Regulatory roles of metabotropic glutamate receptors on synaptic communication mediated by gap junctions

Abstract

Variations of synaptic strength are thought to underlie forms of learning and can functionally reshape neural circuits. Metabotropic glutamate receptors play key roles in regulating the strength of chemical synapses. However, information within neural circuits is also conveyed via a second modality of transmission: gap junction-mediated synapses. We review here evidence indicating that metabotropic glutamate receptors also play important roles in the regulation of synaptic communication mediated by neuronal gap junctions, also known as ‘electrical synapses’. Activity-driven interactions between metabotropic glutamate receptors and neuronal gap junctions can lead to long-term changes in the strength of electrical synapses. Further, the regulatory action of metabotropic glutamate receptors on neuronal gap junctions is not restricted to adulthood but is also of critical relevance during brain development and contributes to the pathological mechanisms that follow brain injury.

Introduction

Synapses are complex multi-molecular structures at which information between two neurons is either indirectly or directly delivered via a chemical neurotransmitter (chemical synapse) or cell-cell channels (electrical synapses), respectively. The presence of synapses establishes networks of neurons, or ‘neural circuits’, which constitute avenues at which information within the nervous system is processed. As a consequence of their strategic position, changes of strength at synapses modify the operations performed by these circuits, thereby revealing their relevance to nervous system function. The phenomena underlying changes in synaptic strength are known as ‘synaptic plasticity’. Sustained, long-term, change in synaptic strength is considered to be one of the cellular mechanisms contributing to learning and memory. In addition, changes in synaptic strength can quickly and reversibly rewire neural circuits to mediate adaptation to varying physiological states (Bloomfield and Völgyi, 2009; O’Brien and Bloomfield, 2018).

Electrical transmission is mediated by trans-cellular structures known as ‘gap junctions’, which are aggregates of aqueous intercellular channels formed by the docking of two ‘hemichannels’ (Fig. 1) each contributed by a neighboring cell (Goodenough and Paul, 2009). Hemichannels can be formed by different proteins, named ‘connexins’ in vertebrates and ‘innexins’ in invertebrates. Although unrelated in sequence, connexins and innexins have similar membrane topology and contribute to form comparable intercellular structures with remarkably overlapping functions in the nervous system (Baker and Macagno, 2016; Pereda and Macagno, 2017). Although gap junctions are ubiquitous in the organism, only a few of the about 20 connexin genes identified in humans are expressed in neurons (Söhl et al., 2005; Yoshikawa et al., 2017). Moreover, the vast majority of electrical synapses are formed by connexin 36 (C×36) and its vertebrate homologs (Söhl et al., 2005; O’Brien, 2014; Miller and Pereda, 2017). However, electrical synapses are not made of only connexins or innexins. Rather, in addition to channel-forming proteins, electrical synapses are now considered to be true synaptic structures where their functions rely on the contribution of multiple other molecules, such as scaffolding, trafficking and regulatory proteins (Lynn et al., 2012; Miller and Pereda, 2017).

Figure 1. Regulation of electrical synapses by neurotransmitters.

A, Glutamatergic synaptic activity can shape the strength of electrical synapses via activation of either NMDARs or mGluRs. Modified with permission from Pereda, 2014. B, Amine neuromodulators, generally released from nearby synaptic terminals, regulate electrical transmission via activation of Class A GPCRs. C, Like all GPCRs, mGluRs contain seven transmembrane domains. mGluRs have a distinct ligand-binding pocket in their extracellular (N-) region known as the Venus flytrap domain and a G-protein-interacting domain at the intracellular (C-) region (CT). mGluRs are divided in three groups: Group I (mGlu1, mGlu5), Group II (mGlu2, mGlu3) and Group III (mGlu4, mGlu6, mGlu7, mGlu8), and they signal through different G-proteins. Modified with permission from Julio-Pieper et al. 2011 (Julio-Pieper et al., 2011). D, Involvement of different mGluRs groups in the regulation of neuronal gap junctions.

