Introduction
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and has been linked to a wide range of physiological processes including perception, emotion, cognition, and motor function. To date, glutamate receptors are classified into two major categories: ionotropic glutamate receptors, which are ligand-gated ion channels, and metabotropic glutamate (mGlu) receptors that are coupled to G-proteins (Spooren et al., 2003). Because of their signaling pathways and pharmacological characteristics, mGlu receptors have received increasing attention in excitatory amino acid research. In particular, the physiological roles of mGlu2/3 receptors have been studied thoroughly and growing body of evidence suggests that mGlu2/3 receptor agonists show beneficial effects in animal models of neuropsychiatric diseases, stroke, and epilepsy (Chaki, 2010).
In addition to glutamate, serotonin (5-hydroxytryptamine, 5-HT) has also been implicated in the pathophysiology of a number of psychiatric disorders, such as schizophrenia and depression. In this respect, several studies have shown the induction of positive symptoms, such as delusions, and cognitive impairments by hallucinogenic drugs that are known to function as 5-HT2A receptor agonists or partial agonists. Therefore, the therapeutic effects of atypical antipsychotics have been partially attributed to a blockade of 5-HT2A receptors (Meltzer et al., 2003; Lieberman et al., 2008; González-Maeso and Sealfon, 2009; Ibrahim and Tamminga, 2011).
A striking overlapping laminar distribution of 5-HT2A and mGlu2/3 receptors in the prefrontal cortex (PFC) has been reported by a number of studies and led to the hypothesis that 5-HT2A and mGlu2/3 receptors share close functional interactions with physiological relevance (Aghajanian and Marek, 1999; Gewirtz and Marek, 2000; Marek et al., 2000). Although the exact anatomical localization of both receptor subtypes has still not been fully assessed, several lines of evidence indicate that 5-HT2A receptors are localized predominantly postsynaptically, whereas the majority of mGlu2 receptors are found on the presynapse (Conn and Pin, 1997; Barnes and Sharp, 1999). Nevertheless, there is now considerable evidence showing a close functional antagonistic interaction between 5-HT2A and mGlu2/3 receptors. For example, head-twitch responses (Gewirtz and Marek, 2000) and excitatory postsynaptic currents (Klodzinska et al., 2002) induced by administration of the 5-HT2A receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) are reversed by simultaneous activation of the mGlu2/3 receptor subtype. The reduction of 5-HT2A receptor-mediated responses has generally been attributed to synaptic mechanisms and the autoreceptor function of mGlu2/3 receptors. Interestingly, recent studies have provided further evidence for a direct molecular interaction between 5-HT2A and mGlu2 receptors within the cortex. This review will focus on electrophysiological, biochemical, and behavioral evidence underpinning the close functional crosstalk between 5-HT2A and the mGlu2/3 receptor. In addition, we will outline how these receptor interactions may be implicated in behavioral function, hallucinogenic, and antipsychotic drug actions as well as in psychiatric disorders, especially schizophrenia.
5-HT2A and mGlu2/3 receptors: structure, function, distribution
The 5-HT2 receptor family consists of three receptor subtypes, the 5-HT2A, 5-HT2B, and 5-HT2C receptors, which are comparable in their molecular structure, pharmacology, and signal transduction pathways (Barnes and Sharp, 1999). Among all 5-HT2 receptors, the 5-HT2A receptor subtype is probably the one studied most widely and has been linked to a variety of distinct physiological processes in both the CNS and the periphery (Gray and Roth, 2001). In general, activation of 5-HT2A receptors is associated with a decrease in potassium conductance and leads to neuronal excitation in several brain regions (Barnes and Sharp, 1999). The most prominent and well-understood signaling pathway mediated by 5-HT2A receptors is the Gq/11-protein-mediated activation of phospholipase C (PLC), which hydrolyzes phosphatidylinositol membrane lipids and generates inositol-1,4,5-triphosphate (IP3) as well as diacylglycerol (Willins et al., 1997). The enhanced formation of DAG stimulates protein kinase C activity and ultimately results in the expression of the immediate early gene (IEG) c-fos. IP3 induces the release of Ca2+ from intracellular stores, which in turn induces multiple responses in the cell, including activation of mitogen-activated kinase and the Ca2+/calmodulin complex. Activation of 5-HT2A receptors also leads to the stimulation of phospholipase A2 (PLA2), which hydrolyzes arachidonic acid-containing phospholipids producing free arachidonic acid and lipophospholipid. The PLA2 pathway is completely independent of PLC-mediated signaling and appears to be far more complex than the phosphoinositide (PI) turnover cascade, involving multiple G-proteins as well as ERK1, ERK2, and mitogen-activated protein kinases. It is now clear that specific hallucinogenic ligands interact with 5-HT2A receptors to activate PLC-mediated and PLA2-mediated signaling pathways to different extents. Moreover, the two signaling pathways appear to have different receptor reserves in that activation of a much smaller fraction of the 5-HT2A receptor is required for the stimulation of the PLA2-mediated cascade versus PLC activation (Kurrasch-Orbaugh et al., 2003a, 2003b).
