPI deficits in C57BL/6J mice (Ralph and Caine, 2005). A plausible explanation for this outcome is that activation of D1 receptors alone might not be sufficient enough to disrupt PPI function in rats but the ability of D2 receptors to induce PPI dysfunction is dependent upon the tonic activity of D1 receptors. Hence, based on our current findings, it appears that D1 receptors might be interacting with D2 receptors in the modulation of IVM-mediated PPI alterations and the tendency of raclopride to block IVM’s behavioral effects might be dependent upon tonic activity of D1 receptors in C57BL/6J mice.

To gain insights into the molecular mechanisms by which IVM can induce PPI dysfunction, we investigated its interaction with dopaminergic drugs on DARPP-32 phosphorylation in the ventral striatum. Our findings suggest that positive modulation of P2X4Rs increased DARPP-32 phosphorylation in a D1 receptor dependent manner (Figure 6A). Although, raclopride was able to prevent IVM from further increasing DARPP-32 phosphorylation, it failed to reverse IVM-mediated increase in DARPP-32 phosphorylation, suggesting that the actions of IVM on DARPP-32 are D1-dependent. Enhanced phosphorylation of DARPP-32 at Thr 34 has been linked to PPI deficits mediated by various psychostimulants such as amphetamine, phencyclidine (PCP), lysergic acid diethylamide (LSD) and that these effects were significantly diminished in genetically modified mice wherein DARPP-32 phosphorylation at Thr34 is compromised (Svenningsson et al., 2003). D1 receptor activation on the striatonigral neurons (D1-expressing neurons) of the basal ganglia circuitry stimulates the cAMP/protein kinase-A (PKA) pathway which leads to increased phosphorylation of DARPP-32 at Thr34 (Svenningsson et al., 2004; Girault, 2012) (Figure 6) and possibly manifestation of PPI deficits in mice as demonstrated by selective D1 agonists (Ralph-Williams et al., 2003; Ralph and Caine, 2005). Taking these findings into perspective, the reversal of IVM-mediated changes in DARPP-32 phosphorylation by SCH 23390 may underlie the attenuation of IVM-mediated PPI deficits. Moreover, the failure of raclopride to reverse IVM-induced increase in DARPP-32 phosphorylation might explain the discrepancy between SCH 23390 and raclopride in restoration of IVM-induced PPI deficits. However, this inference cannot be supported by the current evidence that SKF 82958 failed to elicit PPI dysfunction in presence of IVM despite the synergistic interaction between SKF 82958 and IVM in augmentation of DARPP-32 phosphorylation in the ventral striatum. This discrepancy can be attributed to the notion that DARPP-32 alone cannot mediate PPI function considering that multiple other neural substrates may contribute to the alleviation of IVM-induced PPI disruption by SCH 23390. Furthermore, the high dose of SCH 23390 used in this study may involve pharmacological activity at other receptor targets such as agonism at 5-HT2C receptors (Briggs et al., 1991; Millan et al., 2001). Notably, 5-HT2C receptor agonists can rescue PPI deficits induced by psychomimetics (Marquis et al., 2007). Thus, the multi-pharmacological profile of SCH 23390 in amelioration of IVM-induced PPI deficits cannot be disregarded. Moreover, although the ventral striatum [which comprises of nucleus accumbens (NAc) core] is ascertained as an important site for PPI mediation; this behavior is regulated by the cortico-pallido-striatal-thalamic circuitry (Wan et al., 1995; Wan and Swerdlow, 1996; Swerdlow et al., 2008, 2011). Considering that DA receptor agonists induce PPI dysfunction and DA receptor antagonists reverse PPI deficits, it is presumably thought that increased DAergic function in the NAc (Swerdlow et al., 2001a), leads to inhibition of GABAergic neurons projecting toward the ventral pallidum (VP), which has a tonic control over the pedunculopontine nucleus (PPTg), which mediates PPI function (Fendt et al., 2001). Thus, disruption of underlying signaling cascades in any of these brain structures of this complex circuitry could possibly be linked to the antagonistic interaction between IVM and SCH 23390 which warrants further investigation. Detailed delineation of signaling pathways within the PPI circuitry maybe necessitated in elucidating the discrepancy between SCH 23390 and SKF 82958 in regulation of IVM-mediated behavioral effects.

