Biphasic Modulation of Noxious Heat Sensitivity in Sensory Neurons by Peripheral Metabotropic Glutamate Receptors

Release of glutamate during inflammation and injury changes sensitivity and transmission efficiency of noxious sensory information via glutamate receptors. We found that activation of metabotropic glutamate receptor type 5 (mGluR5) transiently increased, and then subsequently decreased, noxious heat sensitivity. Similarly, mGluR5 activation in cultured sensory neurons potentiated intracellular calcium elevation mediated by transient receptor potential channel, subfamily V, member 1 (TRPV1), a noxious heat receptor; subsequent cessation of mGluR5 activation depressed intracellular calcium levels. The underlying mechanisms were potentiation of TRPV1 current in the presence of mGluR5 ligands and persistent inhibition of voltage-gated calcium channels (VGCC), even after mGluR5 ligand washout. Thus, mGluR5 biphasically modulates TRPV1-mediated cellular responses in sensory neurons, which contributes to heat hyper-and hypoalgesia. These phenomena may contribute to changes in noxious heat sensitivity during inflammation and healing. To cite this article: Takayoshi Masuoka, et al. Biphasic modulation of noxious heat sensitivity in sensory neurons by peripheral metabotropic glutamate receptors. Inflammation results in the release of many chemical mediators, such as bradykinin, histamine, serotonin, prostaglandins etc., which act on local tissues [1,2]. These compounds activate a variety of specific receptors and channels on peripheral sensory nerve terminals, which induce pruritus and pain. Glutamate, a dominant neurotransmitter of central nervous system, is also one of the mediators released from neurons, glia, and damaged cells in peripheral tissue [3-5]. It has been proposed that this released glutamate acts in part on group I metabotropic glutamate receptors, consisting of mGluR1 and mGluR5, on peripheral unmyelinated sensory afferents, which contributes to generation and/or potentiation of inflammatory pain [6]. The mechanism of acute pain induced by peripheral group I mGluRs, especially mGluR5, has been gradually elucidated by several studies [6,7]. Peripheral mGluR5 potentiates and/or directly activates the transient receptor potential cation channel, subtype V member 1 (TRPV1), which results in elevation of noxious heat sensitivity and spontaneous pain [7,8]. We additionally identified a novel hypesthesia system for noxious heat, by which mGluR5 mediates persistent inhibition of voltage-gated calcium channels (VGCC) in sensory neurons [8]. It was previously reported that peripheral mGluR5 activity is closely related to TRPV1 function [7,9]. We observed that the presence of mGluR5 ligands potentiated intracellular calcium elevation and current response induced by the TRPV1 agonist capsaicin, in cultured sensory neurons, without changing the capsaicin-sensitive population [8]. Hu et RESEARCH HIGHLIGHT

Inflammation results in the release of many chemical mediators, such as bradykinin, histamine, serotonin, prostaglandins etc., which act on local tissues [1,2] .These compounds activate a variety of specific receptors and channels on peripheral sensory nerve terminals, which induce pruritus and pain.Glutamate, a dominant neurotransmitter of central nervous system, is also one of the mediators released from neurons, glia, and damaged cells in peripheral tissue [3-5]   .It has been proposed that this released glutamate acts in part on group I metabotropic glutamate receptors, consisting of mGluR1 and mGluR5, on peripheral unmyelinated sensory afferents, which contributes to generation and/or potentiation of inflammatory pain [6] .The mechanism of acute pain induced by peripheral group I mGluRs, especially mGluR5, has been gradually elucidated by several studies [6,7]   .Peripheral mGluR5 potentiates and/or directly activates the transient receptor potential cation channel, subtype V member 1 (TRPV1), which results in elevation of noxious heat sensitivity and spontaneous pain [7,8] .We additionally identified a novel hypesthesia system for noxious heat, by which mGluR5 mediates persistent inhibition of voltage-gated calcium channels (VGCC) in sensory neurons [8]   .
It was previously reported that peripheral mGluR5 activity is closely related to TRPV1 function [7,9] .We observed that the presence of mGluR5 ligands potentiated intracellular calcium elevation and current response induced by the TRPV1 agonist capsaicin, in cultured sensory neurons, without changing the capsaicin-sensitive population [8] .Hu et