There is still an unresolved paradox as to why some 5-HT2A receptor agonists [such as lysergic acid diethylamide (LSD) and DOI] show hallucinogenic activity whereas other structurally comparable ligands (i.e. lisuride and ergotamine) with similar affinity and agonist action lack such psychoactive effects. In this context, it has been shown that hallucinogenic and nonhallucinogenic 5-HT2A receptor agonists induce distinct gene expression patterns. Although c-fos-induction was observed in response to both nonhallucinogenic as well as hallucinogenic ligands, increased expression of the early genes egr-1 and egr-2 was elicited only by hallucinogenic 5-HT2A receptor agonists. Hence, c-fos has been suggested to represent a marker for nonspecific 5-HT2A receptor activation, whereas induction of egr-1 and egr-2 seems to be hallucinogen specific (González-Maeso et al., 2007). The distribution of 5-HT2A receptors has been investigated extensively using autoradiography, in-situ hybridization, or immunohistochemical methods. Within the CNS, 5-HT2A are abundantly expressed, but particularly high densities have been found in cortical areas (including layers I, IV, and Va of the neocortex as well as the entorhinal and piriform cortices), in parts of the limbic system (i.e. amygdala and hippocampus) as well as in the basal ganglia (Pazos et al., 1985; Lopez-Gimenez et al., 1997). The majority of 5-HT2A receptors are localized postsynaptically on somata and apical dendrites of pyramidal projecting neurons whereas only a limited population of GABAergic cells and cholinergic interneurons have been found to express this receptor subtype (Xu and Pandey, 2000).
The mGlu receptors were discovered in the mid-1980s and represent a family of class C G-protein-coupled receptors. To date, eight receptor subtypes have been identified, which are divided into three groups on the basis of their molecular structure, pharmacological characteristics, and signal transduction pathways. Group I mGlu receptors (consisting of mGlu1 and mGlu5) are positively coupled to Gq/11 proteins and stimulate PLC activity, whereas group II (including mGlu2 and mGlu3) and group III (comprising mGlu4, mGlu6, mGlu7, and mGlu8) are linked to Gi/o and inhibit adenylate cyclase activity (Swanson et al., 2005; Fell et al., 2012). Moreover, group II mGlu receptors also couple to other signaling pathways, including activation of mitogen-activated protein kinases and phosphatidylinositol 3-kinase pathways, lending further complexity to the mechanisms by which these receptors can regulate synaptic transmission (Niswender and Conn, 2010; Nicoletti et al., 2011). The functional unit of mGlu2/3 receptors appears to be a homomeric dimer with a large amino (N)-terminal extracellular domain – a coiled structure that functions as a ‘Venus Flytrap’ and that is converted from the open into the closed configuration upon agonist binding – and an intracellular carboxy (C)-terminal with numerous phosphorylation sites (Pin and Acher, 2002). Selective group II mGlu receptor agonists described so far include (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate, LY354740 [(1S,2S,5R,6S)-2-aminobicyclo [3.1.0]hexane-2,6-dicarboxylate] as well as the more potent and selective mGlu2/3 receptor agonist LY379268 [(1R,4R,5S,6R)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate]. In contrast, MGS0039 and LY341495 show group II mGlu receptor antagonist activity, with the latter also having some affinity for the mGlu8 receptor subtype (Monn et al., 1997; Kingston et al., 1998).
Neuronal group II mGlu receptors are expressed widely throughout the CNS, with moderate to high levels of expression within the PFC, dorsal, and ventral striatum, thalamus, hippocampus, and amygdala (Conn and Pin, 1997; Cartmell and Schoepp, 2000). In-situ hybridization studies and autoradiography in mice lacking either the mGlu2 and/or the mGlu3 receptor showed that mGlu2 receptors are highly, but discretely, expressed in almost all regions of the limbic system and cortical areas including layer I and Va of the PFC, intralaminar and midline nuclei of the thalamus, regions associated with the perforant pathway as well as in the lateral and basolateral amygdala. At the cellular level, mGlu2 receptors are predominantly localized presynaptically at the periphery of the synapse and distant from the active zone, where they function as autoreceptors and provide negative feedback to reduce glutamate release. Their heterogeneous distribution on glutamatergic and nonglutamatergic cells allows for a complex modulation of neurotransmission, ion channel activity, and synaptic plasticity (Moldrich et al., 2003). By contrast, mGlu3 receptors show a more diffuse and widespread localization and are found presynaptically as well as postsynaptically on neurons, and certain glia cells (Tamaru et al., 2001).
5-HT2A and mGlu2/3 receptor interactions
Evidence from electrophysiology and immunohistochemistry
Electrophysiological studies have shown that 5-HT exerts various modulatory effects on glutamatergic neurotransmission depending on the brain region and the 5-HT receptors involved. As such, activation of 5-HT2A receptors increases the release of glutamate in the PFC and enhances the frequency of spontaneous (nonelectrically evoked) excitatory postsynaptic currents (EPSCs) in apical dendrites of cortical pyramidal cells. The induction of enhanced EPSC frequencies by 5-HT is completely blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonists, pointing toward a crucial involvement of glutamate (Aghajanian and Marek, 1997, 1999).
The electrophysiological studies carried out by Marek et al. (2000) and Aghajanian and Marek (1999) were probably among the first to report a close functional coupling between 5-HT2A and mGlu2/3 receptors in rat cortical brain slices. These studies showed that the frequency and amplitude of 5-HT2A-induced EPSCs were markedly increased by the selective group II mGlu receptor antagonist LY341495, whereas selective mGlu2/3 receptor agonists (i.e. LY354740 and LY379268) induced a robust suppression of DOI-elicited EPSCs (Aghajanian and Marek, 1997; Marek et al., 2000). These findings have been confirmed and extended in a subsequent study by Klodzinska et al. (2002), who showed that activation of mGlu2/3 receptors completely blocked the DOI-induced EPSCs in layer V pyramidal cells in mice cortical slices. Overall, these observations have been interpreted in the sense that activation of 5-HT2A receptors may induce the release of glutamate from thalamocortical terminals upon which group II mGlu receptors are believed to function as autoreceptors. Therefore, excessive glutamate release induced by 5-HT2A receptor agonists such as DOI can be counteracted by the simultaneous activation of presynaptic mGlu2/3 receptors (Marek et al., 2000, 2001; Klodzinska et al., 2002). In support of this notion, activation of mGlu2 receptors by a selective allosteric modulator has also been shown to reduce enhanced excitatory neurotransmission occurring in response to serotonergic hallucinogens (Benneyworth et al., 2007). The suppressant actions of mGlu2 receptors appeared to be more pronounced in prefrontal compared with frontoparietal regions, which may suggest differential effects of the 5-HT2A–mGlu2 interactions in limbic versus nonlimbic-related cortical areas (Marek et al., 2000).