Both chemically- and electrically-mediated synapses undergo changes of their strength via a range of mechanisms (for review see (Pereda et al., 2013; Pereda, 2014; Harris and Littleton, 2015; Monday et al., 2018; O’Brien and Bloomfield, 2018)). In particular, metabotropic glutamate receptors (mGluRs) have been frequently reported to contribute to mechanisms of synaptic modification at chemical synapses (for review see (Gladding et al., 2009; Heifets and Castillo, 2009; Collingridge et al., 2010; Lüscher and Huber, 2010; Cosgrove et al., 2011). In this article, we review evidence indicating that activation of mGluRs can also modify the strength of electrical synapses. While examples for this role are still limited, the available evidence indicates that mGluRs exert important regulatory roles on neuronal gap junctional communication in various functional contexts. We highlight here a set of examples that illustrate the participation of mGluRs in mediating plasticity of electrical synapses and the emergence of neural circuits during brain development, as well as the consequences of increased gap junctional communication following neuronal injury.

Regulation of electrical transmission by neurotransmitters

Electrical synapses have been reported to be under two main regulatory influences: the ongoing activity of neural circuits and the action of neuromodulatory transmitters. Regulation by ongoing circuit activity can occur via activation of ionotropic glutamate receptors, more specifically, NMDA receptors (NMDARs). Activation of NMDARs leads to potentiation of electrical transmission at mixed synapses on the teleost Mauthner cell (Yang et al., 1990; Pereda and Faber, 1996; Pereda et al., 1998), at which glutamatergic synapses and gap junctions co-exist within the same terminal (Pereda et al., 2004). The interaction between the co-existing glutamatergic synapse and gap junctions occurs postsynaptically and requires of an increase in postsynaptic Ca⁺⁺ that is mediated by NMDARs and leads to the activation of Calcium-calmodulin kinase II (CaMKII) (Pereda et al., 1998). However, because this postsynaptic interaction is short-ranged (within a few microns; (Pereda et al., 2003)) it can also be exerted heterosynaptically (Fig. 1A) when glutamatergic synapses and/or extrasynaptic NMDARs are located in close proximity to a neuronal gap junction. Examples of the latter have been reported between cells of the inferior olive (Mathy et al., 2014; Turecek et al., 2014) and between AII amacrine cells of the retina (Kothmann et al., 2012). Since glutamate constitutes the main excitatory neurotransmitter in the nervous system, electrical transmission is likely to be continuously shaped by network activity via interactions with glutamate receptors, a mechanism that was suggested to generate heterogeneity of electrical coupling between cells of an extensively coupled neural network (Hoge et al., 2011; Kothmann et al., 2012). Moreover, heterogeneity of electrical coupling has been proposed to underlie relevant network functions between cerebellar Golgi cells (Vervaeke et al., 2010).

Rather than being involved in the rapid transfer of information within neural circuits, neuromodulators (i.e.: dopamine, noradrenaline, serotonin) work instead as regulatory neurotransmitters that can modify the strength of electrical transmission, often in concert with modifications of chemical synapses and neuronal excitability. By acting on multiple targets, neuromodulators organize specific organismal responses and modifications of network states in response to changing physiological contexts. Although multiple regulatory transmitters were reported to target electrical synapses (Bloomfield and Völgyi, 2009; Pereda et al., 2013; O’Brien and Bloomfield, 2018), dopamine was found to be involved in the regulation of electrical transmission at a wide range of cells types and nervous systems (Lasater et al., 1987; DeVries and Schwartz, 1989; Pereda et al., 1992; Johnson et al., 1993; Xia and Mills, 2004; Mills et al., 2007; Kothmann et al., 2009; Li et al., 2013). In contrast to regulation by glutamate via NMDARs, neuromodulators exert their actions via activation of G-coupled receptors (Rosenbaum et al., 2009) and involves in most cases regulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathways. This feature allows them to recruit a wide range of biochemical cascades, greatly expanding their mechanisms of regulatory action (Fig. 1B).

