Although the behavioral consequences of mPFC-mGluR5 upregulation have been revealed as such, the cellular roles in these circuits remain elusive. Normally, excitatory manipulation of mPFC pyramidal neuronal activity induces an analgesic effect and ameliorates pain. Administration of ionotropic glutamate receptor (iGluR) agonists to the mPFC activated the endogenous analgesic action of the PAG (Ong et al., 2018). Alternatively, mPFC pyramidal neuronal activation increased mPFC-Nucleus accumbens (NAc) activity to reduce pain perception (Lee M. et al., 2015). The action of mGluR5 activation is generally excitatory to neurons; however, there is a gap between the pain-suppressing actions of mPFC-iGluR5 activation and mPFCmGluR5 deactivation.

One possible explanation is that increased prelimbic mGluR5 levels eventually result in a reduction in the firing of excitatory pyramidal mPFC neurons, which project to the PAG or the NAc. Previous studies have reported that the neuronal excitability of mPFC-PAG projection neurons is decreased in chronic pain states, and the increased activity of inhibitory interneurons is responsible for the reduced activity of pyramidal neurons (Zhang et al., 2015; Cheriyan and Sheets, 2018). Interestingly, glutamate stimulation elicited inhibitory inputs to mPFC-PAG projection neurons (Cheriyan and Sheets, 2018). Although the causal relationship between mGluR5 upregulation and increased inhibitory neuronal activity has not been extensively studied, the blockade of mPFC-mGluR5, or of inhibitory neuronal activity, drives mPFC circuits in the same direction, i.e., they induce the analgesic and anti-depressive effects.

There are several conceivable hypotheses. First, mGluR5 upregulation in the chronic pain state may occur predominantly in inhibitory neurons in the mPFC (**Figure 1A**). Second, apart from this explanation, mGluR5 may be upregulated in excitatory neurons that preferentially excite inhibitory interneurons within the local circuits (**Figure 1B**). In the mPFC, the activity of local GABAergic neurons is influenced by the glutamatergic excitatory inputs they receive, and the frequency – but not the amplitude – of the excitatory transmission to the GABAergic neurons were increased in the neuropathic pain state (Zhang et al., 2015). Third, alternatively, activated mGluR5 in the pyramidal synapse may induce glutamatergic long-term depression in pyramidal neurons via interaction with other molecular signals (Otani et al., 1999; Zhong et al., 2008; Ghoshal et al., 2017; **Figure 1C**). For example, a recent study found that coactivation of mGluR5 and the M1 muscarinic acetylcholine receptor in the mPFC exerted a long-lasting decrease in excitatory transmission and concurrent enhancement of GABAergic inhibitory tone (Ghoshal et al., 2017). In this scenario, pyramidal neuronal increase in mGluR5 would not be able to modulate the presynaptic release of GABA because mGluR5 signaling in the mPFC pyramidal neurons fails to engage 2-AG mediated endocannabinoid signaling in chronic pain states (Kiritoshi et al., 2016). It has been reported that in the chronic pain state, mGluR5 activation could not increase pyramidal neuronal activity unless the inhibition from the GABAergic neuron is blocked by treatment with a GABA antagonist or activation of the CB1 receptor in GABAergic neurons (Kiritoshi et al., 2016). Fourth, mGluR5 upregulation may occur in glial cells surrounding the synapses of a specific subpopulation of mPFC neurons (**Figure 1D**). Glial mGluR5 serves to evoke complex and bi-directional effects on the modulation of neuronal activity, and upregulated mGluR5 in cortical astrocytes contributes to synaptic remodeling during the chronic neuropathic pain state (Kim et al., 2016; Ishikawa et al., 2018).

Either way, upregulated mGluR5 in the chronic pain state may be working in a direction that increases inhibitory influences on mPFC pyramidal neurons. In fact, mPFC glutamatergic neuronal activity was increased in response to the mGluR5 antagonist treatment in vivo (Lee K.W. et al., 2015). mPFC pyramidal neurons comprise several subtypes with different firing properties, expressing receptors, and projecting brain regions, and subserve distinct functions. Therefore, the subpopulationspecific upregulation of mGluR5 and its relevance to pain and depression should be investigated in future studies.

pyramidal neurons. (A) mGluR5 is upregulated in the GABAergic inhibitory interneurons. As a result, GABAergic neuronal activity is increased. (B) mGluR5 is increased in the specific subpopulation of pyramidal neurons that preferentially excite local GABAergic neurons. (C) Layer 5/6 pyramidal neurons express more mGluR5. However, the increased mGluR5 suppresses, rather than facilitates, pyramidal neuronal activity. This is achieved by an enhancement of long-term depression in excitatory synapses and concurrent loss of presynaptic modulation by mGluR5. (D) mGluR5 upregulation occurs in astrocytes surrounding the synapses of a specific subpopulation of mPFC neurons. The increased glial mGluR5 serves to enhance excitatory influences on GABAergic neurons. (GLU, glutamatergic neuron, GABA, GABAergic neuron).

### BIOLOGICAL PROCESSES UNDERLYING mPFC-mGluR5 UPREGULATION: POSSIBLE MOLECULAR MECHANISMS

The biological processes that mediate the alteration of mPFC circuits and the cellular mechanisms underlying upregulation of mPFC-mGluR5 are largely unknown. A previous study revealed that depression is critically related to mPFC-mGluR5, and the expression level is regulated by protein p11 (Lee K.W. et al., 2015). As mentioned, the lentiviral knock-down of mGluR5 in excitatory neurons in the mPFC facilitated depressive-like behavior, whereas inhibitory neuron-specific mGluR5 knockdown exerted an anti-depressive effect. Similar results were obtained from the conditional knockout of protein p11 in excitatory or inhibitory neurons. It is worth noting that although p11 could be a strong candidate to be a mediator of mPFCmGluR5 alteration, p11-mediated changes would predominantly occur in a specific subtype of prelimbic layer 2/3 pyramidal neurons, with decreased expression in response to chronic stress (Seo et al., 2017). Thus, the molecular mechanisms underlying upregulated mGluR5 in the prelimbic area of animals with chronic pain (Chung et al., 2017) remain vague.

A possible candidate is dopamine 2 receptor (D2R) mediated control of mGluR5 expression. From the perspective of a reinforcement learning paradigm, dopamine signaling plays a key role in motivated approach or avoidance behavior following aversion. Because D2Rs have a strong binding affinity compared with the dopamine 1 receptor (D1R), persistent dopamine would be mainly occupied by D2R in the nervous system. When negative prediction error occurs by salient event (aversion), dopaminergic neurons would cease firing, resulting in transient dopamine depletion in the relevant brain regions (Bromberg-Martin et al., 2010; Glimcher, 2011). Theoretically,

the reinforcement learning mechanisms induced by these types of negative prediction errors are to correct one's expectancies and behaviors to avoid determinants responsible for aversion. However, some determinants, such as intractable somatic pain or psychological distress are essentially unavoidable. Longterm experiences of unavoidable, persistent distress would alter the prefrontal circuits that assess the internal state to decide coping strategies (Roy et al., 2014; Shrestha et al., 2015; Aliczki et al., 2016; Zhang et al., 2017). It is well established that chronic pain, as well as depression, leads to hypodopaminergic tone in the brain (Niikura et al., 2010; Belujon and Grace, 2017; Ong et al., 2018). Because the baseline occupancy of dopamine is higher in D2Rs, the reduction in spontaneous dopamine level predominantly affects the D2R, leading to receptor deactivation (Bromberg-Martin et al., 2010). The deactivation of D2R due to somatic pain or psychological distress has been implicated in both animal and human studies. Interestingly, D2R deactivation could increase mGluR5 availability in the striatum (Mao and Wang, 2016). According to the previous study, pharmacological deactivation of D2R resulted in an increase in mGluR5 trafficking to the synaptic membrane, which is mediated by the Src kinase family Fyn (Mao and Wang, 2016). The researchers reported that the same regulation of Fyn, which is induced by D2R deactivation, was also observed in the mPFC (Mao and Wang, 2017). Hence, it is plausible that mPFC-mGluR5 increase in chronic neuropathic pain is related to D2R deactivation, although further study is needed to elucidate the occurrence of the phenomenon in the mPFC.

#### REFERENCES

### CONCLUDING REMARKS

Experiencing unavoidable stress, such as somatic pain or psychological distress, alters mPFC circuitry and distorts the perception of the self-state and ensuing decisions regarding coping strategies. We propose that mGluR5 in the mPFC may be a common mediator for both pain and depression. mGluR5 levels would regulate the gain for the assessment of internal state, guide the appraisal of the external state, and be involved in updating the expectancies of behavioral outcome in the mPFC. Increased mPFC-mGluR5 levels during the pathological state would inhibit the activity of pyramidal neurons and reduce the flexibility of the system, resulting in the loss of control of the appropriate processing of the information.

### AUTHOR CONTRIBUTIONS

GC conceived the idea, performed the bibliographical research, wrote the draft, and revised the manuscript. SJK and SKK supervised and revised the manuscript.

## FUNDING

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Government (NRF-2018R1A6A3A11045706 to GC, NRF-2017M3C7A1029611 to SJK, and NRF-2017M3A9E4057926 and NRF-2017M3C7A1025604 to SKK).

**Conflict of Interest Statement:** The 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.

Copyright © 2018 Chung, Kim and Kim. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Group II Metabotropic Glutamate Receptors: Role in Pain Mechanisms and Pain Modulation

Mariacristina Mazzitelli1 , Enza Palazzo2 , Sabatino Maione2 and Volker Neugebauer1,3 \*

1 Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, Lubbock, TX, United States, 2 Section of Pharmacology L. Donatelli, Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy, 3 Center of Excellence for Translational Neuroscience and Therapeutics, Texas Tech University Health Sciences Center, Lubbock, TX, United States

Glutamate is the main excitatory neurotransmitter in the nervous system and plays a critical role in nociceptive processing and pain modulation. G-protein coupled metabotropic glutamate receptors (mGluRs) are widely expressed in the central and peripheral nervous system, and they mediate neuronal excitability and synaptic transmission. Eight different mGluR subtypes have been identified so far, and are classified into Groups I–III. Group II mGluR2 and mGluR3 couple negatively to adenylyl cyclase through Gi/Go proteins, are mainly expressed presynaptically, and typically inhibit the release of neurotransmitters, including glutamate and GABA. Group II mGluRs have consistently been linked to pain modulation; they are expressed in peripheral, spinal and supraspinal elements of pain-related neural processing. Pharmacological studies have shown anti-nociceptive/analgesic effects of group II mGluR agonists in preclinical models of acute and chronic pain, although much less is known about mechanisms and sites of action for mGluR2 and mGluR3 compared to other mGluRs. The availability of orthosteric and new selective allosteric modulators acting on mGluR2 and mGluR3 has provided valuable tools for elucidating (subtype) specific contributions of these receptors to the pathophysiological mechanisms of pain and other disorders and their potential as therapeutic targets. This review focuses on the important role of group II mGluRs in the neurobiology of pain mechanisms and behavioral modulation, and discusses evidence for their therapeutic potential in pain.

#### Edited by:

Sung Jun Jung, Hanyang University, South Korea

#### Reviewed by:

Temugin Berta, University of Cincinnati, United States Barbara Wroblewska, Georgetown University, United States

#### \*Correspondence:

Volker Neugebauer<EMAIL_ADDRESS>
Received: 04 August 2018 Accepted: 24 September 2018 Published: 09 October 2018

#### Citation:

Mazzitelli M, Palazzo E, Maione S and Neugebauer V (2018) Group II Metabotropic Glutamate Receptors: Role in Pain Mechanisms and Pain Modulation. Front. Mol.

### METABOTROPIC GLUTAMATE RECEPTORS

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The glutamatergic system provides excitatory neurotransmission throughout the central nervous system (CNS), and dysfunction of this system seems is correlated with several disorders such as schizophrenia, depression, and pain states. The interaction of glutamate with its ligand-gated cation channels (NMDA, AMPA, and kainate) mediates fast transmission and cell signaling while the activation of metabotropic glutamate receptors (mGluRs) stimulates intracellular pathways linked to various effector systems involved long-lasting modifications. First evidence for the ability of glutamate to stimulate the production of inositol phosphate (Sladeczek et al., 1985) formed the foundation for the cloning of the first mGluR (Masu et al., 1991). Numerous studies have greatly expanded the field. mGluRs belong to the G-protein coupled receptors (GPCRs) superfamily, and are classified in three groups based on sequence homology, signal transduction pathways and pharmacological agent selectivity (Schoepp et al., 1999; Neugebauer, 2007, 2015; Niswender and Conn, 2010; Nicoletti et al., 2011; Yin et al., 2014).

The activity of those receptors can be modulated by pharmacological manipulations of the orthosteric binding sites with agonists, antagonists, or inverse agonists. The issue with the orthosteric ligands is the conservation of the binding site that makes it difficult to develop selective molecules for specific receptors. Another approach is the use of allosteric modulators that bind to specific sites of the receptors different from the orthosteric ones, and as a consequence they modulate the affinity of the receptor for its endogenous ligand. This binding is saturable such that no further effect is possible when all sites are occupied; better selectivity can be achieved because the allosteric binding sites present a lower evolutionary conservation compared to the orthosteric sites, and allosteric modulation can produce positive or negative effects based on the intrinsic activity of the compound (Conn et al., 2009; Montana and Gereau, 2011; Wood et al., 2011). Current research efforts are focused on the development of new positive and negative allosteric modulators (PAM and NAM) as potential therapeutic tools and strategies.

### GROUP II METABOTROPIC GLUTAMATE RECEPTORS

The group II mGluRs are found throughout the nervous system, including regions and circuits critically involved in nociceptive signaling and pain modulation as well as in emotional processing (Gu et al., 2008; Wright et al., 2013). They contribute to and modulate synaptic transmission and neuroplasticity, acting at the preterminal region away from the active zone of the synapse as autoreceptors on glutamatergic neurons or heteroreceptors on GABAergic neurons mediating a negative feedback signal (Nicoletti et al., 2011). As perisynaptic receptors, they are located on the pre-synaptic membrane distant from the synaptic cleft, where they can be activated by substantial synaptic glutamate release or astrocytic glutamate (Muguruza et al., 2016; Maksymetz et al., 2017). However, some evidence suggests that mGluR2 and mGluR3 are also expressed post-synaptically. Whereas mGluR2 seems to be present exclusively on neurons, mGluR3 is also found on glia cells (Muguruza et al., 2016). It is becoming clear now that group II mGluRs interact closely with other mGluRs, which has important functional implications. For example, mGluR2 forms a heterodimeric complex with mGluR4 that regulates the efficacies of mGluR2 and mGlu4 allosteric modulators (Yin et al., 2014), while mGluR3 and mGluR5 interact synergistically in the CNS through cross-talk of signaling pathways rather than by heterodimer interactions (Di Menna et al., 2018).