RESEARCH HIGHLIGHT
al. demonstrated that mGluR5 in the peripheral terminals of sensory neurons mediate synthesis of prostaglandins via the phospholipase C (PLC) cascade, and then directly activate or sensitize neighboring TRPV1 [7] .It has also been reported that mGluR5 activation facilitates TRPV1 trafficking and activity in other cell lines [10] .Protein kinase C (PKC) activation induced by mGluR5 causes rapid delivery of functional TRPV1 channels to the plasma membrane and potentiates TRPV1 currents in oocytes co-expressing TRPV1 and mGluR5 [10] .Bhave et al. reported that phosphorylation of TRPV1, induced by PKC activation, sensitizes TRPV1 channels [11] .Furthermore, increased levels of phosphatidylinositol 4, 5-bisphosphate (PIP2) resulted in inhibition of TRPV1 channels [12] , suggesting that PLC activation by mGluR5 may be a consequence of PIP2-mediated TRPV1 channel inhibition.
Several studies indicate that mGluR5 activation also causes an elevation in intracellular calcium concentration ([Ca 2+ ]i) in cultured rodent dorsal root ganglion (DRG) neurons [7,9,13] .Hu et al. reported that mGluR5-induced calcium responses were observed in 38 % of capsaicin-sensitive neurons [7] , although we were unable to detect mGluR5-induced calcium responses in cultured mouse DRG neurons.We hypothesized that these inconsistencies may be caused by differences in membrane excitability, expression of molecules in culture, and/or recording conditions.Kim et al. demonstrated that the mGluR5-induced calcium responses were mediated by activation of TRPV1 subsequent to diacylglycerol production [9] , which acts in the central terminals of sensory neurons in the spinal cord.Interestingly, in the central nervous system, such as the cerebellum, striatum etc., mGluR1/5 are functionally coupled with other TRP channels, such as TRPC1 and TRPC3/7 [14,15] , which are also expressed by DRG neurons [16] .It is likely that the interactions between group I mGluRs and TRP channels have low specificity, which may enable easy switching between coupled molecules with variable cellular conditions, such as channels, enzyme and scaffold protein expression.
Our study clarified that agonist removal after activation of mGluR5 caused a significant decrease in the degree of TRPV1-mediated calcium elevation, without decreasing the TRPV1 current [8] .This is the result of persistent inhibition of VGCC currents by mGluR5 activation in capsaicin-sensitive DRG neurons, even after cessation of mGluR5 activation.Several studies have reported that Gq/11 protein-coupled metabotropic receptors contribute to VGCC current modulation.For instance, the application of mGluR agonists reduced L-type VGCC currents in cultured hippocampal and neocortical neurons [17,18] .Gamper et al. reported that the depletion of PIP2, by muscarinic M1 receptor activation, resulted in the suppression of N-type calcium currents in superior cervical ganglion (SCG) neurons [19]   .Liu et al. reported that diacylglycerol lipase activation inhibited L-and N-type calcium channels induced by M1 receptor activation in SCG neurons [20] .Interestingly, mGluR5a activation can also cause biosynthesis of 2-arachidonoylglycerol, an endocannabinoid, resulting in N-type calcium channel inhibition via the activation of cannabinoid receptor type 1 (CB1 receptor) in cultured SCG neurons [21] .However, it is likely that the mechanism by which mGluR5 inhibits VGCC currents may be different from those described in previous reports, because VGCC currents were temporarily inhibited during ligand application in these reports, while they were sustainably inhibited in our study.Recently, we have come to recognized that mGluR1/5 activation can regulate internalization and translation of several molecules via at least two signal transduction pathways (extracellular signal-regulated kinase (ERK) and mammalian target of rapamycin (mTOR)), resulting in long-term synaptic depression (LTD) in the hippocampus [22,23]   .Therefore, it is possible that mGluR1/5 also regulate internalization or translation of VGCC regulating molecules in DRG neurons over an extended period.
Scroggs and Fox reported that the distribution of VGCC subtypes was dependent on cell size in rat DRG neurons [24] .For instance, the percentage of L-type calcium channels was significantly larger in small diameter neurons (20-27 μm; 52.9% of total whole cell calcium current) than in medium and large diameter neurons (33-38 and 45-51 μm; 6.6% and 19.4% of total whole cell calcium current, respectively).Contrastively, the percentage of N-type calcium channels was similar in small, medium, and large diameter neurons.Almost all TRPV1-expressing neurons in the DRG are classified as small and medium sized neurons [25] .Therefore, mGluR5 activation may inhibit a type of VGCC current that is predominantly expressed in capsaicin-sensitive neurons.In future studies, it is necessary to clarify the subtype of VGCC inhibited by mGluR5 activation.
We determined that mGluR5 activation in sensory nerve terminals induced heat hyperalgesia, followed by heat hypoalgesia in mouse hind paws.These changes in thermal sensitivity correlate with modulation of intracellular calcium elevation by mGluR5, through TRPV1 activation [8] (Figure 1).In other words, mGluR5 activation produced acute heat hyperalgesia in vivo, which correlated with sensitization of TRPV1 responses in the presence of glutamate in primary sensory neurons in vitro (Figure 1B).Jung et al. also reported that an intraplantar injection of DHPG, a selective mGluR1/5 agonist, facilitated thermal and mechanical sensitivity in rats [26]   .Our novel and unique observations were found during the chronic phase after mGluR5 stimulation, when heat hypoalgesia was observed [8] .The heat hypoalgesia in vivo may correlate with the reduction in TRPV1-mediated [Ca 2+ ]i elevation after mGluR5 agonist washout seen in vitro (Figure 1C), perhaps due to the metabolism or dilution of ligand within the paw tissue.It is possible that mGluR5 contributes to both potentiation of heat hyperalgesia during inflammation and its improvement following resolution of inflammation.The other interesting observation is that mGluR5 has a weak effect on mechanical pain sensitivity.Mechanoreceptive neurons co-express degerin/epithelial Na + channels and TRP (TRPV3, TRPV4 or TRPA1) channel proteins that together, form mechano-electrical transduction (MeT) channels [27] , which are independent of TRPV1.
In conclusion, mGluR5 activation had biphasic effects on TRPV1-mediated [Ca 2+ ]i elevation in mouse DRG neurons [8]   .These effects were due to the transient potentiation of TRPV1 currents and the persistent inhibition of VGCC currents induced by TRPV1-mediated depolarization.These phenomena may contribute to the changes in noxious heat sensitivity during inflammation and its healing.

Figure 1 .
Figure 1.Schematic summary of the modulation of TRPV1-mediated cell responses induced by mGluR5 in sensory neurons.(A)Activation of TRPV1 channels causes an inward-current response, followed by VGCC activation induced by depolarization, which resulted in a large intracellular calcium elevation.(B) Activation of mGluR5 potentiates TRPV1 channel activity, although VGCC is inhibited.In total, TRPV1-mediated intracellular calcium elevation is potentiated, which contributes to the elevation of noxious heat sensitivity.(C) Following cessation of mGluR5 activation, TRPV1 activity enhancement is terminated, while VGCC are persistently inhibited.Therefore, TRPV1-mediated intracellular calcium elevation is reduced, which contributes to the decline in noxious heat sensitivity.