The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 μM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 μM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of −70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.
BACKGROUND AND PURPOSEElevation of glutamate, an excitatory amino acid, during inflammation and injury plays a crucial role in the reception and transmission of sensory information via ionotropic and metabotropic receptors. This study aimed to investigate the mechanisms underlying the biphasic effects of metabotropic glutamate mGlu5 receptor activation on responses to noxious heat. EXPERIMENTAL APPROACHWe assessed the effects of intraplantar quisqualate, a non-selective glutamate receptor agonist, on heat and mechanical pain behaviours in mice. In addition, the effects of quisqualate on the intracellular calcium response and on membrane currents mediated by TRPV1 channels, were examined in cultured dorsal root ganglion neurons from mice. KEY RESULTSActivation of mGlu5 receptors in hind paw transiently increased, then decreased, the response to noxious heat. In sensory neurons, activation of mGlu5 receptors potentiated TRPV1-mediated intracellular calcium elevation, while terminating activation of mGlu5 receptors depressed it. TRPV1-induced currents were potentiated by activation of mGlu5 receptors under voltage clamp conditions and these disappeared after washout. However, voltage-gated calcium currents were inhibited by the mGlu5 receptor agonist, even after washout. CONCLUSIONS AND IMPLICATIONSThese results suggest that, in sensory neurons, mGlu5 receptors biphasically modulate TRPV1-mediated intracellular calcium response via transient potentiation of TRPV1 channel-induced currents and persistent inhibition of voltage-gated calcium currents, contributing to heat hyper-and hypoalgesia.
Damaged tissues release glutamate and other chemical mediators for several hours. These chemical mediators contribute to modulation of pruritus and pain. Herein, we investigated the effects of long-term activation of excitatory glutamate receptors on functional expression of transient receptor potential vaniloid type 1 (TRPV1) in dorsal root ganglion (DRG) neurons and then on thermal pain behavior. In order to detect the TRPV1-mediated responses in cultured DRG neurons, we monitored intracellular calcium responses to capsaicin, a TRPV1 agonist, with Fura-2. Long-term (4 h) treatment with glutamate receptor agonists (glutamate, quisqualate or DHPG) increased the proportion of neurons responding to capsaicin through activation of metabotropic glutamate receptor mGluR1, and only partially through the activation of mGluR5; engagement of these receptors was evident in neurons responding to allylisothiocyanate (AITC), a transient receptor potential ankyrin type 1 (TRPA1) agonist. Increase in the proportion was suppressed by phospholipase C (PLC), protein kinase C, mitogen/extracellular signal-regulated kinase, p38 mitogen-activated protein kinase or transcription inhibitors. Whole-cell recording was performed to record TRPV1-mediated membrane current; TRPV1 current density significantly increased in the AITC-sensitive neurons after the quisqualate treatment. To elucidate the physiological significance of this phenomenon, a hot plate test was performed. Intraplantar injection of quisqualate or DHPG induced heat hyperalgesia that lasted for 4 h post injection. This chronic hyperalgesia was attenuated by treatment with either mGluR1 or mGluR5 antagonists. These results suggest that long-term activation of mGluR1/5 by peripherally released glutamate may increase the number of neurons expressing functional TRPV1 in DRG, which may be strongly associated with chronic hyperalgesia.
Activation of muscarinic acetylcholine receptors M1 (M1-mAChRs) is known to facilitate the induction of long-term potentiation (LTP) in hippocampal CA1. We previously reported that M1-mAChRs are functionally expressed in the intracellular membranes, such as endoplasmic reticulum and Golgi apparatus, as well as cell-surface, in neuroblastoma cells and neurons in central nervous system. In this study, we took the extracellular field recording in acute hippocampal slice preparation to examine how cell-surface and intracellular M1-mAChRs regulates hippocampal LTP. LTP in hippocampal CA1was observed 60 min after high frequency stimulation (HFS) at Schaffer collaterals. Pretreatment with carbachol (CCh) significantly enhanced LTP beyond 1h after HFS, and the facilitation was thoroughly inhibited by pirenzepine, a cell-permeable M1 antagonist. On the other hand, a cell-impermeable M1 antagonist, MT7, inhibited the early part of facilitation (5-15 min) without affecting the late part (50-60 min). Tetraethylammonium (TEA), an acetylcholine uptake inhibitor, also selectively suppressed the late part of facilitation. Next, we examined the effect of diisopropyl fluorophosphate (DFP), an irreversible cholinesterase inhibitor thereupon. Pretreatment with DFP also significantly and persistently enhanced LTP after HFS, and the facilitation was also completely inhibited by pirenzepine. In addition, MT7 selectively suppressed the early part of facilitation, while TEA attenuated the late part of facilitation. These results suggest that an exogenous cholinergic agonist or endogenously released acetylcholine acts on both the cellsurface and the intracellular M1-mAChRs, resulting in augment of hippocampal LTP.
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