Therapeutic usefulness of compounds acting on group II mGluRs has been suggested for amyotrophic lateral sclerosis (Battaglia et al., 2015), schizophrenia, depression, anxiety (Patil et al., 2007; Fell et al., 2011; O'brien et al., 2014; Muguruza et al., 2016), drug addiction (Moussawi and Kalivas, 2010), Parkinson's disease (Dickerson and Conn, 2012), and pain states (Neugebauer, 2007; Montana and Gereau, 2011; Neugebauer, 2013; Chiechio, 2016). The specific contribution of mGluR2 or mGluR3 subtypes has been challenging to determine because of the close similarity of the proteins that makes it difficult for pharmacological approaches to target these subtypes selectively. In order to better understand the role of individual mGluR subtypes, considerable effort has been dedicated to the development of more selective and CNS penetrant NAMs and PAMs, together with the development of mGluR knockout (KO) mice. While there is strong evidence for an over-activation of the glutamatergic system in pain states (Neugebauer, 2007; Zhou et al., 2011; Guida et al., 2015), and mGluRs in particular (Neugebauer, 2007; Montana and Gereau, 2011; Kolber, 2015), the role of the group II mGluRs and their subtypes in pain mechanisms and pain modulation is less well understood.

## PHARMACOLOGICAL AGENTS TARGETING mGluR2/3

### Orthosteric Ligands

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A number of molecules, classified according to their intrinsic activity and receptor selectivity, have been developed and tested in order to clarify the contribution of group II mGluRs to disease mechanisms and pathological conditions (**Table 1**). Most of the currently available compounds have effects also on other glutamate receptors (ionotropic or metabotropic) and do not differentiate between mGluR2 and mGluR3. For example, DCG-IV is a very potent and selective group II mGluR agonist but has also NMDA agonist effects (Zhou et al., 2011), while L-CCG-I is a potent but not very selective group II mGluR agonist and (2R,4R)-APDC (APDC) is highly selective group II mGluR2/3 agonist (Brabet et al., 1998; Schoepp et al., 1999). (1S,3S)-ACPD (ACPD) is the most selective mGluR2/3 agonist among the isomers of (±)-cis-ACPD (Hölscher et al., 1997). More selective and highly potent group II agonists, such as LY2934747, LY389795, LY354740, LY404039, LY379268 (also in the form of disodium salt), have been developed with good efficacy in animal models (Nicoletti et al., 2011, 2015; Caulder et al., 2014; Yin and Niswender, 2014; Maksymetz et al., 2017) including for pain states (Neugebauer et al., 2000; Simmons et al., 2002; Li and Neugebauer, 2006; Neugebauer, 2007; Chiechio and Nicoletti, 2012; Yin and Niswender, 2014; Johnson et al., 2017). SLx-3095-1 is the racemate (± isomers HCl salt) of the agonist LY379268 (– isomer) (Yamamoto et al., 2007). EGLU, LY341495 (also produced as disodium salt), and APICA are selective mGluR2/3 antagonists (Niswender and Conn, 2010; Yin and Niswender, 2014). Recently discovered LY3020371 seems to be even more selective for mGluR2/3 among all the mGluRs with potent effects in rat and human synaptosome preparations as well as in in vivo assays (Witkin et al., 2017).

### Allosteric Modulators

The development of selective NAMs and PAMs is now beginning to allow the targeting of mGluR2 and mGluR3 (Dhanya et al., 2010; Sheffler et al., 2011b; Bollinger et al., 2017). Only PAMs selectively binding to mGluR2, but not mGluR3, are available such as BINA, LY487379 hydrochloride, and CBIPES hydrochloride (Johnson et al., 2005; Dhanya et al., 2010; Sheffler et al., 2011b). These mGluR2 PAMs attenuated the ketamine-induced release of histamine in the medial prefrontal cortex (mPFC) (Fell et al., 2010) and decreased ketamine- or phencyclidine-induced hyperlocomotion (Sheffler et al., 2011b), suggesting antipsychotic activity of mGluR2. Selective NAMs are available for mGluR3 (LY2389575, VU0477950, and VU0650786) and more recently for mGluR2 (VU6001966) (Bollinger et al., 2017). LY2389575 established a key role of mGluR3 in neuroprotection against β-amyloid induced toxicity (Caraci et al., 2010; Sheffler et al., 2011a), suggesting that pharmacological activation of mGluR3 with PAMs may be a possible therapeutic strategy in Alzheimer's disease. VU0477950 revealed a crucial role of mGluR3 in cognitive functions in mPFC-dependent fear extinction learning (Walker et al., 2015). VU0650786, an even more selective mGluR3 NAM, implicated the synergistic interaction of mGluR3 and mGluR5 in the generation of synaptic plasticity (long-term depression of excitatory transmission in cortical neurons) (Engers et al., 2015; Di Menna et al., 2018). Recently, VU6001966 emerged as a NAM for mGluR2 without any activity at the other mGluRs and with high CNS penetration (Bollinger et al., 2017; Di Menna et al., 2018).

#### PHARMACOLOGICAL MANIPULATION OF mGluR2/3 IN PAIN: BEHAVIORAL STUDIES

Pharmacological activation of group II mGluRs generally has antinociceptive effects in preclinical studies in rats and mice (Varney and Gereau, 2002; Neugebauer, 2007; Montana and Gereau, 2011; Chiechio, 2016). The activation of mGluR2/3 by systemically (intraperitoneally, i.p.) applied agonists (LY354740, LY379268, and LY389795) decreased nociceptive behavior in the late phase of the formalin test, a relatively acute pain model, in a dose-dependent manner, and the effect was reversed by a group II antagonist (LY341495) (Simmons et al., 2002). Systemic mGlu2/3 activation also decreased mechanical allodynia of neuropathic rats (spinal nerve ligation model, SNL), but had no effect in the tail flick test or the paw withdrawal latency test (acute thermal pain models) (Simmons et al., 2002). Importantly, the antinociceptive effect of systemically (i.p.) applied LY379268 in the formalin pain model was lost in mGluR2, but not mGluR3, knock-out mice (Zammataro et al., 2011), suggesting an important role of mGluR2. Recently, oral application of a prodrug (LY2969822) for the selective mGluR2/3 agonist LY2934747 has been reported to have antinociceptive effects in various preclinical models of inflammatory (formalin, capsaicin, complete Freund's adjuvant [CFA]), postsurgical (plantar incision), visceral (colorectal distension), and neuropathic (SNL) pain (Johnson et al., 2017).

### Peripheral

Subcutaneous injection of a group II mGluR agonist (APDC) into the plantar surface of the hindpaw did not change baseline mechanical and thermal sensitivity but blocked prostaglandin E2 (PGE2)-induced thermal hyperalgesia, PGE2- or carrageenaninduced mechanical allodynia, and nociceptive responses in both phases of the formalin test; these antinociceptive effects were inhibited by co-application of a group II mGluR antagonist (LY341495) (Yang and Gereau, 2002, 2003; Yamamoto et al., 2007). Interestingly, group II antagonists (LY341495 and APICA) alone prolonged the mechanical allodynia in the PGE2 and carrageenan models and increased nociceptive behaviors in the capsaicin model, supporting the hypothesis of endogenous mGluR2/3 activation in inflammatory pain conditions (Yang and Gereau, 2002, 2003; Carlton et al., 2011). The data are consistent with antinociceptive effects of peripheral group II mGluR activation, although there may be species differences (Sheahan et al., 2018; see the sections "Peripheral" and "Clinical Trials and Potential Clinical Uses").

#### TABLE 1 | Drugs acting on mGluR2/3 tested in pain models.

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(Continued)

See the sections "Pharmacological Manipulation of mGluR2/3 in Pain: Behavioral Studies" and "Pharmacological Manipulation of mGluR2/3 in Pain: Electrophysiological Studies" for references.

#### Spinal

Intrathecal (i.th.) administration of a selective group II mGluR agonist (APDC) in the absence of tissue damage had no effect on mechanical thresholds (von Frey test) and thermal paw withdrawal latencies but inhibited capsaicin-induced mechanical allodynia without affecting thermal hyperalgesia (Soliman et al., 2005). However, intrathecal APDC had no effect in an inflammatory pain model induced by subcutaneous (s.c.) CFA injection into the hindpaw (Zhang et al., 2009). In neuropathic rats (SNL model) intrathecal application of a group II mGluR agonist (DCG-IV) decreased mechanical allodynia (von Frey test) and mechanical hyperalgesia (paw withdrawal threshold to noxious pressure stimuli) in a dose-dependent way, and these antinociceptive effects were blocked by a group II mGluR antagonist (EGLU) (Zhou et al., 2011). Interestingly, intrathecal application of DCG-IV had a pronociceptive effect in sham rats, which was reversed by an NMDA receptor antagonist (AP-5), suggesting that this effect was mediated by the activation of NMDA receptors (Zhou et al., 2011). Intrathecal application of a group II mGluR antagonist (LY341495) ameliorated mechanical allodynia, but not thermal hyperalgesia, in the CFAinduced inflammatory pain model; the antinociceptive effect was potentiated by a glial cells inhibitor (fluorocitric acid) (Zhang et al., 2009). These mixed and somewhat inconsistent effects of group II mGluR compounds may be due to a lack of subtype-specificity and/or reflect rather complex functions of group II mGluRs in spinal nociceptive processing.

#### Brainstem

In the periaqueductal gray (PAG), activation of group II mGluRs had pronociceptive effects under normal conditions but antinociceptive effects in an acute pain model. Microinjection of a group II agonist (L-CCG-I) into the dorsolateral PAG dose-dependently inhibited the nociceptive responses (lifting, licking, shaking and flicking the injected paw) in the late phase of the formalin test (acute pain model) (Maione et al., 2000), but had a dose-dependent pronociceptive effect in the hot plate test, decreasing the latency of the nociceptive responses (licking the paw; jumping) (Maione et al., 1998). Both effects were counteracted by the intra-PAG administration of a group II mGluR antagonist (EGLU) (Maione et al., 1998, 2000). EGLU alone had no effect in the hotplate test (Maione et al., 1998). Together with microdialysis data showing that L-CCG-I increased serotonin release in the PAG in a GABAA receptor dependent way (Maione et al., 1998), these results were interpreted to suggest that group II mGluRs in the PAG promote an antinociceptive effect mainly by decreasing GABA release to potentiate the activity of the descending antinociceptive pathway following persistent noxious stimulation (Maione et al., 2000).

#### Brain

In the thalamus group II mGluRs mediate the presynaptic inhibition of GABAergic inhibitory transmission from the reticular thalamic nucleus to the somatosensory ventrobasal

Mazzitelli et al. Group II mGluRs and Pain

thalamus (VB) (Salt and Turner, 1998) to facilitate sensory processing through an action on mGluR2 (Copeland et al., 2012) possibly on astrocytes (Copeland et al., 2017). Stereotaxic administration of a group II mGluR antagonist (EGLU) into the reticular thalamic nucleus, but not other thalamic nuclei, had an antinociceptive effect in an arthritis pain model (complete Freund's adjuvant-induced monoarthritis in the ankle joint), reducing the ankle-bend test scores, possibly through a mechanism that involves blocking the disinhibition of somatosensory thalamic relay neurons (Neto and Castro-Lopes, 2000).

#### Subtype Selective Interventions

Behavioral effects of negative and positive allosteric modulators for mGluR2 and mGluR3 remain to be determined in pain conditions, but the contribution of individual subtypes is being addressed using alternative approaches.

N-acetylcysteine (NAC) has been used to probe mGluR2 function in pain models. NAC promotes the activity of the L-cystine/L-glutamate membrane exchanger (Sxc-), a crucial antiporter for the release of glutamate from astrocytes for the endogenous activation of perisynaptic mGluR2/3 (Kalivas, 2009). NAc may therefore be used to increase endogenous activation of these receptors (Chiechio and Nicoletti, 2012). Systemic (i.p.) application of NAC inhibited nocifensive behaviors in the tail flick test (Truini et al., 2015) and in the second phase of the formalin test (Bernabucci et al., 2012), and decreased mechanical hypersensitivity in an inflammatory pain model (subcutaneous CFA in the hindpaw) and in a neuropathic pain model (chronic constriction injury, CCI) (Bernabucci et al., 2012). The effects of NAC were blocked by an mGluR2/3 antagonist (LY341495) (Bernabucci et al., 2012). The antinociceptive effect of NAC in the formalin pain model was lost in mGluR2, but not mGluR3, knockout mice (Bernabucci et al., 2012), which points to an action on mGluR2.

Drug-induced potentiation of the transcription of GRM2, the gene encoding for mGluR2, has been used to assess antinociceptive effects of increased expression of mGluR2 in dorsal root ganglia and spinal dorsal horn (see Chiechio and Nicoletti, 2012). Indeed, epigenetic drugs such as LAC (Chiechio et al., 2002) and HDAC inhibitors (Chiechio et al., 2009) showed antinociceptive effects in different pain models. Systemic (s.c.) application of LAC decreased mechanical and thermal hypersensitivity in a neuropathic pain model (CCI) through increased expression of mGluR2 but not mGluR3 (Chiechio et al., 2002). Systemic (s.c.) application of HDAC inhibitors reduced the nociceptive response in the second phase of the formalin test by up-regulation of mGluR2 expression (Chiechio et al., 2009). Spinal (i.th.) administration of HDAC inhibitors attenuated the pronociceptive effect of estrogen on visceral sensitivity (increased visceromotor response to colorectal distension) and increased mGluR2 but not mGluR3 expression (Cao et al., 2015), which is consistent with a predominant action on mGluR2.