G-protein-coupled receptors and metabotropic glutamate receptors

G-protein-coupled receptors (GPCRs) (Rosenbaum et al., 2009) constitute some of the most prominent eukaryotic signaling pathways and are conserved from unicellular organisms to mammals (Krishnan and Schioth, 2015). These receptors are capable of sensing a wide variety of environmental cues ranging from ions and photons to complex molecules, such as proteins, lipids and nucleotides. Structurally, GPCRs are made of seven transmembrane domains and they form a ligand-binding pocket in the extracellular region and a G-protein-interacting domain in their intracellular region. G-proteins represent heterotrimeric complexes made of alfa, gamma, and delta subunits, that become disassembled following GPCR activation (Rosenbaum et al., 2009). That is, G-proteins are bound to guanosine 5’-diphosphate (GDP) in their inactive state, and their activation leads to the exchange of guanosine 5’-triphosphate (GTP) for GDP in the alfa subunit leading to disassembly, thereby transducing the signal to various downstream effectors.

Five main families of GPCRs are recognized in mammals: rhodopsin (Family A), secretin (Family B), glutamate (Family C), frizzled, and adhesion (Krishnan and Schioth, 2015). The glutamate and rhodopsin families participate in various regulatory processes in the nervous system, including synaptic transmission, axon guidance, synapse formation and the wiring of neuronal circuits. GPCRs that mediate the action of neuromodulators (serotonin, dopamine, acetylcholine, histamine, adrenaline and norepinephrine) are part of the rhodopsin family, which also includes neuropeptides that serve as modulators (Krishnan and Schioth, 2015). In contrast, GPCRs activated by glutamate constitute a distinct class, known as the ‘metabotropic glutamate receptor family’ (mGluRs) (Niswender and Conn, 2010). In contrast to the GPCRs of Family A, mGluRs exhibit a large extracellular N-terminal domain known as the ‘Venus flytrap domain’ that contains the endogenous glutamate-binding site whereas the C-terminus contains important regions for modulating G-protein coupling (Fig 1C). They are divided in 3 groups (Fig. 1C): Group I (mGlu1, mGlu5), Group II (mGlu2, mGlu3) and Group III (mGlu4, mGlu6, mGlu7, mGlu8). mGluRs can signal through different G-proteins: Group I signal though Gq, which activates PLC thus increasing inositol triphosphate (IP3) and diacylglycerol (DAG), while Group II and III signal through Gi or Go, which inhibits adenylyl cyclase (AC), therefore reducing the production of cAMP (Niswender and Conn, 2010). mGluRs are considered mandatory dimers and to exclusively form homodimers. However, while their existence in native cells remains elusive, it is possible that some mGluRs could be formed by heterodimers. With respect to their subcellular localization, Group I mGluRs are believed to be predominantly postsynaptic, Group II are pre- and postsynaptic, and Group III can be localized either pre- or postsynaptically, depending on the variant (Niswender and Conn, 2010). As we will see, neuronal gap junctions can be regulated by Type I and II mGluRs.

Metabotropic glutamate receptors and plasticity of electrical synapses

Involvement of mGluRs in regulating electrical transmission was first inferred from intracellular recordings of individual thalamocortical cells in slices of the cat dorsal lateral geniculate nucleus. (Hughes et al., 2002) observed that following bath application of either the mGluR group I/II agonist trans-ACPD or the group I agonist DHPG, there was i) an increase in the detection of ‘spikelets’, small brief responses that represent gap junction-mediated postsynaptic manifestations of presynaptic action potentials, and ii) an increased incidence of dye coupling. Consistent with the interpretation that the observed responses represented spikelets, their frequency was reduced by TTX, a voltage-dependent Na⁺ channel blocker which blocks action potentials, and by carbenoxolone, a compound known to block gap junction channels. The requirement of mGluR agonists in the observed phenomenon led to the suggestion that electrical synapses between thalamocortical cells could be upregulated by activation of these receptors.