However, the idea that 5-HT2A receptors on thalamocortical afferents primarily mediate the effects of hallucinogenic drugs on glutamatergic activity in the PFC has been challenged by more recent in-vivo electrophysiological studies. For example, Puig et al. (2003) reported that DOI markedly affected the firing rate of cortical pyramidal cells and increased prefrontal 5-HT release from dorsal raphe (DR) neurons. Disinhibition of the mediodorsal and centromedial thalamic nuclei resembled the effects of DOI as cortical pyramidal cell firing and 5-HT release in the PFC were increased. However, selective activation of prefrontal μ-opioid and mGlu2/3 receptors counteracted the effects of thalamic disinhibition, but failed to attenuate the effects of DOI on 5-HT release. Therefore, the authors concluded that DOI may act on postsynaptic 5-HT2A receptors unrelated to thalamocortical afferents to increase the activity of the mPFC-DR circuit (Puig et al., 2003). It needs to be mentioned though that activation of mGlu2/3 receptors has itself been shown to increase 5-HT release and this effect may have masked the decrease in 5-HT following a reduction in excitatory inputs (Cartmell and Schoepp, 2000). Moreover, and as outlined by Béïque et al. (2007), 5-HT2A receptors appear to directly modulate recurrent intrinsic network activity through the stimulation of a subpopulation of cortical pyramidal cells. Accordingly, overstimulation of 5-HT2A receptors following hallucinogenic drug use might destabilize PFC recurrent circuits and hence give rise to the sensory effects of hallucinogens. In contrast to the ‘historical’ thalamocortical hypothesis, hallucinogen-induced disturbances of cognitive function may not result from excessive stimulation of thalamocortical innervation, but rather from altered function of intrinsic PFC networks (Béïque et al., 2007). In addition, subsequent in-vivo studies by Celada et al. (2008) have shown that DOI led to a disruption of cortical network activity by reducing low-frequency oscillations (0.3–4 Hz) and desynchronizing pyramidal discharge from active phases of low oscillations. The DOI-induced alterations of network activity were similar in thalamic lesioned and control rats, which further supports the notion of an involvement of intracortical 5-HT2A receptors rather than thalamocortical 5-HT2A receptors in mediating the effects of hallucinogenic substances (Celada et al., 2008).
IEGs are the first group of genes to be expressed following synaptic activation and neural stimulation. They do not require de-novo protein synthesis for expression and encode a variety of different proteins, including regulatory transcription factors, structural proteins, and signal transduction proteins as well as growth factors, proteases, and enzymes. Owing to their quality as potential activity markers in the CNS, IEGs have been used extensively in functional mapping studies. In this context, a number of studies have shown that administration of the 5-HT2A receptor agonist DOI induces a widespread expression of the IEG c-fos and its protein product in several cortical regions (Tischmeyer and Grimm, 1999; Hegab and Wei, 2014). Supporting the electrophysiological findings mentioned previously, pretreatment with AMPA/kainate receptor antagonists inhibited DOI-elicited Fos immunoreactivity (Fos IR), suggesting that cortical Fos expression in response to DOI is determined by a 5-HT2A receptor-mediated release of glutamate. Moreover, DOI appeared to induce Fos expression predominantly in cells that lack the 5-HT2A receptor subtype, whereas Fos IR was found in only a few cortical cells that were also positive for 5-HT2A receptors (Mackowiak et al., 1999; Scruggs et al., 2000, 2003). On the basis of these findings, Scruggs et al. (2000) hypothesized that, instead of directly interacting with 5-HT2A receptor-positive cortical pyramidal cells, the effect of DOI on Fos expression may be mediated by binding to 5-HT2A receptors on thalamocortical neurons that impinge on glutamatergic cells in the apical dendritic field of layer V neurons. Subsequently, the 5-HT2A receptor-mediated release of glutamate may then result in the induction of c-Fos protein expression in cortical cells of layers V and III.
The first demonstration that mGlu2/3 receptors can modulate DOI-induced c-fos expression was provided by Zhai et al. (2003). Specifically, the authors showed that the selective mGlu2/3 receptor agonist LY379268 reduced the DOI-elicited increase in c-fos mRNA levels in rat mPFC and this attenuated response was prevented by the mGlu2/3 receptor antagonist LY341495. However, LY379268 did not alter the effect of DOI on Fos expression in either the frontoparietal or the somatosensory cortices, suggesting that mGlu2/3 receptors modulate c-fos induction in a region-specific manner (Zhai et al., 2003). These findings have been confirmed and extended by other studies reporting a decrease in DOI-elicited Fos IR by mGlu2/3 receptor stimulation within frontocortical areas as well as in limbic brain regions such as the basolateral and central amygdala (Benneyworth et al., 2007; Wischhof and Koch, 2012). In addition, activation of mGlu2/3 receptors has been shown to attenuate the 5-HT2A receptor-mediated hydrolysis of PI in the frontal cortex of wild-type mice, as well as in mice lacking either the mGlu2 or the mGlu3 receptor subtype. In contrast, systemic administration of the mGlu2/3 receptor agonist LY379268 was ineffective in double mGlu2/3 receptor knockout mice (Molinaro et al., 2009), raising the possibility that both group II mGlu receptors may interact with 5-HT2A receptors. Strikingly, opposite results were obtained in cortical slice preparations, where LY379268 resulted in an amplified 5-HT2A receptor-mediated PI hydrolysis (Molinaro et al., 2009). However, under the conditions described by González-Maeso et al. (2008), stimulation of mGlu2 receptors did not influence the 5-HT2A receptor-mediated canonical pathway, which increases the formation of IP3 and DAG and ultimately results in enhanced Fos protein expression (González-Maeso et al., 2008). The discrepancy between these findings may be related to the different experimental approaches used (i.e. in vivo vs. in vitro) and overall highlight the importance of in-vivo models for studying the interactions between 5-HT2A and mGlu2/3 receptors.