For the study of mGluR3 function, the neuropeptide N-acetylaspartylglutamate (NAAG) has been tested as a preferential activator of mGluR3 (Neale et al., 2000; Neale, 2011). Consistent with its wide distribution throughout the nervous system, local peripheral (s.c.) application of NAAG inhibited mechanical allodynia in the carrageenaninduced hindpaw inflammatory pain model (Yamamoto et al., 2007) and intracerebroventricular (i.c.v.) administration was antinociceptive in both phases of the formalin pain test (Yamamoto et al., 2008). Another strategy to target mGluR3 is to increase NAAG levels with NAAG peptidase inhibitors such as ZJ-11, ZJ-17 and ZJ-43, ZJ-45 or 2-PMPA, to block NAAG degradation (Neale et al., 2005). NAAG peptidase inhibitors administered systemically or peripherally or locally into CNS regions had antinociceptive effects in models of inflammatory and neuropathic pain. Systemic (intravenous, i.v., or i.p.) application decreased nociceptive behaviors (flinching) in both phases of the formalin pain test (Yamamoto et al., 2004; Nonaka et al., 2017) and had antiallodynic effects in a neuropathic pain model (partial sciatic nerve ligation) without affecting baseline mechanical and thermal sensitivity in the von Frey and hot plate tests, respectively (Yamamoto et al., 2004). Peripheral (s.c.) injection also decreased both phases of the formalin pain test and had anti-allodynic effects in the carrageenan pain model (Yamamoto et al., 2007). Spinal (i.th.) application inhibited nocifensive responses (flinching) in both phases of the formalin pain test and mechanical allodynia in the partial sciatic nerve ligation model, but had no effect in the von Frey and hot plate tests (Yamamoto et al., 2004). Microinjections into PAG or rostral ventromedial medulla (RVM) inhibited nociceptive behaviors (flinching) in both phases of the formalin pain test but had no effect in the hot plate test (Yamada et al., 2012). Injections into the locus coeruleus had similar antinociceptive effects in the formalin test (Nonaka et al., 2017). Intracerebroventricular administration also reduced nociceptive behaviors in both phases of the formalin pain test response (Yamamoto et al., 2008). Antinociceptive effects were blocked with a group II mGluR antagonist (LY341495) where tested in these studies. While there has been some controversy regarding the selective activation of mGluR3 with NAAG and peptidase inhibitors (for Discussion, see Neale, 2011) studies from mGluR2 and mGluR3 knockout mice provide strong evidence for mGluR3 mediated effects (Olszewski et al., 2017).

#### PHARMACOLOGICAL MANIPULATION OF mGluR2/3 IN PAIN: ELECTROPHYSIOLOGICAL STUDIES

Effects of group II mGluR agonists or antagonists on pain-related neuronal activity were studied in primary sensory neurons, spinal dorsal horn, and a few brain regions (amygdala and mPFC). Drugs were typically administered locally and there is surprisingly little information available for neuronal effects of systemic drug application and their site(s) of action. To the best of our knowledge, only a recent study showed inhibitory effects of a systemically (i.v.) applied mGluR2/3 agonist (LY2934747) on extracellularly recorded background activity and electrically evoked (C-fiber stimulation) wind-up discharges of dorsal horn

Mazzitelli et al. Group II mGluRs and Pain

neurons in neuropathic rats (SNL model) (Johnson et al., 2017).

#### Peripheral

A group II agonist (APDC) inhibited extracellularly recorded activity (action potentials) of nociceptive fibers evoked by capsaicin or by an inflammatory soup, and blocked the inflammatory soup- or forskolin-induced sensitization of heat responses in an in vitro rat skin-nerve preparation (Du et al., 2008). APDC had no effect on baseline heat or mechanical thresholds or discharges (Du et al., 2008; Carlton et al., 2011). APDC reversed the PGE2-induced hyperexcitability of cultured mouse and human primary sensory (dorsal root ganglia, DRG) neurons (Davidson et al., 2016). Interestingly, APDC blocked the PGE2-induced sensitization of capsaicin responses (calcium influx) in cultured mouse, but not human, DRG neurons (Yang and Gereau, 2002; Sheahan et al., 2018) through a mechanism that involved Gi dependent inhibition of adenylyl cyclase (Yang and Gereau, 2002).

Group II mGluR antagonists (LY341495 or APICA) enhanced the capsaicin-induced action potentials of nociceptive fibers in the skin nerve preparation or calcium signals in DRG neurons, but had no effect alone, suggesting that group II mGluRs act endogenously to reverse hypersensitivity (Carlton et al., 2011). In presence of excess extracellular glutamate in the skin nerve preparation, the blockade of the group II mGluRs also increased activity and heat responses of nociceptive fibers, which was interpreted to suggest that GluR2/3 activation by the exogenous glutamate decreased nociceptor activity and this activitydependent autoinhibition of nociceptive signal transmission to the CNS would modulate pain sensitivity (Carlton et al., 2011).

#### Spinal

Intrathecal administration of a group II agonist, ACPD, inhibited electrically evoked C-fiber responses of dorsal horn neurons recorded extracellularly in anesthetized rats with a carrageenaninduced hindpaw inflammation (3 h postinduction), but had mixed (excitatory or inhibitory) effects in normal animals (Stanfa and Dickenson, 1998). Administration of group II mGluR agonists (LY379268 and L-CCG-I) into the spinal dorsal horn by microdialysis decreased the central sensitization of primate (Macaca fascicularis) spinothalamic tract cells induced by intradermal capsaicin (30 min postinduction), but had no effect on the responses of non-sensitized neurons to innocuous and noxious cutaneous mechanical stimuli (Neugebauer et al., 2000). Information about the role of spinal group II mGluRs and their subtypes nociception and pain models is surprisingly thin, and given the mixed results of behavioral studies (see the section "Spinal") the spinal cord may not be the main target of their overall beneficial effects related to pain.

#### Brain

Actions of group II mGluR compounds have been studied in the amygdala, a key player in emotions, emotional aspects of pain and pain modulation (Neugebauer et al., 2004; Thompson and Neugebauer, 2017), and in the mPFC, a center for executive functions and behavioral control related to negative emotions and pain (Neugebauer et al., 2009; Ong et al., 2018). In anesthetized rats, stereotaxic administration (microdialysis) of a group II mGluR agonist (LY354740) into the central nucleus of the amygdala (CeA) targeting its laterocapsular division (CeLC), which is also referred to as the "nociceptive amygdala," decreased the responses of CeLC neurons to innocuous and noxious mechanical stimuli under normal conditions, but became more potent in an arthritis pain model (kaolin-carrageenan-induced monoarthritis in the knee) (Li and Neugebauer, 2006). The agonist effects were blocked by co-administration of a group II antagonist (EGLU), which by itself had no effect under normal conditions but increased the evoked responses to noxious stimulation of the arthritic knee in the pain model (Li and Neugebauer, 2006). This would be consistent with endogenous activation and gain of function of mGluR2/3 in the amygdala in a pain condition. Patch-clamp recordings of CeLC neurons in rat brain slices showed that a group II mGluR agonist (LY354740) inhibited excitatory synaptic inputs (EPSCs) from the parabrachial area, which provide nociceptive information to the amygdala (Neugebauer et al., 2004; Thompson and Neugebauer, 2017), under normal conditions, but became more potent in the arthritis pain condition (Han et al., 2006; Kiritoshi and Neugebauer, 2015). LY354740 decrease frequency, but not amplitude, of miniature EPSCs in the presence of TTX, suggesting a presynaptic site of action on the glutamatergic terminals (Han et al., 2006; Kiritoshi and Neugebauer, 2015). EGLU blocked the agonist effect, but had no effect on its own, which is similar to the lack of significant effects of another group II mGluR antagonist (LY341495) at the presumed parabrachial (PB)-CeLC synapse in mouse brain slices (Adedoyin et al., 2010), suggesting that the endogenous activation observed in the in vivo condition (see above) may be lost in the reduced brain slice preparation (Han et al., 2006; Kiritoshi and Neugebauer, 2015).

In the infralimbic mPFC, rat brain slice physiology experiments found that group II mGluRs decreased the output of principal layer V pyramidal cells as the result of an inhibitory action on glutamatergic synapses under normal conditions and in an arthritis pain model (kaolincarrageenan-induced monoarthritis in the knee), and that this system was tonically active under both conditions (Kiritoshi and Neugebauer, 2015; Thompson and Neugebauer, 2017). Specifically, a selective group II mGluR agonist (LY379268) decreased synaptically evoked spiking of pyramidal cells in brain slices from normal and arthritic rats by inhibiting direct excitatory inputs (EPSCs) as well as glutamate-driven feedforward inhibitory transmission (IPSCs) (Kiritoshi and Neugebauer, 2015; Thompson and Neugebauer, 2017). Abnormally enhanced synaptic inhibition of mPFC output in pain conditions has been linked to cognitive dysfunction (Ji et al., 2010; Kiritoshi et al., 2016) and loss of cortical control of amygdala function (Ji and Neugebauer, 2014; Kiritoshi and Neugebauer, 2018). Effects of LY379268 on EPSCs preceded those on IPSCs, resulting in a net inhibitory effect on pyramidal output. Spontaneous and miniature (in TTX) analyses of EPSCs and IPSCs showed that LY379268 acted presynaptically on glutamatergic, but not GABAergic, terminals. The effects of LY379268 were blocked by a selective group II mGluR antagonist

(LY341495) that by itself increased synaptically evoked spiking of pyramidal cells under normal conditions and in the pain model (Kiritoshi and Neugebauer, 2015; Thompson and Neugebauer, 2017), suggesting endogenous activation of mGluR2/3. It has been speculated that failure to release this inhibitory tone to enhance mPFC output could be a mechanism of pain persistence due to a lack of cortical control (Kiritoshi and Neugebauer, 2015; Thompson and Neugebauer, 2017).

#### Subtype Selective Interventions

In mouse amygdala brain slices, exogenous NAAG and a NAAG peptidase inhibitor (ZJ-43) (see the section "Subtype Selective Interventions") have been tested in order to elucidate the specific contribution of mGluR3. Under normal conditions, NAAG and ZJ-43 inhibited excitatory transmission (EPSCs) at the presumed PB-CeLC synapse (see the section "Brain") similarly to an mGluR2/3 agonist (SLx-3095-1) (Adedoyin et al., 2010), which is consistent with an mGluR3 effect. The effect of ZJ-43 was blocked by a group II mGluR antagonist (LY341495). In the formalin pain model (brain slices taken 24 h postinduction), ZJ-43 was much less efficacious than SLx-3095-1 in inhibiting EPSCs, suggesting a decreased release of NAAG or an increased contribution of mGluR2 rather than mGluR3 in the pain condition (Adedoyin et al., 2010).

#### CLINICAL TRIALS AND POTENTIAL CLINICAL USES

Pain conditions affect millions of people, and pain management can be challenging and often is insufficient with currently available tools. Based on several lines of evidence from preclinical studies, drugs acting on mGluR2/3 may be useful for pain relief, but so far have not advanced to clinical trials as analgesic candidates, perhaps because of concerns about the translation from animal models to the human condition (Davidson et al., 2016). However, despite concerns for example about the development of tolerance in some rodent studies, there is no evidence for a loss of efficacy on repeat dosing of group II mGluR agonists in humans (Johnson et al., 2017). In fact, significant anxiolytic efficacy of an oral prodrug (LY544344) of an mGluR2/3

#### REFERENCES

agonist (LY354740) was observed in patients with generalized anxiety (Dunayevich et al., 2008). An oral prodrug (LY2140023, pomaglumetad methionil) of an mGluR2/3 agonist (LY404039) had significant antispychotic efficacy in schizophrenia patients (Patil et al., 2007) or in subgroups of schizophrenia patients (Kinon et al., 2015; Nisenbaum et al., 2016). In these studies, drug effects were maintained or enhanced following several weeks dosing (Johnson et al., 2017). It should be noted that NAC, which has been linked to the endogenous activation of mGluR2/3 (see the section "Subtype Selective Interventions"), given orally to healthy human subjects, decreased thermal pain ratings to laser stimuli and amplitudes of laser-evoked brain potentials without affecting thermal pain thresholds (Truini et al., 2015).

### CONCLUSION

Preclinical studies suggest that group II mGluRs play a significant role in the modulation of nociception and pain conditions. There is some evidence to suggest distinct roles of mGluR2 and mGluR3 subtypes in different neural circuits and regions, but this remains to be determined more thoroughly with the availability of more selective compounds such as allosteric modulators. These new tools are also useful for the analysis of the pathophysiological mechanisms of pain conditions. Effectiveness of mGluR2/3 compounds in clinical studies on conditions other than pain may support their therapeutic potential for the management of pain (Johnson et al., 2017).

#### AUTHOR CONTRIBUTIONS

MM collected information and provided first draft of manuscript. EP and SM provided valuable input to the manuscript. VN conceived the project and finalized the manuscript.

#### FUNDING

Work in the authors' laboratory was supported by National Institute of Health (NIH) grants NS038261, NS081121, and NS106902.

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**Conflict of Interest Statement:** The 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.

Copyright © 2018 Mazzitelli, Palazzo, Maione and Neugebauer. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Epigenetic Alterations in Prenatal Stress Mice as an Endophenotype Model for Schizophrenia: Role of Metabotropic Glutamate 2/3 Receptors

#### Francesco Matrisciano1 \*, Erbo Dong2 , Ferdinando Nicoletti 3,4 and Alessandro Guidotti 1,2

1 Department of Psychiatry, Psychiatric Institute, College of Medicine, University of Illinois Chicago, Chicago, IL, United States, 2 Department of Psychiatry, Center for Alcohol Research in Epigenetics College of Medicine, University of Illinois Chicago, Chicago, IL, United States, 3 Department of Physiology and Pharmacology, University of Rome "Sapienza", Rome, Italy, 4 IRCCS, Neuromed, Pozzilli, Italy

Mice subjected to prenatal restraint stress (PRS mice) showed biochemical and behavioral abnormalities consistent with a schizophrenia-like phenotype (Matrisciano et al., 2016). PRS mice are characterized by increased DNA-methyltransferase 1 (DNMT1) and ten-eleven methylcytosine dioxygenase 1 (TET1) expression levels and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Activation of group II metabotropic glutamate receptors (mGlu2 and−3 receptors) showed a potential epigenetically-induced antipsychotic activity by reversing the molecular and behavioral changes observed in PRS mice. This effect was most likely caused by the increase in the expression of growth arrest and DNA damage 45-β (Gadd45-β) protein, a molecular player of DNA demethylation, induced by the activation of mGlu2/3 receptors. This effect was mimicked by clozapine and valproate but not by haloperidol. Treatment with the selective mGlu2/3 receptors agonist LY379268 also increased the amount of Gadd45 β bound to specific promoter regions of reelin, BDNF, and GAD67. A meta-analysis of several clinical trials showed that treatment with an orthosteric mGlu2/3 receptor agonist improved both positive and negative symptoms of schizophrenia, but only in patients who were early-in-disease and had not been treated with atypical antipsychotic drugs (Kinon et al., 2015). Our findings show that PRS mice are valuable model for the study of epigenetic mechanisms involved in the pathogenesis of schizophrenia and support the hypothesis that pharmacological modulation of mGlu2/3 receptors could impact the early phase of schizophrenia and related neurodevelopmental disorders by regulating epigenetic processes that lie at the core of the disorders.