More direct evidence of the regulation of electrical coupling by mGluRs was obtained during paired recordings from rat thalamic reticular neurons (Landisman and Connors, 2005), at which electrical coupling can be assessed by measuring changes in membrane potential produced by current pulses injected in a presynaptic cell while monitoring changes in the second, postsynaptic cell (Fig. 2A,B). Pharmacological activation of mGluRs (1S,3R-ACPD) evoked a long-term depression of electrical synaptic transmission as evidenced by a reduction in coupling coefficient (CC), a ratio calculated by dividing the amplitudes of the postsynaptic coupling over the presynaptic depolarization, and the estimated junctional conductance (GJC) (Landisman and Connors, 2005). Moreover, the effect was also observed during tetanic stimulation of corticothalamic axons (Fig. 2B, C), an outcome that was prevented by superfusion with the mGluR antagonist MCPG (Fig. 2D), indicating that long-term changes in electrical coupling were induced by synaptic activation of mGluRs. These observations not only lend further support to the notion that pharmacological activation of mGLURs can modulate electrical synaptic transmission but, more importantly, indicate that these effects can be long-lasting and triggered in an activity-dependent fashion by glutamatergic synapses. Electrical transmission between cells of the thalamic reticular nucleus is mediated by C×36 containing gap junction channels (Landisman et al., 2002), although a significant amount of coupling is observed in the C×36 −/− mouse (Lee et al., 2010; Zolnik and Connors, 2016). It is still unclear whether this coupling represents the existence of residual channels formed by other connexins or a compensatory mechanism.

Figure 2. Synaptic activation of mGluRs leads to long-term depression of electrical transmission.

A, Whole cell paired recording between two neurons (inset) of the reticular nucleus of the thalamus (Cell Pre and Cell Post). An extracellular stimulating electrode (Stim. Electrode) was used to activate cortico-thalamic inputs to these cells. B, Left, the hyperpolarization (bottom) evoked by intracellular injection of negative current (600 ms pulse) in the presynaptic cell (Cell Pre) evoked a corresponding coupling potential (top) in the postsynaptic cell (Cell Post). Center, Right: the coupling potential was found reduced after tetanic activation of cortico-thalamic afferents (2 and 40 minutes). C, Time course of normalized coupling coefficients (CC) and estimates of gap junctional conductance (GJC) obtained before and after tetanic activation of cortico-thalamic afferents. D, The effect of activation of cortico-thalamic afferents was blocked by the mGluRs antagonist ACPD. Modified with permission from Landisman and Connors, 2009 (Landisman and Connors, 2005).

These observations, obtained using non-selective pharmacological agents, were followed by studies in which the involvement of specific mGluR receptor groups was tested. By using more receptor-selective pharmacological tools during paired recordings of rat thalamic reticular neurons, activation of both mGluR group I and II were found to induce changes in electrical synaptic transmission, interestingly, in opposite directions. Activation of mGluR group I by DHPG caused long-term depression of electrical coupling, and this effect required activation of adenylyl cyclase which by increasing the availability of cAMP in turn stimulated the PKA pathway. This regulatory pathway was shown to be independent from a second form of depression of electrical transmission at these cells triggered by neuronal cellular activity which, although converging on a common downstream endpoint, is dependent of calcium entry (Sevetson et al., 2017). On the other hand, activation of mGluR group II by NAAG resulted in a long-term potentiation of electrical transmission via inhibition of adenylyl cyclase activity, decreased levels of cAMP and reduced activation of PKA (Wang et al., 2015). Although all the observed changes were small in magnitude, it was argued that the changes would be sufficient to impact network activity.

The observations in thalamic cells suggest that mGluR regulation of electrical transmission can be complex, and that their activation can lead to either depression or potentiation of electrical coupling. Another example of this complexity is the participation of mGluRs in activity-dependent changes of synaptic transmission at auditory mixed synapses on the teleost Mauthner cells. Electrical transmission at these mixed synaptic contacts is mediated by heterotypic channels formed by C×36 homologs, namely C×35 and C×34 (Rash et al., 2013). We previously mentioned that activation of these afferents with high-frequency (500Hz) bursts led to potentiation of electrical and chemical transmission via activation of NMDARs and CaMKII (Yang et al., 1990; Pereda et al., 1998). However, potentiation of electrical and chemical transmission at these terminals can also be induced via a different stimulating protocol, namely a train of 100Hz for one second (Cachope et al., 2007). Remarkably, this activity protocol required endocannabinoid formation and cannabinoid receptor 1 (CB1R) activation. More detailed analysis revealed the mechanism by which synaptic activity leading to the synthesis and release of the endocannabinoid 2-arachydonoylglycerol involved the activation of mGLuR1 receptors, which are present at these synaptic contacts (Fig. 3A–C), and of postsynaptic depolarization. Neither puffed application of the mGluR agonist DHPG (Fig. 3D) nor postsynaptic depolarization (Fig. 3E) by themselves were able to trigger potentiation. However, when DHPG application was immediately followed by postsynaptic depolarization, there was a resultant long-term potentiation of the synaptic response (Fig. 3G), which was prevented by bath application of CB1R antagonists (Fig. 3G). The potentiating effect of 2-arachydonoylglycerol on chemical and electrical transmission was indirect, mediated by CB1Rs associated with dopaminergic varicosities which led to the release of dopamine. Dopamine, in turn, led to potentiation of electrical and chemical transmission via a postsynaptic mechanism, which required activation of dopamine D1/5 receptors and PKA (Cachope et al., 2007). This interplay between multiple signaling systems suggests that mGluRs can engage in complex interactions with other messenger and neurotransmitter systems to regulate electrical transmission. Thus, mGluRs can regulate electrical transmission in an activity-dependent fashion and the complexity of some of its mechanisms suggest their functional role can be expressed via other similarly complex interactions.