Neurotrophic growth factors such as nerve growth factor, neurotrophin-3, and brain-derived neurotrophic factor (BDNF) not only promote cell survival and differentiation of neurons during development but also influence the function and plasticity of neuron populations in the adult brain. In the rodent brain, BDNF is the most ubiquitous neurotrophin and has been implicated in a variety of distinct functions such as memory, circadian regulation, and the pathophysiology of neuropsychiatric disorders (Gilmore et al., 2005; Bredy et al., 2007; Castrén, 2014). In this context, 5-HT2A receptor stimulation has been shown to increase BDNF mRNA in the midlayer of the neocortex and the claustrum while decreasing BDNF expression in the dentate gyrus of the hippocampus. To date, two models have been postulated to explain how 5-HT2A receptor activation may enhance neocortical BDNF levels. First, the increase in BDNF mRNA expression might result directly from the stimulation of 5-HT2A receptors on cortical pyramidal cells, with the subsequent depolarization and elevation of intracellular Ca2+ stores. Another possibility is that BDNF mRNA levels are increased by the 5-HT2A receptor-mediated release of glutamate from thalamocortical afferents. In support of the latter explanation, cortical BDNF expression has been shown to depend on glutamate release and more specifically, on the glutamatergic stimulation of AMPA receptors. Moreover, the selective mGlu2/3 receptor agonist LY354740 suppressed the DOI-induced increase of cortical BDNF mRNA levels, whereas the mGlu2/3 receptor antagonist LY341495 induced an increase in DOI-mediated BDNF expression in the mPFC. Neither LY354740 nor LY341495 alone produced any significant effects on cortical BDNF levels (Vaidya et al., 1997; Zetterström et al., 1999; Gewirtz et al., 2002; Angelucci et al., 2005). Using in-situ hybridization and western blotting, Di Liberto et al. (2010) reported an upregulation of BDNF levels following LY379268 treatment within the cerebral cortex and the hippocampal formation, suggesting that the neuroprotective and trophic effects of mGlu2/3 receptor ligands could be brain region specific.
5-HT2A and mGlu2/3 receptor interactions on a molecular level
Although interactions between 5-HT2A and mGlu2/3 receptors have been the focus of considerable research over the last two decades, it was not until 2008 when González-Maeso et al. (2008) reported for the first time that the mGlu2 receptor subtype can directly interact with 5-HT2A receptors by specific transmembrane domains and under the formation of a heteroreceptor complex on cortical pyramidal. The existence of the heteroreceptor complex was shown by a variety of different in-vitro methods, including coimmunoprecipitation of human cortical brain samples and HEK-293 cells transfected with epitope-tagged receptors, as well as bioluminescence and fluorescence resonance energy transfer in transfected cells. Also, competition binding studies in mouse somatosensory cortex membranes showed that uncoupling of the G-protein receptor complex decreased agonist affinities for 5-HT2A and mGlu2/3 receptor ligands. Within these 5-HT2A–mGlu2 receptor complexes, both receptor subtypes appear to allosterically influence each other in their binding properties. Specifically, the mGlu2/3 receptor agonist LY379268 increased the affinity for hallucinogenic drugs at the 5-HT2A receptor-binding site, whereas DOI reduced the affinity of glutamate receptor binding. In addition, the 5-HT2A–mGlu2 heteroreceptor complex appeared to trigger a unique cellular response when targeted by hallucinogenic substances and activation of the mGlu2 receptor subtype abolished hallucinogen-specific signaling. Besides identifying the 5-HT2A–mGlu2 receptor complex as a possible site of action for hallucinogenic drugs, the authors suggest that the heteroreceptor complex may serve to integrate 5-HT and glutamate signaling to regulate cortical sensorimotor function, a process that is disturbed in psychosis (González-Maeso et al., 2008).
The formation of a 5-HT2A–mGlu2 heteroreceptor complex was confirmed recently in in-vitro studies by Delille et al. (2013). However, these studies also showed that mGlu2 receptors can form complexes with 5-HT2B as well as mGlu5 receptors, indicating that complex formation is not limited to the 5-HT2A–mGlu2 receptor pair. In this study, hallucinogenic 5-HT2A receptor agonists induced signaling by Gq/11 (the canonical 5-HT2A receptor-mediated pathway), but not by Gi/o (the proposed hallucinogenic pathway), and hence, did not alter intracellular cAMP levels. In contrast to the findings reported by González-Maeso et al. (2008), DOI also did not influence [3H]LY341495-binding competition for the mGlu2/3 receptor agonist LY354740. Overall, the authors concluded that the formation of G-protein-coupled receptor heterocomplexes does not necessarily translate into second messenger effects. Although the results do not question the well-documented functional interactions between 5-HT2A and mGlu2 receptors, they do challenge the biological relevance of a 5-HT2A–mGlu2 heteroreceptor complex (Delille et al., 2012, 2013). Nevertheless, the in-vitro studies clearly show that 5-HT2A and mGlu2 receptors can be coprecipitated from brain lysates and are found in close proximity; however, these findings do not prove whether 5-HT2A–mGlu2 heteroreceptor complexes can indeed be found in the intact brain (see Delille et al., 2013 for a detailed review).