Keywords: mGlu2/3 receptors, schizophrenia, clozapine, epigenetics, prenatal stress

#### Edited by:

Volker Eulenburg, Universitätsklinikum Leipzig, Germany

#### Reviewed by:

Anna Maria Pittaluga, Università di Genova, Italy Bashkim Kadriu, National Institute of Mental Health (NIMH), United States

> \*Correspondence: Francesco Matrisciano<EMAIL_ADDRESS>
Received: 14 August 2018 Accepted: 30 October 2018 Published: 30 November 2018

#### Citation:

Matrisciano F, Dong E, Nicoletti F and Guidotti A (2018) Epigenetic Alterations in Prenatal Stress Mice as an Endophenotype Model for Schizophrenia: Role of Metabotropic Glutamate 2/3 Receptors. Front. Mol. Neurosci. 11:423. doi: 10.3389/fnmol.2018.00423

## HIGHLIGHTS

## INTRODUCTION

Schizophrenia is a major psychotic disorder which affects one percent of the world's population and usually leads to a severe mental disability (Ribe et al., 2015). All marketed antipsychotic drugs antagonize D2 dopamine and 5-HT2A serotonin receptors, showing good clinical efficacy in improving positive symptoms, and moderate activity in improving negative symptoms of schizophrenia. None of these drugs has significant activity on cognitive symptoms associated with schizophrenia, with the possible exception of clozapine (Lieberman et al., 2005). Treatment of cognitive and negative symptoms remains an unmet need in the treatment of schizophrenia, and this encourages the identification and validation of novel drug targets. Etiology of schizophrenia is still unknown despite the recent progresses made possible by molecular genetics and functional neuroimaging. It is generally believed that schizophrenia is not caused by a single factor, but results from the convergence of genetics and environmental factors. Recently, an imbalance between GABA and glutamate neurotransmission has been suggested as a key mechanism underlying the pathophysiology of schizophrenia. Drugs that block the activity of NMDA receptors present on cortical-limbic GABAergic interneurons, such as ketamine or phencyclidine (PCP) are capable to replicate the full range of psychotic symptoms, including hallucinations (Kristiansen et al., 2007; Meltzer et al., 2011). Thus, novel antipsychotic drug development should focus on the GABA and glutamate systems, which act upstream of the dopamine circuit, and are primarily involved in the pathophysiology of the disorder (**Figure 1**). A hypofunction of the NMDA receptors on GABAergic interneurons, particularly fast-spiking, parvalbuminpositive chandelier and basket cells, leads to an overactivity of pyramidal neurons and to an impairment of network oscillations that underlie multiple domains of cognitive function (Homayoun and Moghaddam, 2007; Gonzalez-Burgos and Lewis, 2008). The release of GABA is crucial for the normal firing of pyramidal neurons in the prefrontal cortex and for the equilibrium of the subcortical regions fundamental for optimizing cognitive and emotional function (Benes et al., 2007). Schizophrenia is a chronic devastating disorder that leads to a severe disability at relatively young age. It has a peculiar pathological course starting with the prodromal phase followed by a first episode, which occurs around adolescence or young adult age (Millan, 2012). Evidence suggests that epigenetic changes, occurring during early development as a result of the combination of a predisposing genetic background, in shaping the premorbid phase of the disease, and environmental factors, acting as "second hits," precipitate the onset of schizophrenia (Guidotti et al., 2005). We and others have studied the epigenetic hallmarks of schizophrenia in postmortem human brain tissue. Moving from these findings, we have investigated whether the same epigenetic changes occur in the brain of mice subjected to prenatal stress at different stages of postnatal development. The purpose of this review is to provide an update (i) of our current findings and knowledge of the topic of neuroepigenetics in schizophrenia, (ii) of the role of metabotropic glutamate 2/3 receptors in prenatally stressed mice (PRS mice) as potential targets for novel antipsychotics; and (iii) to show our more recent observations on the epigenetic effects induced by the mGlu2/3 receptors agonist, LY379268, and by clozapine.

### EPIGENETIC CHANGES IN SCHIZOPHRENIA

Neuroepigenetic dysregulations were detected in the hippocampus and cortex of brain of patients affected by schizophrenia (Numata et al., 2014; Dong et al., 2015). Epigenetics is defined as modifications of the genome, heritable during cell division, that do not involve a change in DNA sequence. Epigenetic mechanisms are considered to mediate gene-environment interplay during the entire lifespan. Several clinical evidence support a role of altered epigenetic mechanisms underlying embryonic, postnatal, and adult neurogenesis (Roth et al., 2011). Aberrations in the epigenetic regulation machinery have been hypothesized in neurodevelopmental disorders, such as schizophrenia and autism spectrum disorders (Zhubi et al., 2017). A growing body of evidence from Dr. Guidotti's group (Matrisciano et al., 2012, 2013, 2016) and other researchers (Meaney and Szyf, 2005; Benes et al., 2007; McGowan and Szyf, 2010) suggest that epigenetic modifications of DNA (promoter methylation) and chromatin remodeling induced by environmental factors, including stress, may contribute to the complex phenotypes of neuropsychiatric disorders, such as schizophrenia. DNA methyltransferases (DNMT1 and 3a) (the enzymes that transfer a methyl group from S-adenosylmethionine to carbon 5 of the cytosine pyrimidine ring embedded in cytosine-phosphoguanine [CpG] islands containing promoters), and ten-eleven translocation hydroxylase (TET 1,2,3), (the enzymes that catalyze the conversion of 5MC to 5HydroxyMC), are important components of the DNA- methylation/demethylation pathways regulating the expression of key molecules involved in brain development and maturation. Importantly, the prefrontal cortex GABAergic interneurons of schizophrenia patients express an increase in DNMT1 and 3a, and an increase in TET1 associated with deficits in GABAergic function (Guidotti et al., 2011). This includes the downregulation of the

glutamic acid decarboxylase 67 (GAD), reelin, GABA reuptake transporters and brain derived neurotrophic factor (BDNF), which are essential for neurogenesis, neurodevelopmental neuronal migration and synaptic plasticity. In addition to alterations of GABAergic transmission (Veldic et al, 2007; Lewis and González-Burgos, 2008; Kundakovic et al., 2009; Marek, 2010; Niswender and Conn, 2010; Marín, 2012), there are evidence of altered glutamatergic transmission involving both ionotropic glutamate receptors such as NMDA receptors present post-synaptically on GABAergic interneurons (**Figure 1**) and metabotropic glutamate receptors such as mGlu2/3 receptors located at least in part pre-synaptically on thalamocortical glutamate axon terminals [Benes et al., 2007; Stansley and Conn, 2018; **Figure 1**] where they modulate the glutamatergic transmission by restoring the hyperactivity of the cortical pyramidal neurons (**Figure 1**). Clinically, while NMDA receptor agonists are not fully considered as antipsychotic drugs due to their potential excitotoxicity effects and neuronal damage, metabotropic glutamate 2/3 receptors represent a suitable target for glutamatergic tone regulation.

Taken together, these observations suggest that the epigenetic hypothesis of schizophrenia is supported by the following evidence obtained in postmortem human brain: (i) alterations of glutamic acid decarboxylase 67 (gene symbol = GAD1), DNMT and reelin in GABAergic neurons, and (ii) of brain derived nerve growth factor (BDNF), vesicular glutamate transporter (VGLUT1) and (iii) alterations in mGlu2/3 receptors in glutamatergic neurons (Guidotti et al., 2011; Nicoletti et al., 2011).

#### THE PRS AS A SUITABLE ANIMAL MODEL TO STUDY NEURODEVELOPMENTAL DISORDERS

Prenatal or early-life stress, through changes in the epigenetic mechanisms, has been considered a predisposing factor for major neuropsychiatric disorders including schizophrenia, bipolar disorders, and autism spectrum disorder. Time- and spatialdependent neurodevelopmental cues associated with neuronal differentiation and synaptic plasticity support the hypothesis that these disorders might originate even before birth. Interestingly, we have reported that adult offspring of mice exposed to repeated episodes of restraint stress during pregnancy, named PRS mice, exhibit a schizophrenia-like behavioral phenotype characterized by hyperactivity, stereotyped and compulsive behavior, deficits in social interaction and pre-pulse inhibition (PPI), altered fear conditioning, object recognition, and hypersensitivity to N-methyl D-aspartate (NMDA) receptor blockers (Matrisciano et al., 2016). This behavioral phenotype recapitulates positive and negative symptoms, as well as cognitive dysfunction displayed by patients affected by schizophrenia (Matrisciano et al., 2013). PRS mice also show a deficit in cortical GABAergic innervation, which is expected to cause abnormal synchronization of the firing rate of pyramidal neurons, a putative electrophysiological substrate of cognitive dysfunction in psychotic patients and neurodevelopmental animal models of SZ (Gonzalez-Burgos and Lewis, 2008). In addition to alterations of GABAergic system, PRS mice show molecular disruption in chromatin remodeling at genes expressed in glutamatergic neurons, such mGlu2/3 receptors (**Figure 1**). These molecular changes in PRS mice are similar to those observed in the brain of schizophrenia patients, suggesting a strong correlation between the aberrant epigenetic GABAergic/glutamatergic mechanisms and psychotic symptoms. **Table 1** summarize the behavioral and molecular features observed in PRS mice and Schizophrenia patients.

#### ROLE OF METABOTROPIC GLUTAMATE 2/3 RECEPTORS IN SCHIZOPHRENIA

Disruption in the glutamatergic system is considered to play a key role in the pathophysiology of schizophrenia (Akbarian et al., 1995). It has been reported that patients affected by schizophrenia respond only partially to standard "monoaminergic" antipsychotic drugs (Lieberman et al., 2008; Meltzer, 2013). The lack of a full recovery from negative and cognitive symptoms gave the impetus to investigate different molecular targets including mGlu2 and mGlu3 receptors. mGlu receptors, which belong to class C of the G proteincoupled receptors form a family of eight subtypes traditionally subdivided into three groups based on sequence homology, intracellular signaling and pharmacological profile. mGlu1 and mGlu5 receptors (group I) are coupled to Gq/11 and their activation stimulates polyphosphoinositide hydrolysis with ensuing formation of inositol-1,4,5-trisphosphate and

TABLE 1 | Epigenetic and behavioral deficits found in schizophrenia patients and in PRS mice.

(+): presence. Abbreviations: BDNF, brain-derived neurotrophic factor; DNMT1, DNA-methyltransferase 1; TET1, ten-eleven methylcytosine dioxygenase 1; SI, social interaction, NMDA, N-methyl D-aspartate; PPI, pre-pulse inhibition; GAD67, glutamic acid decarboxylate; 5HMC, 5-hydroxymethylcytosine; 5MC, 5methylcytosine

diacylglycerol. These receptors are localized in the peripheral portions of postsynaptic densities [reviewed by (Nicoletti et al., 2011)]. mGlu2 and mGlu3 receptors are coupled to Gi/o and their activation inhibits adenylyl cyclase activity and modulate the activity of calcium and potassium channels. Both receptors are localized in axon terminals where they negatively modulate neurotransmitter release [(Nicoletti et al., 2011); **Figure 1**]. However, recent findings indicate that mGlu3 receptors are also localized in postsynaptic densities, where they boost mGlu5 receptor signaling (Di Menna et al., 2018). mGlu4, mGlu7, and mGlu8 receptors (group III) are also coupled to Gi/o and are found in presynaptic terminals close to the active zone of neurotransmitter release (Nicoletti et al., 2011). Symptoms of schizophrenia are thought to be associated, at least in part, with hyperactive and dysregulated glutamatergic neurotransmission in key brain regions, such as the thalamus, prefrontal cortex, and limbic system. Based on this evidence, pharmacological activation of mGlu2/3 receptors may ameliorate the schizophrenia symptoms through a decrease in glutamate release thereby reducing synaptic firing due to the particular synaptic distribution of these receptors and causing neuroprotective effects. Interestingly, PRS mice showed a decrease in the expression of mGlu2 and mGlu3 receptor mRNA and proteins in the frontal cortex. This decrease manifest at birth and, at least for mGlu2 receptors, persisted in adult life associated with an increase in the DNMT binding to the gene promoter [for more details see (Matrisciano et al., 2013)] suggesting an epigenetic regulation of the receptors induced by prenatal stress and it may reflect a key factor for the pathogenesis of the disease.

In schizophrenia research, particular attention has been paid to group-II mGlu receptors on the basis of genetic and pharmacological data (Gregory and Conn, 2015).