Figure 3. Involvement of mGluRs in potentiation of electrical synapses.

A, Presence of mGluR1 at mixed, electrical and glutamatergic, synapses on the Mauthner cell known as ‘Club endings. B-C, Unambiguously identified because of their larger size, the contact areas of Club endings (arrowheads) on the lateral dendrite of the Mauthner cell exhibit marked labeling for anti-mGluR1. D-G, Normalized time course of the electrical and glutamatergic components of the mixed synaptic response evoked by activation of Club endings. Both mGluR1 activation and dendritic depolarization are required for endocannabinoid release, which leads to potentiation of electrical and glutamatergic transmission at Club endings. When applied alone, neither application the mGluR1 agonist DHPG (D) nor dendritic depolarization (‘depo’) of the Mauthner cell (E) were sufficient to trigger potentiation of the mixed synaptic response. F, Combined application of DHPG and dendritic depolarization evoked long-lasting potentiation of the mixed synaptic response. G, The potentiation evoked mGluR1 activation and dendritic depolarization was prevented by the cannabinoid Type 1 receptor (CB1R) antagonists AM and SR. H, Cartoon summarizes the mechanisms underlying activity-dependent potentiation of electrical (and glutamatergic) transmission and Club ending mixed synapses on the Mauthner cell. Synaptic activity (100 Hz) activates mGluR1s and depolarizes the Mauthner cell, leading to the production of endocannabinoids. Modified with permission from Cachope et al., 2007 (Cachope et al., 2007). Activation of CB1Rs triggers release of dopamine from nearby dopaminergic varicosities. Dopamine acts postsynaptically via D1/5 receptors and PKA to potentiate electrical and glutamatergic transmission. Modified with permission from Pereda et al., 2014 (Pereda, 2014).

Metabotropic glutamate receptors and electrical synapses during development

Neuronal circuits are established during brain development by the emergence of synapses. While in adult mammalian central nervous system chemical and electrical synapses co-exist, they appear at different developmental times and mature with dramatically different time courses. Electrical synapses are first to emerge in the first two postnatal weeks. However, the presence of electrical synapses follows an interesting developmental time course. While initially pervasive they later decrease in number following a stereotyped spatiotemporal and specific cell type pattern (Fig. 4A) to remain in only some cell types and structures of the adult brain (Belousov and Fontes, 2013a). Chemical synapses in contrast develop more gradually and go through a process of initiation, maturation and pruning via synapse elimination, and show adult morphology by the third and fourth weeks (López-Bendito and Molnár, 2003; Farhy-Tselnicker and Allen, 2018).

Figure 4. Contribution of mGluRs to synaptic formation during development and brain injury.

A, Activation of Group II mGluRs enhance (red arrows) neuronal coupling and C×36 expression during early development (P1, postnatal day one). The effect is counterbalanced by activation of GABAARs (blue arrow). Activation of NMDARs at later developmental stages leads the developmental uncoupling of neuronal gap junctions and down-regulation of C×36 (blue arrow). P1 and P30: post-natal days 1 and 30. B, Although with different time course, activation of Group II mGluRs (red arrow) similarly enhance neuronal coupling and C×36 expression following neuronal injury (dark pink bar). Modified with permission from Belousov, 2012 (Belousov, 2012).