In addition to DARPP-32, we also evaluated the effects of IVM on other signaling molecules that may contribute to alterations in sensorimotor gating such as nNOS and its upstream regulator, CaMKIIα. CaMKIIα can phosphorylate nNOS at Ser897, leading to reduction in functional activity (Hayashi et al., 1999; Komeima et al., 2000). IVM decreased CaMKIIα phosphorylation in a D2-dependent manner since its effects were reversed by raclopride, but not by SCH 23390. Nevertheless, SCH 23390 was able to prevent IVM from further decreasing CaMKIIα phosphorylation suggesting that SCH 23390 might be reversing IVM-mediated effects on signaling pathways upstream of CaMKIIα phosphorylation. Interestingly, at a lower dose (5 mg/kg), IVM significantly potentiated the effects of SKF 82958 on CaMKIIα phosphorylation. A mechanistic explanation for this outcome is the synergistic interaction between IVM and SKF 82958 in DARPP-32 phosphorylation which could lead to up-regulation of CaMKIIα phosphorylation via inhibition of protein phosphotase-1 (PP-1) (Strack et al., 1997; Shioda and Fukunaga, 2017). Overall, these findings suggest that the dopaminergic control of IVM over CaMKIIα phosphorylation can be dose-dependent. While there is no direct evidence that links CaMKIIα to PPI regulation; results from our investigation mirror PPI disruptive agents such as indirect DA agonists (methamphetamine) (Suemaru et al., 2000) and N-Methyl-D-aspartate receptor (NMDAR) antagonists (PCP, ketamine) (Molteni et al., 2008; Cui et al., 2009) that have been reported to decrease CaMKIIα phosphorylation in mice and that this effect was fully restored upon D1 or D2 receptor antagonism. Additionally, CaMKIIα has been implicated in information processing (Coultrap and Bayer, 2012) and pathophysiology of psychiatric disorders characterized by cognitive abnormalities (Frankland et al., 2008; Yamasaki et al., 2008; Matsuo et al., 2009). If PPI deficiency can precede fragmentation of cognitive function (McGhie and Chapman, 1961); then alterations in CaMKIIα activity can be linked to PPI deficits in the clinical population.

On account of the significant reduction in CaMKIIα phosphorylation, IVM tended to reduce the phosphorylation of its downstream target, nNOS. Although raclopride, but not SCH 23390, was able to reverse IVM-mediated decrease in CaMKIIα phosphorylation; the pharmacological blockade of both D1 and D2 receptors significantly reversed IVM-mediated changes in nNOS phosphorylation. These results suggest that CaMKIIα could potentially be involved upstream in the effects of D2, but not D1, receptors on IVM-induced changes in nNOS phosphorylation. Although D1 receptor activation can enhance nNOS activity via NMDARs-mediated Ca²⁺ currents which involves CaMKIIα (Cepeda et al., 1998; Lee et al., 2010), stimulation of D1 receptors can also lead to enhanced phosphorylation of nNOS at Ser897 via PKA activation (Missale et al., 1998; Neve et al., 2004) or protein kinase C (PKC) activation (Felder et al., 1989; Undie and Friedman, 1990) which could ultimately result in reduced nNOS activity (Bredt et al., 1992; Dinerman et al., 1994; Lee et al., 2010). In further support of the argument that CaMKIIα may not be involved in the interaction between P2X4Rs and D1 receptors in nNOS phosphorylation, IVM potentiated the effects of SKF 82958 in regulation of CaMKIIα phosphorylation but decreased SKF 82958-induced increase in nNOS phosphorylation.