An initial hypothesis was that activation of mGlu2/3 receptors could improve psychotic symptoms by inhibiting glutamate release (Battaglia et al., 1997), and, therefore, restraining the hyperactivity of pyramidal neurons associated with schizophrenia. However, this mechanism may also amplify the defect in glutamate-mediated activation of GABAergic interneurons, thus worsening the "glutamatergic hypofunction" that underlies cognitive dysfunction in schizophrenia. The evidence that activation of mGlu2 receptors inhibits electrophysiological responses mediated by 5-HT2A receptors at thalamo-cortical synapses (Aghajanian and Marek, 2000) shed new light into the defensive role played by mGlu2 receptors in schizophrenia. Javier Gonzales-Maeso and his Associates have consistently shown that mGlu2 and 5-HT2A receptors form functional multimeric complexes, in which mGlu2 receptors negatively modulate 5-HT2A receptor signaling (González-Maeso et al., 2008). Interestingly, opposite changes in the expression of mGlu2 and 5-HT2A receptors were found in postmortem brain tissue from patients affected by schizophrenia, with the physiological balance between the two receptors being shifted toward 5-HT2A receptors (Muguruza et al., 2013). This is nicely consistent with the reduced expression of mGlu2 receptors in the prefrontal cortex found across the postnatal development of PRS mice, which show a schizophrenia-like phenotype in the adult life (Matrisciano et al., 2016). Interestingly, treatment with atypical antipsychotics down-regulates mGlu2 receptors in the prefrontal cortex as a result of an epigenetic mechanism that, in this particular case, is mediated by histone deacetylation at the Grm2 gene promoter (Kurita et al., 2012). This contributes to explain why schizophrenic patients who had received a previous treatment with atypical antipsychotics failed to respond to pomeglumetad (Kinon et al., 2015), and raise the interesting possibility that acetylating drugs (e.g., inhibitors of histone deacetylases) may boost the activity of mGlu2 receptor agonists or positive allosteric modulators (PAMs) in the clinic. The mGlu2-centric scenario in the treatment of schizophrenia is supported by a large number of preclinical studies showing that the behavioral effects of orthosteric mGlu2/3 receptor agonists in behavioral tests that are predictive of antipsychotic activity are abrogated in mGlu2 receptor knockout mice, and that selective mGlu2 receptor PAMs display robust antipsychotic-like activity in rodents (Gerwirtz and Marek, 2000; Schoepp and Marek, 2002; Egan et al., 2004; Benneyworth et al., 2007; Patil et al., 2007; Conn et al., 2008). However, the following observations bring to a re-evaluation of mGlu3 receptors in schizophrenia research: (i) mice lacking mGlu3 receptors display a psychoticlike phenotype (Lainiola et al., 2014), and show developmental abnormalities in cortical GABAergic transmission (M. Cannella et al., unpublished observations); (ii) polymorphic variants of GRM3 are consistently associated with schizophrenia [reviewed by Maj et al., 2016], whereas no variants of GRM2 have been associated with psychiatric disorders; and, (iii) as reported above, mGlu3 receptors boost mGlu5 receptor signaling (Di Menna et al., 2018), and mGlu5 receptors are candidate drug targets in the treatment of schizophrenia [reviewed by Foster and Conn, 2017]. It is noteworthy that expression of mGlu3 receptors was also reduced in the prefrontal cortex of PRS mice, although this reduction was significant at 1 and 9 days of postnatal life, but not after weaning (Matrisciano et al., 2013). In clinical studies, systemic treatment with pomeglumetad methionyl, an oral prodrug of the mGlu2/3 receptor agonist, LY404039, showed antipsychotic activity in specific subgroups of population investigated, such as early-in-onset episodes and no history of atypical antipsychotic drug treatment which is known to epigenetically down-regulate mGlu2 receptors in mice, supporting the hypothesis that the additional preclinical studies and the identification of an appropriate target subgroup with altered glutamatergic tone are required to study these compounds (Kinon et al., 2015).

### THE EPIGENETIC "ENDOPHENOTYPICAL" MOUSE MODEL FOR SCHIZOPHRENIA (PRS): ROLE OF METABOTROPIC GLUTAMATE 2/3 RECEPTORS

Urged by the need to use a neurodevelopmental animal model to study the epigenetic status at each neurodevelopmental stage of schizophrenia, we investigated the molecular and behavioral abnormalities found in the brain of the offspring of dams stressed during pregnancy (PRS mice). PRS mice showed a marked and long-lasting increase in DNMT enzymes (both 1 and 3a), and TET enzymes and a significant increase in 5 methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) in the promoters of putative schizophrenia-related genes, such as bdnf, gad1, reln, and the early inducible gene, GADD45 (growth arrest DNA demage), associated with an alteration in these gene expression.

To the best of our knowledge, the PRS model represents a promising model to study the natural course of major psychosis including schizophrenia compared, for example, to the phencyclidine (PCP) model which is a well-established pharmacological-induced model for schizophrenia reflecting the positive symptoms through the blockade of the NMDA receptors. Schizophrenia and autism spectrum disorders are considered diseases of neurodevelopment, characterized by a natural course, starting with a prodromal phase, first episode during adolescence or early adulthood, followed by relapses/remitted periods and eventually leading to brain function deterioration that ensues over subsequent adult years. Hence, the epigenetic history of such complex neurodevelopmental disorders cannot be adequately studied only in the postmortem brains of chronic SZ patients. We then have focused on studying the epigenetic signature of schizophrenia in offspring of PRS mice.

We also used PRS mice for the study of the role played by mGlu2 and mGlu3 receptors in the pathophysiology of schizophrenia based on clinical findings with pomeglumetad methionyl, an oral prodrug of the mGlu2/3 receptor agonist, LY404039. This drug showed an efficacy similar to the comparator, olanzapine, on positive and negative symptoms of schizophrenia in a phase-2 clinical trial (Patil et al., 2007), but not in subsequent trials. However, an exploratory analysis of all clinical studies confirmed the antipsychotic activity of pomeglumetad in schizophrenic patients who were early-indisease or had not been treated with atypical antipsychotic drugs (Kinon et al., 2015).

We found that expression of mGlu2 and mGlu3 receptors was reduced in the frontal cortex of PRS mice (Matrisciano et al., 2012; Holloway et al., 2013), as a result of an increased binding of DNMT1 and methylcytosine binding protein-2 (MeCP2) to the Grm2 gene promoter (Matrisciano et al., 2012; **Figure 1**).

As summarized in **Table 1**, PRS adult offspring showed alterations in the epigenetic regulation of schizophreniarelated gene as reelin, GAD67, BDNF, and mGlu2/3 receptors. Behaviorally, adult PRS-mice showed deficits similar to those observed in psychotic patients such as abnormalities in social interaction, locomotor activity, and pre-pulse inibition (PPI). In addition, we found epigenetic abnormalities such as a marked increase in the expression of DNMT1, DNMT3a, and TET, a significant increase in 5-methylcytosine (5MC) and 5 hydroxymethylcytosine (5HMC) in the promoters of putative schizophrenia-related genes, such as bdnf, gad1, reln, and the early inducible gene, GADD45 (growth arrest DNA demage). Interestingly, the biochemical and behavioral abnormalities of PRS mice were corrected by the treatment with LY379268 (Matrisciano et al., 2012; Holloway et al., 2013), an orthosteric agonist of mGlu2/3 receptors, which shows "therapeutic efficacy" in a range of animal models used to predict antipsychotic activity (Cartmell et al., 1999, 2000; Carter et al., 2004).

BDNF, brain-derived neurotrophic factor; DNMT1, DNA-methyltransferase 1; TET1, teneleven methylcytosine dioxygenase 1. Gadd45 (Growth – arrest and DNA damage; MeCP2, Methylcytosine binding protein 2.

In PRS mice, considered by us as a neurodevelopmental endophenotypical model for schizophrenia, expression levels of epigenetic biomarkers can be assessed at different phases of development in order to further elucidate the underlying pathogenetic mechanisms and predicting treatment responses at specific stages of the disease, with particular attention to early detection and possibly early intervention.

Little is known on the action of antipsychotics on specific epigenetic mechanisms in GABAergic or glutamatergic neurons. Thus, PRS mice represent a valid and suitable model for drug testing and development.

#### CLOZAPINE AND THE mGlU2/3 RECEPTOR AGONIST LY379268: EPIGENETIC EFFECTS IN THE PRS MOUSE MODEL FOR SCHIZOPHRENIA

Clozapine, the prototype of atypical antipsychotics, is considered the drug of choice in patients with treatment-resistant schizophrenia due, in our opinion, to its unique chromatin remodeling properties. We have shown that clozapine reversed the behavioral deficits and induced chromatin remodeling in PRS mice that are resistant to haloperidol treatment (Dong et al., 2016). We recently studied the epigenetic mechanisms underlying the efficacy as potential antipsychotic-like activity of the mGlu2/3 receptors agonist, LY379268, as compared to the activity of clozapine, in PRS mice. **Table 2** summarizes the epigenetic effects of clozapine and LY379268 in the frontal cortex of PRS mice. Clozapine reversed promoter hypermethylation of schizophrenia-related genes such as bdnf, reln, and gad1 (Dong et al., 2016). Interestingly, these effects were shared by valproate, an anti-epileptic drug used for the treatment of bipolar disorder, which is chemically unrelated to clozapine, and induces demethylation of gene promoters presumably as a result of histone acetylation and chromatin opening. Both clozapine and LY379268 were able to reduce the overexpression of DNMT1 and TET found in the frontal cortex of PRS mice. This overexpression is similar to that found in brain tissue of patients affected by schizophrenia (Matrisciano et al., 2016). DNMT enzymes are responsible for the conversion of cytosines into 5-methyl-cytosines, whereas TET enzymes convert the 5MC residues into 5-hydroxymethylcytosines by hydroxylation reaction in a sequence of events of cytosines metabolism. Clozapine and LY379268 were also able to reverse the hypermethylation of schizophrenia-like promoter genes such as gad1, bdnf, and reln and the ensuing increase in their mRNA expression levels. In addition, LY379268 induced a decrease of MECP2 binding at the mGlu2, Gad1, and Bdnf gene promoters, whereas clozapine reversed DNMT binding at the promoters of schizophrenia-related genes. Both clozapine and LY379268 reversed the increase in locomotor activity in PRS mice and the deficits showed by these mice in social interaction tasks. These findings are consistent with the previous evidence that a combined treatment with clozapine and valproate reversed the downregulation of GAD67 expression induced by repeated methionine administration in mice (Guidotti et al., 2014). The same authors showed that the effects of clozapine on DNA-demethylation were mimicked by antipsychotic drugs chemically related to clozapine, such as the dibenzodiazepines, quetiapine and olanzapine, but not by the chemically unrelated risperidone (Guidotti et al., 2011). Thus, a more systematic and comprehensive analysis of the effects of different antipsychotics on the epigenetic signature in PRS mice is warranted. We reported that the strong effect of clozapine on DNA methylation in PRS mice and the lack of effect of clozapine in control mice cannot be considered as secondary to changes in dopaminergic or serotonergic genes such as D2, Htr1a, or Htr2a in the cortex of PRS mice. Of note, a correlation between the methylation state of schizophrenia-related genes and behavioral deficits exists (Dong et al., 2016). The increase in DNMT1 binding to selected Gad1, Reln, and Bdnf-ix regulatory regions in PRS mice was considerably reduced by clozapine treatment whereas haloperidol failed to reduce the increased DNMT1 binding in PRS mice, in agreement with previous results (Matrisciano et al., 2013). Clozapine and LY379268 may exert their antipsychotic activity either indirectly by decreasing DNMT and TET expression levels, and/or more directly by interfering with the DNMT1 or MeCP2 DNA-binding domains. In addition, both clozapine and LY379268 increased the expression levels of Gadd45-β (growth arrest and DNA-damage-inducible protein 45), a member of the Gadd45 family of small nuclear acidic proteins, which it was reported to facilitate DNA de-methyation (Ma et al., 2009; Matrisciano et al., 2011). Taken together, we can speculate that both clozapine and mGlu2/3 receptor agonists act in our model as epigenetic de-methylating agents, and because of that they may regulate processes that lies at the core of the pathophysiology of schizophrenia.

#### CONCLUSIONS

This review underlies the concept that the PRS mouse model has construct and face validity as an experimental epigenetic model of vulnerability for neurodevelopmental disorders such as schizophrenia, schizoaffective disorders, and autism. This mouse model is highly reproducible and useful for novel anti-psychotic drug screening acting on altered epigenetic mechanisms. Earlylife stressors, even during pregnancy, in mice lead to alterations of some molecular players of epigenetic mechanisms that are translated into a schizophrenia-like phenotype. A potential glutamate-based pharmacotherapy for schizophrenia remains, at least in part, a possibility that requires the identification of an appropriate subgroup of patients that satisfy specific criteria such as no previous history of atypical antipsychotic treatments and treatment onset in early phases of the disease. For preclinical studies, PRS mice represent a valid epigenetic "endophenotype" model for drug testing and development and for studying the pathogenesis of the disease. In our opinion, mGlu2/3 receptors, based on the peculiar role as modulators of glutamate transmission in the frontal cortex, can still represent a suitable target for novel antipsychotic medications targeting specific

#### REFERENCES

high-risk population with dysregulation of brain glutamatergic tone. Then, ligands acting on mGlu2 and 3 receptors, either orthosteric agonists or PAMs, require further experimental studies in PRS mice and other epigenetic models to identify the optimum receptors target and time window of intervention in the treatment of psychosis.

#### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

#### FUNDING

This work was supported by the Grant RO1MH0933 to AG RO1MH101013 to AG.

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**Conflict of Interest Statement:** The 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.

Copyright © 2018 Matrisciano, Dong, Nicoletti and Guidotti. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Negative Allosteric Modulators of mGlu7 Receptor as Putative Antipsychotic Drugs

Paulina Cieslik, Monika Wo ´zniak, Katarzyna Kaczorowska, Piotr Bra ´ nski, ´ Grzegorz Burnat, Agnieszka Chocyk, Bartosz Bobula, Piotr Gruca, Ewa Litwa, Agnieszka Pałucha-Poniewiera, Agnieszka W ˛asik, Andrzej Pilc and Joanna Wieronska ´ \*

Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland

The data concerning antipsychotic-like activity of negative allosteric modulators (NAMs)/antagonists of mGlu7 receptors are limited. The only available ligands for this receptor are MMPIP and ADX71743. In the present studies, we used stable cell line expressing mGlu7 receptor and it was shown that both compounds dose-dependently potentiated forskolin elevated cAMP concentration in the T-REx 293 cells, showing their inverse agonist properties. Subsequently, pharmacokinetic studies were performed. Both compounds were given intraperitoneally (i.p.) at the dose of 10 mg/kg and reached Cmax 0.25–0.5 h after administration, and then they declined rapidly, ADX71743 being almost undetectable 2 h after administration, while the concentration of MMPIP was still observed, suggesting that the concentration of MMPIP was more stable. Finally, we investigated the role of both mGlu7 receptor NAMs in animal models of schizophrenia. Behavioral tests commonly used in antipsychotic drug discovery were conducted. Both tested compounds dose-dependently inhibited MK-801-induced hyperactivity (MMPIP at 15 mg/kg; ADX at 5 and 15 mg/kg) and DOI-induced head twitches (MMPIP at 5, 10, 15 mg/kg; ADX at 2.5, 5, 10 mg/kg). Moreover, the same effects were noticed in novel object recognition test, where MMPIP (5, 10, 15 mg/kg) and ADX71743 (1, 5, 15 mg/kg) reversed MK-801-induced disturbances. In the social interaction test, antipsychotic activity was observed only for ADX71743 (5, 15 mg/kg). ADX71743 at the dose 2.5 mg/kg reversed MK-801-induced disruption in prepulse inhibition while MMPIP at 10 mg/kg reversed MK-801-induced disruption in spatial delayed alternation. The present studies showed that mGlu7 receptor may be considered as a putative target for antipsychotic drugs, though more studies are needed due to limited number of available ligands.