It is believed that the initial prevalence of electrical synapses serves to outline an initial blueprint of neuronal connectivity which is later established by the emergence of chemical synapses, whose appearance leads in most cases to the elimination of electrical synapses (Mentis et al., 2002; Arumugam et al., 2005). Moreover, it has been shown that early formation of electrical synapses is a requisite for the formation of chemical synapses. This developmental sequence in synapse type formation has been observed in various nervous systems ranging from invertebrates (Szabo et al., 2004; Todd et al., 2010; Bargmann, 2012) to mammals (Yu et al., 2012) and it relies, at least partially, on interactions between neurotransmitter receptors and neuronal gap junctions. However, interactions between receptors and neuronal gap junctions shape and modulate rather than determine the formation of synapses, which likely follows a developmental gene expression plan. The phenomenon has been best characterized in the mammalian nervous system. As illustrated in the cartoon of Fig 4A, both gap junction coupling and expression of the neuronal C×36 dramatically increase during early stages of brain development. Tracer coupling has been reported to be extensive in rat cortex, hippocampus, dentate gyrus, striatum, and spinal cord and the presence of neuronal gap junctions was documented combining tracer and electrical coupling with expression of C×36. Studies in rat cortical and hypothalamic neurons (Park et al., 2011) revealed that the early increase of C×36 coupling is driven by an interplay between group II mGluR receptors. Activation of group II mGluR receptors increases coupling and C×36 expression via a cAMP/PKA-dependent pathway whereas activation of GABA-A receptors, which are depolarizing at this developmental stage, increases intracellular Ca⁺⁺ leading to the activation of PKC and a reduction of coupling and C×36 expression. In-vitro and in-vivo studies showed that the effect of mGluRs requires a two-week chronic activation by agonists whereas shorter application times (1 hour) resulted in only transient effects in coupling without changes in C×36 expression (Song et al., 2012). The increase in C×36 expression mediated by mGluRs depends on transcription and requires of a neuron-restrictive silencer element in the C×36 gene promoter (Park et al., 2011).

Remarkably, these early interactions between neuronal gap junctions and neurotransmitter receptors take place at a time when chemical synaptic transmission is not yet established, suggesting non-synaptic functions of these receptors. Actually, both mGluR and GABA-ARs are evolutionarily ancient and precede the appearance of chemical synapses (see below). The prevalent and widespread distribution of electrical synapses is markedly reduced in the third postnatal week, in coincidence with the maturation of chemical synapses (López-Bendito and Molnár, 2003; Farhy-Tselnicker and Allen, 2018). The reduction in C×36 expression relies on another interaction between neuronal gap junctions and neurotransmitter receptors, in this case with NMDARs. Activation of NMDARs during this period leads to a marked decrease in neuronal coupling and reduction in the expression of C×36 (Arumugam et al., 2005). The actions of NMDARs are mediated by a CREB-dependent down-regulation of C×36 gene expression (Arumugam et al., 2005).

Metabotropic glutamate receptors and electrical synapses following neuronal injury