The ability of IVM to increase nNOS activity (via reduced phosphorylation of nNOS at Ser897) can lead to increased DARPP-32 phosphorylation at Thr34 via the NO/cGMP/guanyl cyclase (GC)/protein kinase G (PKG) signaling cascade (Tsou et al., 1993; Nishi et al., 2005; Threlfell and West, 2013). Additionally, the ability of IVM to dampen SKF 82958-induced decrease in nNOS phosphorylation may provide a mechanistic explanation for the synergy between IVM and SKF 82958 in regulation of DARPP-32 phosphorylation. This could represent a potential mechanism underlying IVM-mediated increased DARPP-32 phosphorylation considering that P2X4Rs are LGICs localized on GABAergic interneurons (Amadio et al., 2007) that are enriched in nNOS (Bredt et al., 1990, 1991) and have been reported to play a role in modulation of NOS activity via the cGMP/GC/PKG pathway (Yang et al., 2015). Hence, it is more logical to assume that cGMP/PKG pathway is involved upstream of P2X4R-mediated effects on DARPP-32 phosphorylation rather than the canonical cAMP/PKA activity which is regulated by G-coupled protein receptors (GPCRs) (Pierce et al., 2002). Based on previous evidence that have supported an interaction between DA neurotransmission and nNOS activity (Sammut et al., 2006, 2007; Hoque et al., 2010; Hoque and West, 2012), the reversal of IVM-mediated decrease in nNOS phosphorylation by DA receptor antagonists may underlie their antagonistic interaction with IVM in regulation of DARPP-32 phosphorylation. However, future investigations testing the effects of IVM on NO and cGMP levels in presence of dopaminergic drugs would be warranted to confirm this hypothesis.

In contrast to CaMKIIα, there is a growing body of evidence that supports role of nNOS in PPI regulation in mice (Klamer et al., 2004; Salum et al., 2006; Fejgin et al., 2008; Issy et al., 2009, 2014). The ability of IVM to decrease nNOS phosphorylation at Ser897 and induce PPI deficits is in corroboration with current theory that increased NO signaling leads to PPI deficits. NOS inhibitors have been observed to attenuate PPI deficits mediated by DA receptor activation (amphetamine, methylphenidate) (Issy et al., 2009) or NMDARs antagonism (PCP) (Klamer et al., 2004; Fejgin et al., 2008) From a disease standpoint, multiple preclinical and clinical findings have linked increased NO signaling to pathophysiology of psychiatric disorders characterized by sensorimotor gating deficits including schizophrenia (Bernstein et al., 2001; Yao et al., 2004; Bernstein et al., 2005; Lauer et al., 2005; Nasyrova et al., 2015). Genetic deficiency of nNOS generates a mouse model that exhibits behavioral and neurochemical abnormalities that are reminiscent of psychiatric disorders (Tanda et al., 2009). Increased nNOS activity has been detected in several brain regions in schizophrenia (Karson et al., 1996; Bernstein et al., 2001; Baba et al., 2004). Furthermore, genetic polymorphisms in nNOS have been detected as risk factor for schizophrenia (Reif et al., 2006; Rovny et al., 2018). Overall, based on findings from previous reports and current investigation, the regulation of nNOS activity can contribute to dopaminergic control of IVM-mediated PPI deficits and P2X4Rs-mediated increase in nNOS activity can represent a novel mechanism underlying psychiatric disorders characterized by sensorimotor gating perturbations especially schizophrenia.