Keywords: schizophrenia, metabotropic glutamate receptor 7, antipsychotic, negative allosteric modulators, MMPIP, ADX71743

#### INTRODUCTION

Metabotropic glutamate receptors (mGluR) are being extensively studied as new pharmacological targets for central nervous system (CNS) disorders such as depression (Mitsukawa et al., 2006), anxiety (Swanson et al., 2005), schizophrenia (Conn et al., 2009; Nickols and Conn, 2014), neurodegenerative disorders (Gu et al., 2014; Litim et al., 2017), and pain (Acher and Goudet, 2015; Chiechio, 2016). Among these receptors, mGluR7 is one of the most conserved mGluR

#### Edited by:

Enza Palazzo, Università degli Studi della Campania Luigi Vanvitelli Naples, Italy

#### Reviewed by:

Danilo De Gregorio, McGill University, Canada Francesco Matrisciano, University of Illinois at Chicago, United States

#### \*Correspondence:

Joanna Wieronska ´<EMAIL_ADDRESS>
Received: 12 June 2018 Accepted: 16 August 2018 Published: 20 September 2018

#### Citation:

Cieslik P, Wo ´zniak M, Kaczorowska K, ´ Branski P, Burnat G, Chocyk A, ´ Bobula B, Gruca P, Litwa E, Pałucha-Poniewiera A, W ˛asik A, Pilc A and Wieronska J (2018) Negative ´ Allosteric Modulators of mGlu7 Receptor as Putative Antipsychotic Drugs. Front. Mol.  The affinity of mGlu7 for glutamate is also relatively low (Ki = 869 µM) (Wright et al., 2000) and thus the receptor might play a modulatory role in the CNS, protecting from glutamate overstimulation (Niswender and Conn, 2010). Recent evidence suggests mGlu7 receptor involvement in the pathology of schizophrenia, as several polymorphisms of gene encoding this receptor have been found in different populations, for example, significant transmission distortion of rs17031835 in intron 1 of GRM7 in Indonesian sib-pair families (Ganda et al., 2009), 14 single nucleotide polymorphisms (SNPs) in GRM7 of Han Chinese population (Li et al., 2016) or synonymus polymorphism (371T/C, rs3749380) in exon 1 of GRM7 in Japanese patients with schizophrenia (Ohtsuki et al., 2008). Due to the lack of highly specific, bioavailable compounds mGluR7 activity is yet poorly understood, especially in the context of schizophrenia.

Mitsukawa et al. described the first selective positive allosteric modulator of mGlu7 receptors – AMN082 (Mitsukawa et al., 2005). It was shown that AMN082 possesses antidepressantlike profile in FST and TST, and anxiolytic properties in four plate test and stress-induced hyperthermia (Palucha et al., 2007; Stachowicz et al., 2008). However, it did not exhibit any antipsychotic-like profile and rather enhanced MK-801- or DOIinduced effects, which may suggest a potential beneficial role of antagonists or negative allosteric modulators (NAMs) in animal models of schizophrenia (Wieronska et al., 2012 ´ ). Additionally, some studies reported off target activity of AMN082 (Sukoff Rizzo et al., 2011).

Up to date, there have been only two mGlu7 receptor NAMs synthetized – MMPIP and ADX71743 (Suzuki et al., 2007; Kalinichev et al., 2013). MMPIP was shown to impair cognition and decrease social interaction in WT mice or rats, bared no effect on spontaneous activity and motor performance (Hikichi et al., 2010) but induced analgesic effects (Palazzo et al., 2016, 2015). Additionally, no antidepressive and anxiolytic effects were described. Moreover, MMPIP did not reverse the pharmacologically induced disruption of prepulse inhibition (PPI, Hikichi et al., 2010). However, the effects of MMPIP might be difficult to explain as it also acts as an inverse agonist (Suzuki et al., 2007).

Another NAM of mGlu7 receptor – ADX71743 – was found to exert an anxiolytic but not antidepressant effect (Kalinichev et al., 2013). When administered to animals, it did not impair their locomotor activity and motor performance. The antipsychotic activity of ADX71743 is not well described and understood as it caused a moderate decrease in amphetamine-induced hyperactivity, but had no effect on DOI-induced head twitches and the conditioned avoidance response (Kalinichev et al., 2013).

Here, we extensively describe the action of MMPIP and AXD71743 both in vitro and in vivo in the context of schizophrenia. Antipsychotic activity of both compounds was evaluated in number of behavioral tests, such as: MK-801 induced hyperactivity, DOI-induced head twitches, modified forced swim test, social interaction test, PPI, and novel object recognition (NOR) test. Their potential effect on motor performance was assessed in rotarod test. Due to better pharmacokinetic properties, the activity of MMPIP was also tested in spatial delayed alternation test. In order to confirm the profile of interaction of MMPIP and ADX71743 with mGlu7 receptor, the intracellular levels of cAMP were measured. Additionally, pharmacokinetic and electrophysiological studies were performed.

### MATERIALS AND METHODS

#### Animals and Housing

Male Albino Swiss mice (20–25 g) were used in most behavioral tests. Male Wistar rats (200–250 g) were used in spatial delayed alternation test and PPI of the acoustic startle response test. Male C57BL/6J WT and mGlu7 KO mice were used in electrophysiological studies. The animals were kept in a room with 12:12 light–dark cycle at a temperature of 21–22◦C. Food and water were provided ad libitum. The animals were used only once, none of the animals has been run multiple experiments. All procedures were conducted according to the guidelines of the National Institutes of Health Animal Care and Use Committee and were approved by the II Local Ethics Committee by the Institute of Pharmacology, Polish Academy of Sciences in Krakow.

#### Drugs

MMPIP [6-(4-Methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo(4,5-c)pyridin-4(5H)-one], ADX71743 [6-(2,4-Dimethylphenyl)-2-ethyl-6,7-dihydro-4(5H)-benzoxazolone], MK-801 [(5S,10R)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate] and DOI (4-Iodo-2,5-dimethoxyα-methylbenzeneethanamine hydrochloride) were purchased from Tocris Bioscience, Bristol, United Kingdom. For behavioral and pharmacokinetic studies, MK-801 and DOI were dissolved in 0.9% NaCl, MMPIP in 0.5% methylcellulose (Sigma– Aldrich, St Louis, MO, United States) and ADX71743 in small amount of DMSO (Sigma–Aldrich) and then titrated in 20% captisol (Cydex Pharmaceuticals, Lawrence, KS, United States). Final concentration of DMSO in the whole solution was 2%. Control groups received appropriate vehicles. All drugs were administered in a volume of 10 ml/kg when given to mice, and 1 ml/kg when given to rats. The doses of the compounds we used were partially chosen on the basis of the other studies (Hikichi et al., 2010), but mostly were established experimentally. Mostly, the compounds were administered up to the dose of 15 mg/kg; however, in some case when the activity was evident at the lower doses, the dose of 15 mg/kg was not investigated.

A homogeneous time-resolved fluorescence (HTRF) cAMP dynamic 2 (Cisbio, Codolet, France) assay was performed as previously described (Chru´scicka et al., 2015) with recombinant cell lines. Briefly, HEK 293 T-REx cells stably expressing mGlu7 receptor, were collected and suspended in Hanks-HEPES buffer. The cell suspension was added to compounds solution with 5 µM of forskolin (final concentration). After 5 min incubation in 37◦C, 5 µl of cAMP-d2 conjugate in lysis buffer was added and mixed with the 10 µl cell suspension by means of an automated pipetting system (Tecan Evo 200, Tecan, Mannedorf, Switzerland). Next, 5 µl anti-cAMP cryptate conjugate was added and the fluorescence at 620 and 665 nm was read after 1 h (Tecan Infinite M1000). The results are shown as the 665 nm/620 nm ratio multiplied by 104 . The detected signal was inversely proportional to the concentration of cAMP in the sample. Antagonist activity of ADX71743 or MMPIP are shown as a percentage of the inhibition of L-Glu activity at its EC80 concentration. Dose response data from ADX71743 or MMPIP were analyzed with Prism Version 7.03 (GraphPad Software Inc.). Each experiment was performed three times (n = 3), and each data point was in triplicate.

#### Pharmacokinetic Studies

The method described below was successfully applied to a pharmacokinetic study of ADX71743 and MMPIP in mouse (Albino Swiss) after i.p. injection. Compound ADX71743 and MMPIP were administered to mice at 10 mg/kg i.p. At 0.25, 0.50, 1.0, 2.0, 4.0, 6.0 h, the mice were anesthetized, and the blood was collected from the portal vein to the tubes containing 5% EDTA. The mice were then perfused with 0.1M PBS to remove remaining blood from the body, and the brains were taken out for the analysis. Blood was centrifuged at 2000 rpm for 10 min at 4◦C, and the plasma was collected and frozen at −80◦C for further analysis.

Plasma and tissue samples from all drug-treated animals were thawed at room temperature prior to use. Standard protocol of sample preparation: 200 µl acetonitrile was added to the eppendorfs with 50 µl of studied plasma samples or tissue homogenate. Samples were mixed for 5 min on a mixer at 25◦C and 1400 rpm. Tubes were then centrifuged at 2000 × g for 15 min at 4◦C. About 180 µl of each supernatant was transferred into a plate well. Finally, each sample was injected into the column.

In calibration curve – serial dilution method, plasma was spiked with standard at different concentration levels. Acetonitrile was added. Mixed, centrifuged supernatant was taken.

## LC-MS Analysis

#### Chromatographic Conditions

Plasma and tissue samples from all drug-treated animals at selected time points are analyzed using previously developed a non-validated liquid chromatography-tandem mass spectrometry (LC-MS)/MS method. A sensitive and highly selective LC-MS method was used to determine drug concentration in mouse plasma samples or tissue homogenate.

LC/MS analysis was carried on a Bruker amaZon SL mass spectrometer using positive/ negative ion ESI mode. Chromatographic separation was achieved on a Ascentis Express C18 column, (5 cm × 2.1 mm, 2.7 µM, Supelco Technologies) at room temperature with a thermostatted column oven. A gradient elution of eluents A [acetonitrile (LiChrosolv, Reag. Ph Eur) +0.1% formic acid (Sigma–Aldrich, 98–100%)] and B (water +0.1% formic acid) was used for separation. The flow rate was set at 1 ml/min. The injection volume was 20 µl, and the time of injection was 4 min.

#### Mass Spectrometric Conditions

An Ion trap mass spectrometer (Bruker amaZon SL) was equipped with an electrospray source, operating in the positive/negative ion mode. Data were collected and processes using Bruker Quant Analysis software. Quantification of analytes was performed in SIM mode.

### Electrophysiology

Mice (wild and KO, approx. 25 g) were housed under a controlled light/dark cycle (light on: 0700–1900) and had free access to standard food and tap water. Mice were anesthetized with isoflurane (Aerrane, Baxter) decapitated, their brains were dissected and immersed in an ice-cold artificial cerebrospinal fluid (ACSF) of the following composition (in mM): NaCl (130), KCl (5), CaCl2 (2.5), MgSO4 (1.3), KH2PO4 (1.25), NaHCO3 (26), and glucose (10), bubbled with a mixture of 95% O2/5% CO2 to pH 7.4. Frontal cortical slices (bregma 1.9–1.4, 380 µm) were cut in a coronal plane using a vibrating microtome and they were stored at 32◦C. A single slice was next transferred to the recording chamber (32◦C ± 0.5◦C) and superfused at 2.5 ml/min with a ACSF.

A bipolar stimulating electrode (FHC) was placed approx. 2 mm lateral to the midline and approx. 1.0 mm below the pial surface (in layer V) (**Figure 1**). Stimuli (duration: 0.2 ms) were applied at 0.033 Hz using a constant-current stimulus isolation unit (WPI). Field potentials (FPs) were recorded using glass micropipettes filled with ACSF (1–3 M), which were placed approx. 0.2 mm below the cortical surface (in layer II/III). FPs were amplified (Axoprobe 1A, Axon Instruments), A/D converted at 10 kHz and stored using Micro1401 interface and Signal 4 software (CED).

The stimulus–response curves obtained for each slice were fit with the Boltzmann equation: Vi = Vmax/(1 + exp[(u − Uh)/−S]), where Vmax is the maximum FP amplitude; u is the stimulation intensity; Uh is the stimulation intensity evoking FP of half-maximum amplitude; S is the factor proportional to the slope of the curve; "exp" is exponentiation – mathematical operation. The results are expressed as the means ± SEM. Statistical analyses were carried out using t-test (Tokarski et al., 2011).

For each slice, at the beginning of the experiment an inputoutput curve was generated in ACSF. A stimulus-response (input-output) curve was made for each slice. To obtain the curve, stimulation intensity was gradually increased stepwise

(16 steps; 0–100 µA). One response was recorded at each stimulation intensity. Next, standard ACSF was replaced by a solution containing MMPIP or ADX71743, for 20 min, and input-output curves were generated again. Statistical analyzes were carried out using paired t-test and ANOVA.

### MK-801-Induced Hyperactivity

The locomotor activity was recorded individually for each animal in OPTO-M3 locomotor activity cages (Columbus Instrument) linked online to a compatible PC activity, as described previously by Wozniak et al., 2016b ´ . Each cage (13 cm × 23 cm × 15 cm) was surrounded with an array of photocell beams. Interruptions of these photobeams resulted in horizontal activity defined as ambulation counts. The mice were placed in the locomotor activity cages for acclimatization for 30 min Then, MMPIP (10, 15 mg/kg) or ADX71743 (5, 10 mg/kg) were administered i.p. Both drugs were given 30 min prior to MK-801 injection (0.35 mg/kg, i.p.). The locomotor activity was measured for 60 min immediately after MK-801 administration.

#### DOI-Induced Head Twitches

The experiment was performed according to previously described procedure (Wieronska et al., 2012, 2013 ´ ). Immediately after a 30 min acclimatization period, DOI (2.5 mg/kg, i.p.) was administered in order to induce head twitches. The number of head twitches was then counted for 20 min. MMPIP (5, 10, and 15 mg/kg) or ADX71743 (2.5, 5, and 10 mg/kg) were administered i.p. 30 min before DOI. Subsequently the compounds were administered chronically (for 10 days) each at the two active doses (MMPIP 5 and 10 mg/kg and ADX71743 2.5 and 5 mg/kg). The test was performed on 11th day, 30 min after the last administration.