The involvement of mGluRs in regulating gap junction transmission is not restricted to development and synaptic plasticity in adulthood, as these receptors have been also shown to regulate neuronal gap junctions in neuronal injury. Increase in C×43 expression, likely corresponding to astrocytes, following ischemia was first reported in hippocampus and striatum (reviewed in (Hansson et al., 2000)). A subsequent study in hippocampus revealed that, in addition to astrocytic C×43, elevated levels of C×36 (expressed in neurons) and C×32 (expressed mainly in oligodendrocytes) were also found after global ischemia (Oguro et al., 2001), indicating that neuronal coupling could increase following neuronal injury. Further evidence indicates that C×36 expression increases in the first hours following neuronal injury (Fig. 4B) (Wang et al., 2010). However, the beneficial versus deleterious effects of this increase is a matter of controversy (Belousov and Fontes, 2013a). A group of studies (reviewed in (Belousov and Fontes, 2013b)) indicate that the increase in C×36 is mediated by activation of group II mGluR receptors (Fig. 4B). These studies compared various forms of injury (ischemia, hyperactivity, trauma, and hypoosmotic shock) (Wang et al., 2010; Belousov, 2012), which all lead to an increase in coupling and C×36. The involvement of mGluRs during development, whose activation increases gap junction coupling and neuronal survival (Park et al., 2011) prompted testing if -under pathological conditions- mGluR activation would similarly modulate C×36-mediated neuronal coupling. This possibility was tested using the oxygen-glucose deprivation (OGD) in-vitro model of mouse somatosensory cortical neurons, at which C×36 protein expression and gap junction coupling increased in response to OGD challenge. Similar increase in C×36 and gap junction coupling was evoked by bath application of the group II mGluR agonist LY379268 and was prevented by bath application of the group II mGluR antagonist LY341495 (Wang et al., 2012). The involvement of mGluRs was additionally tested in an in-vivo model of photo-thrombotic focal cerebral ischemia that also exhibited increased levels of C×36 expression, an effect that was mimicked by systemic administration of mGluR agonists in non-ischemic mice. Consistently, systemic treatment with a mGluR antagonist prevented the increase in C×36 observed in response to the ischemic insult (Wang et al., 2012). The changes required cAMP and activation of PKA. Because despite the increase in C×36 protein no changes in mRNA levels were observed, the regulatory effects of mGluRs must likely involve post-transcriptional mechanisms (Wang et al., 2012).

Thus, brain injury is known to produce a massive increase in extracellular glutamate, and blocking group II mGluRs prevented the increases in coupling and C×36 (Wang et al., 2012). Blockade of both mGluRs and neuronal coupling were beneficial for neuronal survival, suggesting that the increase in C×36 actually contributes to the pathological processes that follow injury. The evidence supports the model represented in the cartoon of Figure 5. The excess of glutamate that follows neuronal injury leads to overactivation of NMDARs, which in turn trigger mechanisms of neuronal death in unaffected cells (Wang et al., 2010; Belousov, 2012). The concurrent activation of group II mGluRs leads to a simultaneous increase in C×36. The increased coupling allows NMDA-triggered cell death signals to spread to neighboring cells in the lesion’s penumbra, amplifying the effect of the injury by promoting secondary death. In other words, group II mGLuRs seem to act in synergy to amplify the toxic effect of NMDARs.

Figure 5. Contributions of mGluRs to neuronal death.

Release of glutamate from injured neurons promotes death of additional neurons via NMDARs activation. NMDAR-mediated Ca⁺⁺ overload leads to the generation of molecules that promote cellular death (‘death signals’). Glutamate simultaneously activates Group II mGluRs, which by enhancing gap junctional coupling facilitates the spread of ‘death signals’ to neurons adjacent to the lesion (‘penumbra’). Modified with permission from Belousov and Fontes, 2013 (Belousov and Fontes, 2013a).

Evolutionary perspective of modulation by metabotropic glutamate receptors

Neuronal gap junctions were found to be regulated by glutamate via activation of both NMDARs and Type I and II mGluRs. There are however evolutionary features that likely make regulation of neuronal gap junctions by NMDARs and mGluRs distinct. Recent studies have focused on understanding the evolutionary origin of neurons and synapses. More specifically, the core question centers on determining if neurons, with their distinct anatomy and functional properties, were evolutionarily preceded by synaptic proteins or, alternatively, the formation of synapses followed the origin of neurons (Jorgensen, 2014). Our current knowledge of the proteomic composition of vertebrate glutamatergic synapses enabled applying comparative genomic approaches for the identification of ancestral synaptic components (Ryan and Grant, 2009). The picture emerging from these studies is that some synaptic proteins precede the origin of neurons and the nervous system and are even present in unicellular organisms, generally participating in responses to environmental stimuli. Remarkably, mGluRs were found to be present in organisms lacking a nervous system such as Porifera (sponges) and, therefore, prior to a common ancestor of all synapses or ‘ursynapse’ (Ryan and Grant, 2009).