Deficits in PPI have been linked to a wide spectrum of “perceptual” disorders including schizophrenia (Braff et al., 1978, 1999; Braff, 1993; Kumari et al., 1999, 2000), bipolar disorder (Perry et al., 2001), obsessive–compulsive disorder (Swerdlow et al., 1993) and Tourette’s syndrome (Swerdlow et al., 2001b) as well as to autism spectrum disorders such as autism (Perry et al., 2007) and Fragile X syndrome (Frankland et al., 2004). Mutant mouse models for susceptible genes that have been suggested to be involved in the pathophysiology of aforementioned diseases exhibit PPI dysfunction (Ralph et al., 2001; Duncan et al., 2004; Kinkead et al., 2005; Tanaka et al., 2006; Ohgake et al., 2009; Lipina et al., 2010; Willi et al., 2010; Vuillermot et al., 2011), making PPI a reliable endophenotype in the genetic studies of neuropsychiatric disorders (Braff et al., 2007; Powell et al., 2012). Moreover, several of the clinical typical and atypical anti-psychotics such as haloperidol, risperidone, clozapine, olanzapine, quetiapine have been demonstrated to ameliorate PPI deficits in genetic and pharmacological models of such diseases (Swerdlow and Geyer, 1993; Bast et al., 2001; Geyer et al., 2001; Kumari and Sharma, 2002; Levin et al., 2007). Thus, PPI has been elucidated as a useful behavioral assay in investigating molecular mechanisms of psychiatric diseases as well as screening of potential antipsychotic drugs. The ability of IVM to disrupt PPI function via P2X4R potentiation (Bortolato et al., 2013) as well as the reduced PPI function in the P2X4R KO mouse model (Wyatt et al., 2013) suggests a role for P2X4Rs in pathophysiology of psychiatric disorders characterized by sensorimotor gating perturbations. Additionally, our findings also suggest a role for DA neurotransmission in altering IVM-mediated effects on sensorimotor gating. Considering the link between dysregulated dopaminergic function and sensorimotor gating abnormalities in psychiatric diseases (Swerdlow et al., 1986; Braff and Geyer, 1990; Swerdlow and Geyer, 1993; Braff et al., 2001) has been well consolidated over past decades, our preclinical studies suggest a role for P2X4Rs in pathophysiology of psychiatric diseases. Moreover, we have also demonstrated that IVM can induce dysregulation in signaling molecules including DARRP-32, CaMKIIα and nNOS, all of which have been previously linked to psychiatric diseases characterized by sensorimotor gating abnormalities (Albert et al., 2002; Bernstein et al., 2005; Ishikawa et al., 2007; Frankland et al., 2008; Nasyrova et al., 2015; Wang et al., 2017). However, further investigations at the clinical level would be warranted to link p2rx4 gene mutations (leading to aberrations in functioning) with neuropsychiatric disorders in order to ascertain such a role for P2X4Rs in patients diagnosed with psychiatric illnesses. Until date, single nucleotide polymorphisms in human p2rx4 gene have been only linked to hypertension and age-related macular degeneration (Caseley et al., 2014).

Although the findings from our current investigation provide novel mechanistic insights into role of P2X4Rs in regulation of sensorimotor gating, there are certain limitations of our study that needs to be acknowledged. First, we only identified the effects of IVM in regulation of signaling molecules in one brain region that is part of a complex neural circuitry that regulates PPI function (Swerdlow et al., 2001a, 2008). Hence, future investigations that will test the impact of dopaminergic drugs on IVM-mediated changes in DARPP-32, CaMKIIα and nNOS phosphorylation in other brain regions integral to PPI circuitry including medial prefrontal cortex (mPFC), ventral hippocampus and basolateral amygdala would be warranted. Elucidation of the interaction between P2X4Rs and DA receptors in these specified brain regions would provide a holistic overview of role of P2X4Rs in mechanisms of sensorimotor gating. Another major limitation of our study is the absence of translational applicability due to lack of specific P2X4R antagonists. Based on our results with IVM, antagonism of P2X4R function would alleviate sensorimotor gating deficits and represent a novel therapeutic strategy. An alternative approach is to use viral vectors that would specifically knockdown P2X4R expression in a brain region critical for PPI regulation such as the NAc, mPFC or ventral hippocampus. Lastly, we did not establish a link between DARPP-32, CaMKIIα or nNOS and the dopaminergic control of IVM-mediated PPI deficits. Hence, future experiments would include testing the impact of interaction between P2X4Rs and DA receptors on PPI regulation upon genetic ablation of these signaling molecules by viral vectors or in genetic knockout mouse models. Such investigations would be pivotal for establishing a role for DARPP-32, CaMKIIα and nNOS in IVM-mediated PPI deficits.

Overall, the current investigation provides novel insights into a potential interaction between P2X4Rs and DA receptors in modulation of PPI and that perturbation of this interaction could lead to manifestation of sensorimotor gating deficits and subsequent cognitive fragmentation. Most importantly, our findings support the development of P2X4R antagonists as potential anti-psychotics for treatment of diseases linked to DA-dependent sensorimotor gating dysfunction.

The datasets generated for this study are available on request to the corresponding author.

All experiments were undertaken as per guidelines established by the National Institutes of Health (NIH) and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Southern California, Los Angeles, CA, United States.

SK designed the research study, performed the experiments, analyzed the data, and wrote the manuscript. LA, MJ, and DD reviewed and edited the manuscript.

DD and LA are inventors on a patent for the use of ivermectin for treatment of alcohol use disorders. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.