5-HT2A and mGlu2/3 receptor interactions with relevance to behavior
One of the most commonly used models to assess the hallucinogenic properties of 5-HT2A receptor agonists in rodents is the head-twitch response, which, in rats, is sometimes also called wet-dog shakes. Studies with selective 5-HT2A receptor antagonists have clearly shown the dependence of this behavior on 5-HT2A receptor activation (Fox et al., 2010; Dougherty and Aloyo, 2011; Halberstadt, 2015), and these findings are further supported by the absence of DOI-induced head shakes in 5-HT2A receptor knockout mice. Moreover, microinfusion of DOI into the mPFC is sufficient to induce a head-twitch response, suggesting a crucial involvement of prefrontal 5-HT2A receptors in generating this hallucinogen-induced behavior (Willins and Meltzer, 1997).
In one of the first demonstrations of a physiological interaction between 5-HT2A and mGlu2/3 receptors, Gewirtz and Marek (2000) reported that activation of mGlu2/3 receptors by the selective agonist LY354740 suppressed the frequency of DOI-induced head shakes in rats, whereas administration of the mGlu2/3 receptor antagonist LY341495 had the opposite effect and increased the number of DOI-induced head twitches (Gewirtz and Marek, 2000). Similarly, the mGlu2/3 receptor agonist LY379268 induced a reduction of the DOI-induced head-twitch response in mice (Klodzinska et al., 2002) and these findings have been replicated several times by a number of different research groups in both mice and rats (Benneyworth et al., 2007; Moreno et al., 2011a; Holloway et al., 2013; Chiu et al., 2014).
Interactions of serotonergic and glutamatergic systems, especially by the 5-HT2A receptor subtype, have further been implicated in the regulation of prepulse inhibition (PPI) of the acoustic startle response (ASR), a commonly used test to assess sensorimotor gating mechanisms. Several studies showed that administration of DOI induced a marked reduction of PPI in rats, and these PPI-disruptive effects of DOI have been specifically attributed to its agonistic actions at 5-HT2A receptors (Geyer et al., 1978, 2001; Geyer and Tapson, 1988; Sipes and Geyer, 1995, 1997). Moreover, reversal of pharmacologically induced PPI deficits is considered a model to identify potential antipsychotic properties. In this context, previous work of our laboratory has shown that pretreatment with the mGlu2/3 receptor agonist LY379268 dose dependently attenuated the DOI-induced PPI impairments in Wistar rats (Wischhof et al., 2012). Similarly, stimulation of mGlu2/3 receptors also attenuated the reduction of the ASR following administration of DOI. LY379268 itself did not exert any effects on PPI and only slightly increased ASR amplitudes, suggesting only a minor involvement of mGlu2/3 receptors in the regulation of sensorimotor gating processes per se (Wischhof and Koch, 2012). In addition to glutamate, a variety of the effects of 5-HT are mediated by interactions with the dopaminergic system. However, the PPI-disruptive effects of 5-HT2A receptor activation seem not to involve direct or indirect stimulation of dopamine D2 receptors as they were not reversed by D2 receptor antagonists such as haloperidol and raclopride (Johansson et al., 1995; Padich et al., 1996). Taken together, these data support the notion of an interaction between 5-HT and the glutamatergic system in modulating PPI and further underpin the functional antagonism between 5-HT2A and mGlu2/3 receptors in particular.
Loss of impulse control depends on dopamine receptor activation, particularly within the nucleus accumbens (van Gaalen et al., 2006; Pattij et al., 2007; Pattij and Vanderschuren, 2008), and it has been shown that activation of 5-HT2A receptors facilitates dopamine release (Bortolozzi et al., 2005). The impulsivity enhancing effects of DOI were attenuated by dopamine receptor blockade (Koskinen and Sirviö, 2001; Koskinen et al., 2003), raising the possibility that a 5-HT2A receptor-mediated activation of the dopaminergic system underlies deficient impulse control. However, DOI-induced premature responding in the five-choice serial reaction time task (5-CSRTT), a measure for motor impulsivity and deficient response inhibition, seems to involve dopamine receptors only indirectly as intra-accumbens administration of DOI had no effect on the number of premature responses (Koskinen and Sirviö, 2001). Overall, these findings suggest that other brain regions and/or neurotransmitter systems are primarily targeted by 5-HT2A receptor agonists, whereas changes in dopaminergic activity are possibly secondary. The mGlu2/3 receptor agonist LY379268 has been shown to suppress premature responding induced by phencyclidine (Greco et al., 2005), a noncompetitive NMDA receptor antagonist that increases 5-HT release and facilitates 5-HT2A receptor-mediated glutamate transmission (Chaki, 2010). In this respect, previous findings from our group buttress the possible involvement of 5-HT2A and mGlu2/3 receptors in modulating impulsivity-like behaviors. Specifically, pretreatment with the mGlu2/3 receptor agonist LY379268 attenuated the DOI-elicited increase in premature responding in the 5-CSRTT, suggesting that 5-HT2A receptor-mediated impulsivity-like behaviors may, to some extent, involve enhanced glutamatergic neurotransmission (Wischhof and Koch, 2012). In a subsequent study, we further showed that an intracranial injection of DOI into the mPFC of rats is sufficient to induce impulsive over-responding in the 5-CSRTT, whereas the combined administration with LY379268 attenuated the effect of intra-mPFC DOI on response inhibition (Wischhof et al., 2011). Similarly, administration of DOI into the orbitofrontal cortex of rats has been shown to induce delay aversion in a delay-based decision-making T-maze task, which reflects impulsive decision making or, in other words, cognitive impulsivity (Hadamitzky et al., 2009; Wischhof et al., 2011). Simultaneous activation of orbitofrontal cortex mGlu2/3 receptors attenuated the effects of DOI and prevented the 5-HT2A receptor-mediated increase in impulsive choices (Wischhof et al., 2011). Overall, these findings indicate (a) that the impulsivity-enhancing effects of 5-HT2A receptor agonists may be because