Thus, the appearance of mGLURs preceded that of ionotropic glutamate receptors (NMDARs and AMPARs) found in Cnidaria (which includes jelly fish, anemone, and hydra; (Ryan and Grant, 2009)). Such a disparity in the evolutionary emergence of mGluRs and NMDARs is likely to be reflected in the functional contributions of each of these receptors. For example, upregulation of neuronal gap junctions and coupling by mGluRs during development occurs during the first two postnatal weeks when chemical synapses are still forming (Farhy-Tselnicker and Allen, 2018), suggesting that in this case mGluRs play a more ancient, non-synaptic, evolutionary role. In contrast, the regulatory role of NMDARs on electrical synapses during development takes place once chemical synapses are mature. The differential involvement of these receptors during development reminds us of Ernst Haeckel’s phrase “ontogeny recapitulates phylogeny”, which defined the now refuted ‘recapitulation theory’.

There might be also differences in the way mGluRs regulate electrical transmission in the mature brain. While as with NMDARs the synaptic localization of mGluRs allows them to sense the flow of ongoing information within neural circuits, mGluRs have the distinct capacity of amplifying this information via powerful G-protein-mediated signaling mechanisms. In other words, mGLURs seem to uniquely combine the features of both NMDA (synaptic activation) and amine neuromodulators receptors (GPCR signaling) for the regulation of electrical transmission and cellular functions in general. Moreover, presynaptic boutons are not the only source of glutamate. That is, electrical synapses could be also regulated by release of glutamate (Parpura et al., 1994) or D-serine (Meunier et al., 2017) from nearby astrocytic processes. This mechanism, reported to regulate chemical synapses (Navarrete and Araque, 2008, 2010), could add an additional layer of complexity to the regulatory functions of NMDARs and mGluRs on electrical transmission.

Conclusions

All the examples of regulation of electrical synapses by mGluRs reviewed here involved gap junctions expressing C×36 or its vertebrate homologs. While C×36 was proposed to co-exist with other connexins in neuronal cell types (Li et al., 2008), there is no evidence of mGluRs regulating electrical synapses formed by other neuronal connexins. There is, on the other hand, data indicating that the regulatory roles of mGluRs are not restricted to neuronal gap junctions. In endothelial cells, activation of mGluR5 leads to an increase in C×43 expression in a model of homocysteine-dependent impairment of cerebral endothelial wound repair (Chen et al., 2012). Also, in the heart, mGluRs 1/5 were found to be present in the ventricular myocardium and intercalated disks which contains gap junctions formed by C×43. Activation of mGluRs 1/5 with DHPG in H9c2 cardiomyoblast cells led to phosphorylation on C×43 and reduction of gap junctional communication (Xie et al., 2014). In summary, regulation of gap junctions by mGluRs might be widespread, operating at multiple tissues and targeting many connexin isoforms. Finally, in the nervous system, astrocytes are coupled by abundant C×43- and C×30-containing gap junctions which are influenced by neuronal activity (Rouach et al., 2004; Koulakoff et al., 2008) and they express various types of mGluRs (Spampinato et al., 2018), raising the possibility that their intercellular channels could be similarly regulated by these receptors.

In contrast to chemical synapses, the evidence indicating a regulatory role for mGluRs on electrical synapses is still limited. However, the data suggest that mGluRs play important roles in various functional contexts. Far from only regulating the strength of electrical synapses in the adult brain, mGluRs participate in the formation of neural circuits during development. Moreover, their critical participation in pathological mechanisms that follow neuronal injury makes them candidates for pharmacological intervention (Belousov and Fontes, 2013a). Thus, altogether, the evidence suggests that mGluRs are likely to play critical roles in the regulation of neuronal gap junctions and given the range of fundamental processes that they contribute to, it might only constitute the tip of the iceberg of a fundamental regulatory role.

Highlights.

• Metabotropic glutamate receptors (mGluRs) act as regulators of gap junction-mediated communication between neurons.

• Activity-driven interactions between mGluRs and neuronal gap junctions lead to long-term changes in the strength of electrical synapses.

• mGluR regulation of electrical synapses might involve complex interactions with other neurotransmitter systems.

• mGluR modulation of neuronal gap junctions also play relevant roles during neurodevelopment and contribute and brain injury.