## Modified Forced Swim Test

The modified forced swim test was performed according to the method introduced by Noda (Noda et al., 1995, 1997; Wieronska ´ et al., 2015a; Wozniak et al., 2016a ´ ). The swim tests were performed in a glass cylinder (height, 20 cm; internal diameter, 15 cm) containing 11 cm of water maintained at 23–24◦C. After the acclimation period, the animals underwent the first swim test, where the immobility time was measured during a 3 min period (T1). On the next day, chronic (13 days) MK-801 administration (0.4 mg/kg, i.p.) was started. After a 1-day break, on the 15th day of experiment, the second swim session was performed and the immobility time during 3-min test was measured again (T2). The T2 − T1 difference was reported as the result of the experiment. MMPIP (1, 5, and 15 mg/kg, i.p.) or ADX71743 (5, 10, and 15 mg/kg, i.p.) were administered acutely 30 min before the T2 session.

### Social Interaction Test

The method was adapted from de Moura Linck et al., 2008 and Wozniak et al., 2016b ´ . After the 2-day habituation trial (10 min/day) a pair of mice was placed in the open field for 5 min. The social interactions between two mice were determined based on the total time spent participating in social behavior such as genital investigation, sniffing, chasing, and fighting each other. The total number of social episodes was also measured. The test was video-recorded and viewed by a trained observer. MMPIP (5, 10, and 20 mg/kg, i.p) or ADX71743 (1, 5, and 15 mg/kg, i.p.) were administered 30 min before MK-801 (0.3 mg/kg, i.p.), which was administered 30 min before the test.

### Novel Object Recognition Test

The experiment was performed according to Nilsson et al., 2007 with minor modifications (Wozniak et al., 2016b ´ ). Following a 2-day habituation period (10 min/day), a training trial was performed, where mice were allowed to explore two identical objects for 5 min. About 1 h later, a test trial was conducted, where one of the familiar object was replaced by a novel object. The animals were then allowed to explore the objects for 5 min. MMPIP (5, 10, and 15 mg/kg, i.p.) and ADX71743 (1, 5, and 10 mg/kg; i.p.) were administered 30 min before MK-801 (0.3 mg/kg, i.p.), which was administered 30 min before the training trial. Time spend exploring (i.e., sniffing or touching) the familiar (Tfamiliar) or novel object (Tnovel) was measured by a trained observer and then the recognition index was calculated for each mouse [(Tnovel − Tfamiliar)/(Tfamiliar + Tnovel)] × 100.

### Rotarod Test

The animals were trained for 3 consecutive days at the speed of 18 rpm, one session per day for 3 min. If a mice fell during the habituation period, it was placed back on the apparatus. On the following day, the test trial was performed. After the mice were placed on the apparatus (Mouse Rota-Rod NG, UGO BASILE S.R.L.) moving at the speed of 12 rpm, the accelerating mode was started (maximum speed – 24 rpm). The latency to fall was measured during 3-min test session. Mice were injected with MMPIP (5, 15, and 30 mg/kg, i.p.) or ADX71743 (5, 15, and 30 mg/kg, i.p.) 30 min before the test trial.

#### Spatial Delayed Alternation Test

fnmol-11-00316 September 18, 2018 Time: 19:5 # 5

The spatial delayed alternation test was performed using a wooden T-maze, according to Sławinska et al., 2013 ´ and Wieronska et al., 2015b ´ .

During the adaptation phase, lasting 3 days, the animals were allowed to freely explore the maze for 10 min. For the next 2 days, rats were confined to either of the two end-arms and allowed to drink a 10 % sucrose solution there for 10 min twice daily. On the following day, a 2-week training phase was started. The animals performed one training session per day, which consisted of one forced trial (i.e., one of the end-arms was closed) followed by ten free choice trials. During the free choice trial the animal was placed in the starting arm and after the guillotine door was raised, it was allowed to choose to enter one of the endarms. After the response, the rat was placed back to the starting arm, where it stayed for 10 s. If the chosen end-arm was the opposite to the previously visited one, a correct response was scored, and the animal was closed in the compartment where it was allowed to drink the sucrose solution for 5 s. After an incorrect response, the animal was gently returned to the starting arm. The training phase was carried out until the animals scored 7 correct responses in a training session in 2 consecutive days.

On the day of the test, the animals were injected with MMPIP and/or MK-801, and the aforementioned 10-trial session was repeated. MMPIP was administered at a dose of 5 or 10 mg/kg 30 min prior to MK-801 (0.1 mg/kg) administration. The test was started 30 min after the MK-801 injection.

#### Prepulse Inhibition

The procedure was performed according to Czyrak et al., 2003. On the day before the experiment, the animals were subjected to a single startle session consisting of two trials, each presented 20 times during the session. During the first trial, a 120 dB, 40 ms pulse was presented, and on the second trial this pulse was preceded by a 75 dB, 20 ms prepulse. On the day of the experiment, the animals were habituated to the background white noise (65 dB) for 5 min (which continued throughout the test), after that the startle session was carried out as described above. Startle response amplitude was defined as the difference between the maximum force detected during a recording window and the force measured immediately before the stimulus onset (the threshold was set at 10 g). For each animal, the amplitudes were averaged separately for each type of trial. The PPI was calculated as the difference between the amplitudes of the pulse (P) and the prepulse + pulse (PP+P), divided by the amplitude of the pulse alone [([P − (PP + P)]/P)×100].

MMPIP (5, 10, and 15 mg/kg) and ADX71743 (2.5, 5, and 10 mg/kg) were administered 30 min prior to MK-801 (0.3 mg/kg), which was administered 30 min before the habituation phase.

### Statistical Analysis

Statistical analysis was performed using Statistica 12 package (StatSoft Inc., Tulsa, OK, United States). A one-way ANOVA followed by Newman-Keuls post hoc test was used to analyze the behavioral experiments and Student's t-test for paired samples was used to assess the differences in the amplitude of FPs. Data are presented as mean ± SEM.

### RESULTS

#### cAMP

In order to confirm the NAM profile of ADX71743 and MMPIP, the substances were incubated with 6.26 mM of L-Glu (EC80). Both ADX71743 and MMPIP dose-dependently antagonized L-Glu inhibition of cAMP accumulation in the presence of forskolin, with the IC50 values of 0.44 µM (± 0.13) (n = 3) and 0.38 µM (± 0.15) (n = 3) respectively (**Figure 2A**).

In the second set of experiments the cells were incubated with forskolin with increasing concentration of ADX71743 or MMPIP without agonist in order to analyze their inverse agonist properties. Both compounds dose-dependently potentiated forskolin action, elevating cAMP concentration in the T-REx 293 cells (**Figure 2B**). IC50 of both substances was very similar – ADX71743 0.22 µM (± 0.07) (n = 3) and MMPIP 0.34 µM (± 0.14) (n = 3). This effect was not observed for an antagonist of mGluR7 – XAP044.

#### Pharmacokinetics

The concentration of ADX71743 and MMPIP in mouse plasma and brain are shown in **Table 1**. Cmax was evident in brain and plasma 0.25 h after injection of ADX71743, and 0.5 h after MMPIP administration. **Figure 3** represents comparison between ADX71743 and MMPIP concentrations in the brain in selected time points after administration.

Data presented in **Table 2** showed that ADX71743 and MMPIP had different cytochrome P450 inhibition profile. Weak inhibition (IC50 > 10µM) of cytochrome P450 was observed in case of 1A2, 2B6, 2C9, 2D6 isoforms for both NAM mGluR7 standards. Mild inhibition (3.3 < IC50 < 10) of isoform 2C19 was determined for ADX71743 standard, while strong inhibition (IC50 < 1.1) was observed only for MMPIP in case of isoform 3A4 as well as 2C19.

#### Electrophysiology

Analyses of FPs recorded in slices obtained from WILD mice revealed an increase in the relationship between stimulus intensity and FP amplitude (input–output curve) after MMPIP administration in wild animals (**Figure 4A**), compared to KO (P < 0.001, paired t-test, **Figure 4B**). Parameters characterizing input-output curves of FPs, calculated using the Boltzmann fits, are summarized in **Table 3A**. The amplitude of FPs was markedly higher over a wide range of stimulation intensities (P < 0.001, two-tailed) (**Figure 4A**).

The effect of MMPIP administration (before-after effect) was about 23% higher in WILD group compared to KO animals

The dashed line represents the cAMP level corresponding to 5 µM of forskolin (B). Representative results, data points presented as mean ± SEM.

TABLE 1 | Plasma (A) and brain (B) concentration of MMPIP and ADX71743 after

administration of 10 mg/kg.

Tmax – time at maximum observed concentration Cmax noted in minutes after administration of drug, T1/2 – terminal elimination half-life after administration, Cmax – maximum drug concentration obtained after administration of a drug between the time of doing and the final observed point, and AUC – the area under the concentration-time curve.

(123% vs. 100,1%, P < 0.001, two-tailed, t = 6.544 df = 30, **Figure 4C**).

ADX71743 administration increased the amplitude of recorded FP in wild animals, whereas were ineffective in

Kinetic solubility (µg/ml), metabolic stability mice microsomes (% remaining after 60 min), and clint (ml/min/mg/ protein).

KO group (P < 0.002; P < 0.49, paired t-test). Parameters characterizing input-output curves of FPs, calculated using the Boltzmann fits, are summarized in **Table 3B**. The amplitude of FPs increased over a higher ranges of stimulation intensities (P < 0.001, paired t-test, two-tailed, t = 9.426 df = 23)

FIGURE 4 | The influence of mGlu7-selective antagonist (MMPIP) on the relationship between stimulus intensity and amplitude of field potentials (FP) in WILD (A) and KO mice (B), and the before-after effect of the drug (C). The influence of mGlu7-selective antagonist (ADX71743) on the relationship between stimulus intensity and amplitude of FPs in WILD (D) and KO mice (E), and the before-after effect of the drug (F). Filled circles, quadrates: FPs recorded in slices after 20 min MMPIP/ADX administration prepared from WILD mice, open circles/quadrates: control preparations (n = 18). ∗∗∗P < 0.001.

(**Figures 4D,E**). The effect of ADX71743 administration (beforeafter effect) was higher in WILD group compared to KO animals (116% vs. 106, P < 0.001, two-tailed, t = 5.71 df = 35, **Figure 4F**).

### MK-801-Induced Hyperactivity

One-way ANOVA analysis revealed the statistically significant effects of treatments [F(3.34) = 23.38, P < 0.0001 (**Figure 5A**)

∗∗∗P < 0.001. Two-tailed paired t-test, t = 8.084, df = 9.

∗∗∗P < 0.001. Two-tailed paired t-test, t = 9.426, df = 23. Vmax is the maximum FP amplitude; Uh is the stimulation intensity evoking FP of half-maximum amplitude; S is the factor proportional to the slope of the curve; and n is the number of slices.

n = 10 (A,B,E,F).

and F(3.32) = 21.2, P < 0.0001 (**Figure 5B**)]. Neuman-Keuls post hoc analysis indicated significant increase in the locomotor activity after MK-801 administration when compared to control groups (P < 0.0001) and the significant reversal of MK-801 induced effect after MMPIP administration at the highest dose (15 mg/kg) used in the study (P < 0.05). Both doses of ADX71743 (5 and 15 mg/kg) decreased MK-801-induced hyperactivity in a statistically significant way (P < 0.001 and P < 0.05) when compared to MK-801-treated groups.

#### DOI-Induced Head Twitches

One-way ANOVA analysis revealed the statistically significant effects of treatments [F(3.16) = 13.96, P < 0.0001 (**Figure 5C**) and F(3.18) = 15.75, P < 0.0001 (**Figure 5D**)]. Dunnet's

post hoc analysis indicated that the administration of MMPIP significantly reduced DOI-induced head twitches at all investigated doses 5, 10, and 15 mg/kg (P < 0.001) (**Figure 5C**). The effect of ADX71743 was also significant at 2.5 mg/kg (P < 0.01), 5 mg/kg (P < 0.001), and 10 mg/kg (P < 0.0001) (**Figure 5D**).

The compounds showed similar activity after chronic (10 days) administration: MMPIP at the doses 5 and 10 mg/kg and ADX71743 at the doses 2.5 and 5 mg/kg. One-way ANOVA analysis revealed the statistically significant effect of MMPIP treatment [F(2.19) = 15.67, P < 0.0001] and Dunnet's post hoc comparison revealed statistically significant effect of both doses (P < 0.05 and P < 0.0001). Similarly, the effect of ADX71743 administration was also significant [F(2.17) = 16.56, P < 0.0001] and Dunnet's post hoc comparison revealed the statistical effect of both investigated doses (P < 0.001 and P < 0.0001) (**Figures 5C,D**).

#### Modified Forced Swim Test

One-way ANOVA analysis revealed the statistically significant effects of treatments [F(4.44) = 5.8, P < 0.0007 (**Figure 5E**) and F(4.45) = 4.3, P < 0.005 (**Figure 5F**)]. Neuman-Keuls post hoc analysis indicated significant increase in the immobility time after MK-801 administration when compared to control groups (P < 0.001) and the significant reversal of MK-801-induced effect after MMPIP administration at the dose of 1 mg/kg (P < 0.01) and 15 mg/kg (P < 0.01). The effect of ADX71743 was significant only at the dose of 15 mg/kg (P < 0.05) when compared to MK-801-treated group.

#### Social Interaction Test

One-way ANOVA analysis revealed the statistically significant effects of treatment in the time of interaction [F(4.30) = 9.20, P < 0.0001 and F(4.45) = 7.63, P < 0.0001] (**Figures 6A,C**) and the number of episodes [F(4.30) = 4.2, P < 0.007 and F(4.45) = 10.44, P < 0.0001] (**Figures 6B,D**). Neuman-Keuls post hoc analysis indicated significant reduction of social behaviors after MK-801 administration when compared to control groups (P < 0.01). MMPIP had no effect on both measured parameters (**Figures 6A,B**).

ADX71743 at a dose of 5 and 15 mg/kg reversed the effect of MK-801 on the duration (P < 0.01 and P < 0.05) and number of social episodes (P < 0.001 and P < 0.001) (**Figures 6C,D**).