of a primary increase in glutamate release, especially in the neocortex, which may subsequently activate dopaminergic neurotransmission, ultimately resulting in impaired impulse control, and (b) that a pathological overactivation of 5-HT2A receptors can be normalized by mGlu2/3 receptor agonists, which might therefore be beneficial for the treatment of impulsivity-like disorders. Interestingly, altered 5-HT2A and mGlu2/3 receptor-binding levels have recently also been linked to the differences in impulsive behaviors observed in Roman low-avoidance and Roman high-avoidance (RHA) rats. These rat strains have been bred selectively for their rapid versus extremely poor acquisition of active avoidance behavior in a shuttle box. In addition to opposite emotional and motivational profiles (including differences in novelty seeking, stress sensitivity, and susceptibility to addictive substances), these two phenotypes show clear differences in impulsivity, with RHA showing a more impulsive phenotype as indexed by a significantly increased number of premature responses in the 5-CSRTT (Escorihuela et al., 1999; Steimer and Driscoll, 2003; Klein et al., 2014). Moreover, the higher levels of impulsivity correlated with increased 5-HT2A receptor binding within the frontal cortex, whereas no correlation was found for either 5-HT1A receptor or SERT (5-HT transporter) binding. Strikingly, in addition to the increased 5-HT2A receptor binding, RHA rats also showed a marked reduction of mGlu2 receptor protein levels (Klein et al., 2014). Taken together, both 5-HT2A as well as mGlu2/3 receptors have been implicated independently in impulsivity; however, it remains to be elucidated whether the functional antagonistic relationship between these two receptor subtypes is actually based on heterodimeric interactions or synaptic mechanisms.
Cortical mGlu2/3 receptors have been implicated in habituation processes of simple stimulus-bound behaviors, such as habituation of the ASR or the odor-elicited orienting response. In addition, habituation can also be observed in locomotor activity paradigms as mice and rats usually show a decrease over time in motor activity in an open field, reflecting habituation to the previously unfamiliar environment. Bespalov et al. (2007) studied the effects of the mGlu2/3 receptor antagonist LY341495 on locomotor activity in mice and showed that blockade of mGlu2/3 receptors prevented habituation as motor activity levels basically remained unchanged for the entire 2-h test duration. Interestingly, the effects of LY341495 were reversed by administration of atypical antipsychotic drugs such as clozapine and olanzapine (Bespalov et al., 2007). The locomotor-stimulating effects of mGlu2/3 receptor blockade have further been found to be less pronounced in mice lacking the 5-HT2A receptor subtype (González-Maeso et al., 2008), suggesting an involvement of 5-HT2A–mGlu2/3 receptor interactions in habituation processes. These findings are of particular interest, given that conceptual habituation deficits are commonly observed in schizophrenic patients, and may contribute toward the cognitive symptoms of this mental illness.
Dysregulated expression of 5-HT2A and mGlu2/3 receptors in schizophrenia
In addition to being suspected of involvement in the action of antipsychotic drugs, 5-HT and glutamate have also been linked to the pathophysiology of schizophrenia, and a number of studies have implicated 5-HT2A, 5-HT2C, mGlu2, and mGlu3 receptor genes in psychosis (Egan et al., 2004; Quednow et al., 2008; Abdolmaleky et al., 2011; Lett et al., 2012). As such, many research groups have investigated the expression levels of 5-HT2A receptors in the frontal cortex of schizophrenic patients, either by radioligand binding assays on postmortem tissue samples or by means of PET studies. However, the results obtained are overall quite heterogeneous, with some studies reporting an upregulation of 5-HT2A receptor-binding sites whereas others have found no alterations or even a downregulation of 5-HT2A receptors. The discrepancies have been discussed in relation to demographical and clinical factors, such as age, treatment with antipsychotic drugs, and suicide as cause of death. For example, Muguruza et al. (2013) found that 5-HT2A receptor-binding sites were increased in antipsychotic-free schizophrenic patients, whereas medicated individuals showed no changes. The upregulation of 5-HT2A receptors was not related to suicidal behavior and the authors hypothesized that increased densities of cortical 5-HT2A receptors may be associated with the psychotic symptoms observed in schizophrenic patients (Muguruza et al., 2013). In contrast, Rasmussen et al. (2010), who carried out a large-sample PET study, have reported lower frontocortical 5-HT2A receptor binding in antipsychotic-free first-episode schizophrenic patients. In male patients, the results further showed a significant negative correlation between 5-HT2A receptor binding and positive psychotic symptoms. The authors speculated that downregulation of 5-HT2A receptors could point toward a compensatory mechanism in response to hyperactive second messenger systems and/or overactivity in other neurotransmitter systems on which 5-HT2A receptors have modifying influences (Rasmussen et al., 2010). So far, only a few studies have investigated the expression levels of mGlu2 and mGlu3 receptor genes in postmortem brains of schizophrenic patients. The majority of findings suggest that the expression of mGlu3 receptor mRNA is not altered in schizophrenia, whereas mGlu2 receptor mRNA levels have been found to be higher, lower, or unaffected (Richardson-Burns et al., 2000; Ghose et al., 2008; González-Maeso et al., 2008). Interestingly, though, González-Maeso et al. (2008) reported an upregulation of prefrontal 5-HT2A receptors in postmortem brain samples of unmedicated schizophrenic patients, and these changes were accompanied by a simultaneous downregulation of the mGlu2 receptor subtype. Given that the integration of serotonergic and glutamatergic signaling is crucial for sensorimotor processes, this abnormal expression pattern of 5-HT2A and mGlu2 receptors has been suggested to possibly predispose an individual to the development of psychosis.
Despite the fact that further studies and larger sample sizes are clearly needed to clarify whether mGlu2 receptor levels are altered in schizophrenic patients and whether chronic antipsychotic drug treatment influences expression levels, there are now several lines of evidence obtained from animal studies that suggest a possible involvement of dysregulated 5-HT2A and mGlu2 receptor expression in the pathology of schizophrenia. For example, repeated episodes of prenatal restraint stress have been shown to cause a reduction in mGlu2/3 receptor expression in the hippocampus of juvenile and adult rat offspring (Laloux et al., 2012). Similarly, reduced expression of genes encoding for group II mGlu receptors has been found in the frontal cortex and hippocampus of mice prenatally exposed to restraint stress. The alterations were accompanied by an enhanced binding of MeCP2 (methyl CpG-binding protein 2) to 5-methylcytosines of the respective genes, which indicates increased DNA methylation. Moreover, prenatally stressed mice also showed reduced expression levels of BDNF and GAD67 while frontocortical type-I DNA methyl transferase (DNMT1) expression was increased. These findings are of particular interest, given that activation of mGlu2/3 receptors is believed to exert a strong influence on epigenetic mechanisms, particularly by inhibiting pathological increases in DNMT1 expression and by promoting DNA demethylation. Systemic administration of LY379268 (0.5 mg/kg, twice daily over 5 consecutive days) could correct the schizophrenia-like phenotype in these mice and it has been suggested that epigenetic changes in mGlu2/3 receptor genes may lie at the core of the pathological programming caused by prenatal stress (Matrisciano et al., 2012). An abnormal pattern of 5-HT2A and mGlu2 receptor densities has further been found in the frontal cortex of adult mice born to either influenza virus-infected or poly I:C-infected mothers. The changes were consistent with behavioral abnormalities in the sense that prenatally immune-challenged mice showed an enhanced head-twitch response to DOI, whereas the antipsychotic-like effects of LY379268 were decreased in these animals (Moreno et al., 2011b). Moreover, another study reported that adult rats with a history of early life stress showed enhanced prefrontal network activity following 5-HT2 receptor stimulation as well as increased expression of the IEG Arc. However, these changes occurred without any alterations in 5-HT2A and 5-HT2C receptor mRNA expression and with only a small increase in 5-HT2 receptor binding. The enhanced 5-HT2A receptor-stimulated PFC excitability in animals exposed to early life stress resembles clinical findings indicating that patients with affective disorders also show increased activity in prefrontal networks (Benekareddy et al., 2010). In our hands, adult rats prenatally exposed to lipopolysaccharide showed an enhanced head-twitch response and a greater induction of Fos protein expression in response to DOI. Moreover, pretreatment with LY379268 failed to block the PPI-disruptive effects of DOI in prenatally lipopolysaccharide-exposed rats, whereas activation of mGlu2/3 receptors reversed the DOI-induced PPI deficit in control animals (Wischhof et al., 2015). Taken together, the findings indicate that adverse events (such as maternal infection, prenatal, or early life stress) during early developmental stages may disturb 5-HT2A and mGlu2/3 receptor function and/or expression, and that these changes may, at least partially, contribute toward the pathophysiology of schizophrenia. In support of this notion, a recent study showed that 5-HT2A receptor knockout mice showed reduced expression of mGlu2 receptor mRNA, as well as histone modifications at the promoter region of the mGlu2 receptor gene that correlated with transcriptional repression. Binding of the transcription factor Egr-1 to the mGlu2 promoter was decreased in these mice, whereas viral-mediated overexpression of Egr-1 caused an upregulation of mGlu2 receptor mRNA levels (Kurita et al., 2013).
Hallucinogenic drugs and atypical antipsychotics
Activation of 5-HT2A receptors is believed to be the primary pharmacological mechanism for the action of hallucinogenic drugs. More recently, there has also been interest in mGlu2 receptors as possible contributors toward the cellular and behavioral responses induced by hallucinogenic 5-HT2A receptor agonists, and the demonstration of a 5-HT2A–mGlu2 receptor complex raised the possibility of a functional interaction between these two receptor subtypes that specifically affects hallucinogen-mediated pathways. In this respect, coexpression of mGlu2 decreased the 5-HT2A receptor-dependent activation of Gq/11 whereas activation of Gi/o, and hence, hallucinogenic signaling was increased, and these effects were reversed by the mGlu2/3 receptor agonist LY379268 (González-Maeso et al., 2008; González-Maeso and Sealfon, 2009). In addition, stimulation of mGlu2 receptors by LY379268 has been shown to specifically block the DOI-elicited induction of egr-1, whereas Fos protein expression, which requires the activation of Gq/11, was not altered. Similarly, mGlu2 receptor knockout mice showed a reduced head-twitch response to DOI and LSD, as well as a diminished expression of egr-2. In contrast, mice with diminished mGlu2 receptor signaling did not differ from wild-type animals in the DOI-elicited induction of c-fos. The changes in IEG expression were not because of altered 5-HT2A receptor densities and thus, the findings may indicate that mGlu2 receptors are, to some extent, necessary for mediating hallucinogenic-like effects (Moreno et al., 2011a). The authors further speculated that rather than 5-HT2A alone, the 5-HT2A–mGlu2 heteroreceptor complex could be the actual molecular target responsible for the effects of hallucinogens. In support of this notion, chronic treatment with the mGlu2/3 receptor antagonist LY341494 induced a downregulation of [3H]ketanserin binding in the somatosensory cortex of wild-type, but not mGlu2 receptor knockout mice, and these changes were accompanied by reduced cellular and behavioral responses to LSD (Moreno et al., 2013). Long-lasting changes in 5-HT2A receptor expression and function have further been reported following binge dosing of methamphetamine in mice. In addition to upregulation of frontocortical 5-HT2A receptor levels, the animals showed a greater DOI-induced head-twitch response as well as enhanced c-Fos and Egr-2 expression. Strikingly, these changes were accompanied by a decrease in prefrontal mGlu2 receptor protein expression, suggesting a complex interaction not only between 5-HT and glutamate but also involving the dopaminergic system (Chiu et al., 2014).