#### Novel Object Recognition Test

One-way ANOVA analysis revealed the statistically significant effects of treatments [F(4.37) = 3.7, P < 0.01 (**Figure 7A**) and F(4.44) = 5.99, P < 0.0006 (**Figure 7B**)]. Neuman-Keuls post hoc analysis indicated significant reduction of recognition index after MK-801 administration when compared to control groups (P < 0.01) and the significant reversal of MK-801-induced effect after MMPIP administration at the doses of 10 (P < 0.05) and 15 mg/kg (P < 0.01) (**Figure 7A**) and ADX71743 at the doses 1 mg/kg (P < 0.01), 5 mg/kg (P < 0.01), and 15 mg/kg (P < 0.001) (**Figure 6B**) when compared to MK-801-trated animals.

Number of animals in group varied n = 8–10.

#### Prepulse Inhibition

MK-801 (0.3 mg/kg) enhanced the amplitude of the acoustic startle response and markedly attenuated the prepulse-induced inhibition of the acoustic startle response (up to 16% of control). One-way ANOVA analysis revealed statistically significant effect of treatment [F(4.39) = 22.84, P < 0.0001 (**Figure 7C**) and F(4.39) = 19.96, P < 0.000 (**Figure 7D**)]. Neuman-Keuls post hoc comparison revealed that MK-801 inhibited startle response comparing to control groups (P < 0.0001). The effect of MK-801 on the prepulse-induced inhibition of the acoustic startle response was not antagonized by the selective NAM of mGlu7 receptor MMPIP in all doses (5, 10, and 15 mg/kg). When given alone, MMPIP (10 mg/kg) attenuated the amplitude of the acoustic startle response and markedly enhanced the prepulse-induced inhibition of the acoustic startle response (up to 137% of control), but the effect was not statistically significant (**Figure 7C**). ADX71743 inhibited MK-801-induced disruption in PPI at the lowest dose 2.5 mg/kg (P < 0.05) (**Figure 7D**).

#### Spatial Delayed Alternation Test

One-way ANOVA analysis revealed a statistically significant effect of treatment [F(4.42) = 10.59, P < 0.0001] (**Figure 7E**). Neuman-Keuls post hoc analysis indicated significant reduction of choice accuracy after MK-801 administration (P < 0.0001). MMPIP at a dose of 10 mg/kg rescued the MK-801–induced cognitive impairments, by improving the choice accuracy (P < 0.001) (**Figure 7E**).

#### Motor Coordination

In the rotarod test, neither MMPIP nor ADX71743 did not induce detectable motor impairments when compared to the control group [F(6.63) = 0.919] (**Figure 7F**).

#### DISCUSSION

The present paper constitutes the complex study concerning putative antipsychotic-like activity of mGlu7 receptor NAMs. We used two commercially available compounds, MMPIP and ADX71743. The first reports concerning their pharmacological activity were released several years ago, however, the activity of the compounds is still not fully investigated and established.

Here, we have used a variety of techniques that allowed us to investigate the in vitro and in vivo activity of both compounds, their pharmacokinetics and pharmacological effects.

Using T-REx 293 cell lines (commercially available version of HEK 293 cell line with single FRT site) with inducible expression of mGlu7 receptor the selectivity of the compounds toward mGlu7 receptors was confirmed. Both compounds dose-dependently antagonized L-Glu inhibition of cAMP accumulation in the presence of forskolin and L-glutamate. Similar results were shown earlier for ADX71743 (Kalinichev et al., 2013).

This is the first report showing the activity of MMPIP on HEK line, and thus can be compared with the activity of ADX71743. The earlier studies with MMPIP were performed on CHO lines, and the activity of the compound was investigated in the presence of L-AP4, without the presence of L-glutamate. Additionally, different techniques for intracellular cAMP assessment like AplhaScreen and Phenyx cAMP assays were used to characterize both NAMs which can differ in sensitivity and measurement range (Suzuki et al., 2007; Kalinichev et al., 2013). In the Kalinichev et al.'s (2013) paper, HEK 293 cells and L-glutamate were used. The EC50 for L-Glu and IC50 for ADX71743 in the presence of EC80 the agonist were significantly lower comparing to results obtained by our group. Different method of cAMP measurements and different host cells caused difficulties to collate the biological activity of the two chemicals. Here, we compare the activity of both compounds and our result indicates that the affinity of MMPIP is slightly better than that of ADX71743, although both compounds are very potent. Moreover, we demonstrated inverse agonist action for both MMPIP and ADX71743 in heterologous expression system. Our results and data presented by Suzuki et al. (2007) confirmed the intrinsic activity of mGluR7 that can be showed in the presence of inverse agonist. Moreover, this may have very important biological effect on in vivo studies due to dual way of action of ADX71743 and MMPIP. However, we must keep in mind differences which can be observed between species and even between different cell hosts from the same species. For example, the mGluR7 positive allosteric modulator AMN082 activates the receptor in CHO cells, as well its effect can be observed in behavioral studies. In contrast, human cell line HEK 293 expressing mGluR7 does not respond to this compound (Niswender et al., 2010).

Systemic administration of the compounds confirmed that they reach Cmax rapidly, 0.25–0.5 h after administration, followed by a rapid decline. The concentration of ADX71743 was almost undetectable 2 h after administration, while the concentration of MMPIP was still observed. Similar pharmacokinetic profile was described earlier for the compounds; however, the doses of ADX71743 used were much higher (100– 150 mg/kg) than that used in our studies, and the compound was administered s.c. (Kalinichev et al., 2013), while MMPIP was administered similarly as in the work of Hikichi et al., 2010. The important thing is that in the present studies several time points were analyzed in contrary to the work of Hikichi et al. (2010) where the concentration of the compound was measured only at one time point, 1 h after administration (Hikichi et al., 2010). Comparing the results obtained for both compounds it may be concluded that the most potent NAM of mGluR7 – ADX71743 exhibits high kinetic solubility, low metabolic stability in mice liver microsomes consistent with high clearance, while MMPIP shows better metabolic stability but lower biological activity as well as solubility.

Subsequently, the specificity of compounds was assessed in electrophysiology experiments, in which, with the use of mGlu7 KO mice, we established that the compounds were active only on the slices obtained from wild type animals and not from mGlu7 KO mice. Then, we compared the activity of the compounds in variety of behavioral models. In our earlier studies, the propsychotic effect of mGlu7 PAM, AMN082, was showed (Wieronska et al., 2012 ´ ). Therefore, it could be assumed that NAMs of mGlu7 receptor can be proposed as putative antipsychotic agents. To confirm this hypothesis, both compounds were examined in variety of animal tests and models with high predictive validity toward antipsychotic-like efficacy of drugs.

MK-801 induced hyperactivity and DOI-induced head twitches were used as the tests predictive for positive symptoms of schizophrenia. Both compounds reversed MK-801-induced deficit without exerting own effects on spontaneous locomotor activity in active doses. The activity of ADX71743 was more evident in this test, and lower doses of the compound (5 mg/kg) restored MK-801-induced deficit. MMPIP was active only in the highest administered dose, 15 mg/kg. In previous studies, the activity of ADX71743 was showed in amphetamineinduced hyperactivity, but much higher doses were needed to reach significant effect (100 and 150 mg/kg were active). Also, the different route of administration (subcutaneous) was applied in that studies (Kalinichev et al., 2013). The activity of MMPIP has not been investigated in this paradigm so far.

Both investigated compounds also reversed DOI-induced head twitches. DOI, similar to the other 5-HT2A activating agents (i.e., d-lysergic diethylamide acid, LSD), has hallucinogenic potential in humans (Jacobs and Trulson, 1979; Geyer and Vollenweider, 2008; Vollenweider and Kometer, 2010), and in animals, it induces characteristic head twitches (González-Maeso et al., 2008; De Gregorio et al., 2016a), that are reversed by the administration of both typical and atypical neuroleptics (Marona-Lewicka et al., 2005; De Gregorio et al., 2016b). The activity of ADX71743 was observed in lower doses than that observed after MMPIP administration. Again our results differ from the results presented in the studies of Kalinichev et al. (2013) where ADX71743 was active at the dose of 100 mg/kg and higher.

In the next step, the activity of both drugs was investigated in tests for negative symptoms of schizophrenia, such as social interaction and modified forced swim test. The social interaction test resembles social withdrawal observed in schizophrenia patients while modified forced swim test is considered as a model of depressive-like symptoms of schizophrenia (Noda et al., 1995, 1997; de Moura Linck et al., 2008). In both tests, only atypical (e.g., risperidone), and not typical neuroleptics, effectively reverse MK-801-induced deficits (de Moura Linck et al., 2008). It is in line with clinical efficacy of drugs, where only atypical neuroleptics are potent to reverse negative symptoms, although the efficacy of drugs is not always satisfactory.

Here, both compounds were active in modified forced swim test and only ADX71743 reversed MK-801-induced deficits in social interaction test. So again the activity of ADX71743 was better than the activity of MMPIP.

Novel object recognition (NOR) and spatial delayed alteration (SDA) were used as the models of cognition, while PPI reflects attentional deficit associated with schizophrenia. MK-801 disrupts the ability of animals to discriminate between the known, old object, and the novel one in NOR (Nilsson et al., 2007; Grayson et al., 2015), and to make proper choice to obtain the reward in SDA. This disruption of short working memory is antagonized by atypical, but not typical antipsychotics. In NOR, both drugs were active, but the activity of ADX71743 was more evident. Additionally, MMPIP was tested in SDA test and prevented, in all investigated doses, the disruptive effect of MK-801. In the former studies performed in the work of Hikichi et al. (2010), the drug was shown to reduce the recognition index in the NOR test and decreased the location index in the object location test at the doses 10–30 mg/kg. Therefore, it seems that the compound does not possess any procognitive effect when given alone, on contrary it rather disturbs cognitive behaviors, while when given prior MK-801 it prevents the development of disruptive effects of the drug. The other compound, ADX71743, was not investigated in the models of cognition yet. Therefore, this is the first study showing the pro-cognitive activity of the compound in the models of schizophrenia. Here, we also show for the first time that ADX71743 in low doses is potent to prevent MK-801-induced disruption in PPI, while such an activity was not observed for MMPIP, similarly as in previous studies (Hikichi et al., 2010).

In these studies, the activity of compounds was wellinvestigated in pharmacologically induced animal models of schizophrenia, showing their preventive effect on MK-801-induced disruptions of those behaviors after acute administration. However, it should be taken into consideration that the potential desensitization effect, after repeated administration of two compounds, may be responsible for the failure in neuroleptic efficacy in clinical trials. Therefore both compounds were administered chronically to compare if their efficacy will be similar as after acute administration. The activity of the compounds was tested in DOI-induced head twitches and no tolerance was observed. However, further studies are needed especially with non-pharmacologically induced animal model of schizophrenia to fully characterize the antipsychotic properties of above described compounds.

Considering the putative mechanism of action of mGlu7 NAMs, it must be taken into consideration that mGlu7 receptors are localized mainly on GABAergic terminals (Dalezios et al., 2002). The expression of this receptor on GABAergic neurons is almost 10 times higher than on glutamatergic neurons. Therefore, the receptor predominantly regulates GABA release than glutamate release (Summa et al., 2013). Its activation leads to inhibition of GABA release while its inhibition may contribute to increased release of this neurotransmitter. According to the hypothesis of schizophrenia raised by Conn et al. (2009), the increased release of glutamate due to the loss of inhibitory control over the glutamatergic neurons is the main cause of schizophrenia development. The majority of recent studies concerning antipsychotic activity of mGlu ligands was focused on the inhibition of glutamate release through the activation of the receptors expressed on glutamatergic nerve terminals. Here, it seems that the inhibition of mGlu7 receptors expressed on GABAergic neurons may contribute to the increase of GABA efflux and thus bring back the inhibitory control over the glutamatergic transmission (scheme of the **Figure 8**).

Based on the present studies, it is clear that the investigated compounds may have preventive effect in developing psychotic behaviors. However, to better establish the role of mGlu7 receptor in schizophrenia and putative antipsychotic effects of its inhibition, more work must be undertaken and new ligands with better pharmacokinetic properties acting at mGlu7 receptor should be synthesized. The trend is now open as recently a paper

was released, where new compounds inhibiting mGlu7 receptor were proposed (Reed et al., 2017).

#### ETHICS STATEMENT

fnmol-11-00316 September 18, 2018 Time: 19:5 # 13

All the procedures were conducted in accordance with the European Communities Council Directive of September 22, 2010 (2010/63/EU) and Polish legislation acts concerning animal experimentations.

#### AUTHOR CONTRIBUTIONS

PC and MW performed the DOI-induced head twitches, social interaction, and NOR. PC contributed to the analysis of the results and manuscript writing. KK carried out

#### REFERENCES

the pharmacokinetics studies. PB and GB performed the cAMP analysis. AC performed the PPI. BB performed the electrophysiology experiments. PG and EL performed the SDA. AP-P performed the hyperactivity and modified forced swim test. AW performed the analysis of video recordings from behavioral tests (SI, NOR). AP collected the funds and contributed to the discussion. JW performed the data analysis, coordination, wrote the manuscript, and collected the funds.

#### FUNDING

This work was supported by Grant No. 2015/17/B/NZ7/02984 (OPUS) from the National Science Center (NCN) in Poland to JW and by Grant No. 2012/6/06/A/NZ7/00014 (MAESTRO) given to AP, and by NCBR Grant no. PBS1/B7/8/2012 (Allosterix).

disorders. Neuropsychopharmacology 31, 1112–1122. doi: 10.1038/sj.npp. 1300926

**Conflict of Interest Statement:** The 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.

Copyright © 2018 Cie´slik, Wozniak, Kaczorowska, Bra ´ nski, Burnat, Chocyk, Bobula, ´ Gruca, Litwa, Pałucha-Poniewiera, W ˛asik, Pilc and Wieronska. This is an open- ´ access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Metabotropic Glutamate Receptor 7: A New Therapeutic Target in Neurodevelopmental Disorders

Nicole M. Fisher1† , Mabel Seto1† , Craig W. Lindsley1,2 and Colleen M. Niswender1,3 \*

1 Department of Pharmacology and Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN, United States, 2 Department of Chemistry, Vanderbilt University, Nashville, TN, United States, 3 Vanderbilt Kennedy Center, Vanderbilt University Medical Center, Nashville, TN, United States