In addition to a modest affinity for dopamine D2 receptors, all atypical antipsychotic drugs have a high affinity for 5-HT2A receptors, and their beneficial effects have been partially attributed to a blockade of this receptor subtype (Meltzer et al., 2003). The finding that activation of group II mGlu receptors reduces excessive glutamate release has spurred the search for potent and selective agonists as potential therapeutic agents in the treatment of neurodegenerative and neuropsychiatric disorders. Although antipsychotic-like properties of mGlu2/3 receptor agonists have been reported in a number of animal studies (Spooren et al., 2003; Moghaddam, 2004; Woolley et al., 2008), clinical trials were less successful. As such, the oral prodrug LY2140023 was found to attenuate positive and negative symptoms in schizophrenic patients in a phase 2 clinical trial (Patil et al., 2007), but these findings could not be replicated in follow-up studies (Adams et al., 2013; Walker and Conn, 2015). Considering the wealth of evidence from neurochemical, electrophysiological, and behavioral studies, it seems reasonable to conclude that 5-HT2A–mGlu2 receptor interactions may provide an alternative target for the development of a novel class of antipsychotics in the treatment of neuropsychiatric disorders. Uslaner et al. (2009) treated mice with the mGlu2/3 receptor agonist LY379268 and the 5-HT2A receptor antagonist M100907 at a submaximal dose and showed that the combined administration of both substances produced a significantly greater effect on amphetamine-induced and MK-801-induced hyperlocomotor activity. The authors concluded that a single compound with both mGlu2/3 receptor agonist as well as 5-HT2A receptor antagonist activity, or coadministration of two substances selective of these receptors, may produce greater therapeutic efficacy and possibly also allows for lower doses to be used, hence reducing adverse side effects (Uslaner et al., 2009). In this respect, and as reviewed recently by Ellenbroek and Prinssen (2015), 5-HT3 receptor antagonists may also be effective as ‘add-ons’ to standard antipsychotic medication and have been shown to reduce negative symptoms as well as motor side-effects induced by dopamine D2 receptor antagonism (Ellenbroek and Prinssen, 2015). The notion of a possible involvement of 5-HT2A–mGlu2 receptor interactions in mediating antipsychotic drug effects received further support from a recent study by Kurita et al. (2012). Chronic treatment with atypical antipsychotics induced a downregulation of mGlu2 receptor transcription that was associated with a decreased histone acetylation at the promoter region. Moreover, these epigenetic changes were accompanied by a 5-HT2A receptor-mediated upregulation and increased binding of histone deacetylase 2 to the mGlu2 promotor. Conversely, histone deacetylase inhibitors prevented the repressive histone modifications induced by chronic atypical antipsychotic drug treatment while, at the same time, enhancing their therapeutic-like effects. It is well known that 5-HT2A receptors show a rather atypical regulation pattern as both chronic agonist as well as antagonist treatments result in a downregulation of this receptor. However, the results presented by Kurita et al. (2012) further imply that chronic treatment with atypical antipsychotics, which are 5-HT2A receptor antagonists, also downregulates mGlu2 receptor expression, which, overall, may restrain the therapeutic effects of atypical antipsychotic agents. Conversely, a single pretreatment with the mGlu2/3 receptor agonist LY354740 completely blocked the DOI-induced 5-HT2A receptor downregulation, suggesting that 5-HT2A receptor agonist-induced downregulation may not solely be because of simple occupancy of the receptor by direct agonists (Marek et al., 2006).
Conclusion
Numerous studies have accumulated evidence for a functional antagonistic interaction between 5-HT2A and mGlu2 receptors in the brain. More recent publications suggested the formation of a 5-HT2A–mGlu2 heteroreceptor complex on cortical pyramidal cells, which might mediate the effects of hallucinogenic substances and, at least partially, the effects of atypical antipsychotic agents. Moreover, changes in the expression pattern of 5-HT2A and mGlu2 receptors have been found in schizophrenic patients as well as in rodent models of schizophrenia, possibly indicating a crucial involvement of these two receptor subtypes in the disease pathology. However, the close functional interaction between 5-HT2A and mGlu2 receptors may provide an alternative target for the development of a novel class of antipsychotics in the treatment of neuropsychiatric disorders. However, it remains to be elucidated whether 5-HT2A and mGlu2 receptors function as heteromers in the intact brain and future studies should focus on in-vivo experiments investigating the crosstalk of native receptors with relevance to behavioral function and/or psychosis.
Acknowledgements
The authors apologize to all colleagues whose work could not be cited because of space constraints.
Conflicts of interest
There are no conflicts of interest.
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