Periodontitis promotes bacterial extracellular vesicle-induced neuroinflammation in the brain and trigeminal ganglion.

Gram-negative bacteria derived extracellular vesicles (EVs), also known as outer membrane vesicles, have attracted significant attention due to their pathogenic roles in various inflammatory diseases. We recently demonstrated that EVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) can cross the blood-brain barrier (BBB) and that their extracellular RNA cargo can promote the secretion of proinflammatory cytokines, such as IL-6 and TNF-α, in the brain. To gain more insight into the relationship between periodontal disease (PD) and neuroinflammatory diseases, we investigated the effect of Aa EVs in a mouse model of ligature-induced PD. When EVs were administered through intragingival injection or EV-soaked gel, proinflammatory cytokines were strongly induced in the brains of PD mice. The use of TLR (Toll-like receptor)-reporter cell lines and MyD88 knockout mice confirmed that the increased release of cytokines was triggered by Aa EVs via TLR4 and TLR8 signaling pathways and their downstream MyD88 pathway. Furthermore, the injection of EVs through the epidermis and gingiva resulted in the direct retrograde transfer of Aa EVs from axon terminals to the cell bodies of trigeminal ganglion (TG) neurons and the subsequent activation of TG neurons. We also found that the Aa EVs changed the action potential of TG neurons. These findings suggest that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis.

Depression of Synaptic N-methyl-D-Aspartate Responses by Xenon and Nitrous Oxide.

In "synapse bouton preparation" of rat hippocampal CA3 neurons, we examined how Xe and N2O modulate N-methyl-D-aspartate (NMDA) receptor-mediated spontaneous and evoked excitatory post-synaptic currents (sEPSCNMDA and eEPSCNMDA). This preparation is a mechanically isolated single neuron attached with nerve endings (boutons) preserving normal physiologic function and promoting the exact evaluation of sEPSCNMDA and eEPSCNMDA responses without influence of extrasynaptic, glial, and other neuronal tonic currents. These sEPSCs and eEPSCs are elicited by spontaneous glutamate release from many homologous glutamatergic boutons and by focal paired-pulse electric stimulation of a single bouton, respectively. The s/eEPSCAMPA/KA and s/eEPSCNMDA were isolated pharmacologically by their specific antagonists. Thus, independent contributions of pre- and postsynaptic responses could also be quantified. All kinetic properties of s/eEPSCAMPA/KA and s/eEPSCNMDA were detected clearly. The s/eEPSCNMDA showed smaller amplitude and slower rise and 1/e decay time constant (τ Decay) than s/eEPSCAMPA/KA Xe (70%) and N2O (70%) significantly decreased the frequency and amplitude without altering the τ Decay of sEPSCNMDA They also decreased the amplitude but increased the Rf and PPR without altering the τ Decay of the eEPSCNMDA These data show clearly that "synapse bouton preparation" can be an accurate model for evaluating s/eEPSCNMDA Such inhibitory effects of gas anesthetics are primarily due to presynaptic mechanisms. Present results may explain partially the powerful analgesic effects of Xe and N2O. SIGNIFICANCE STATEMENT: We could record pharmacologically isolated NMDA receptor-mediated spontaneous and (action potential-evoked) excitatory postsynaptic currents (sEPSCNMDA and eEPSCNMDA) and clearly detect all kinetic parameters of sEPSCNMDA and eEPSCNMDA at synaptic levels by using "synapse bouton preparation" of rat hippocampal CA3 neurons. We found that Xe and N2O clearly suppressed both sEPSCNMDA and eEPSCNMDA. Different from previous studies, present results suggest that Xe and N2O predominantly inhibit the NMDA responses by presynaptic mechanisms.

Protein Kinase A Is Responsible for the Presynaptic Inhibition of Glycinergic and Glutamatergic Transmissions by Xenon in Rat Spinal Cord and Hippocampal CA3 Neurons.

The effects of a general anesthetic xenon (Xe) on spontaneous, miniature, electrically evoked synaptic transmissions were examined using the "synapse bouton preparation," with which we can clearly evaluate pure synaptic responses and accurately quantify pre- and postsynaptic transmissions. Glycinergic and glutamatergic transmissions were investigated in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xe presynaptically inhibited spontaneous glycinergic transmission, the effect of which was resistant to tetrodotoxin, Cd2+, extracellular Ca2+, thapsigargin (a selective sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor), SQ22536 (an adenylate cyclase inhibitor), 8-Br-cAMP (membrane-permeable cAMP analog), ZD7288 (an hyperpolarization-activated cyclic nucleotide-gated channel blocker), chelerythrine (a PKC inhibitor), and KN-93 (a CaMKII inhibitor) while being sensitive to PKA inhibitors (H-89, KT5720, and Rp-cAMPS). Moreover, Xe inhibited evoked glycinergic transmission, which was canceled by KT5720. Like glycinergic transmission, spontaneous and evoked glutamatergic transmissions were also inhibited by Xe in a KT5720-sensitive manner. Our results suggest that Xe decreases glycinergic and glutamatergic spontaneous and evoked transmissions at the presynaptic level in a PKA-dependent manner. These presynaptic responses are independent of Ca2+ dynamics. We conclude that PKA can be the main molecular target of Xe in the inhibitory effects on both inhibitory and excitatory neurotransmitter release. SIGNIFICANCE STATEMENT: Spontaneous and evoked glycinergic and glutamatergic transmissions were investigated using the whole-cell patch clamp technique in rat spinal sacral dorsal commissural nucleus and hippocampal CA3 neurons, respectively. Xenon (Xe) significantly inhibited glycinergic and glutamatergic transmission presynaptically. As a signaling mechanism, protein kinase A was responsible for the inhibitory effects of Xe on both glycine and glutamate release. These results may help understand how Xe modulates neurotransmitter release and exerts its excellent anesthetic properties.

Trichloroethanol, an active metabolite of chloral hydrate, modulates tetrodotoxin-resistant Na+ channels in rat nociceptive neurons.

BACKGROUND: Chloral hydrate is a sedative-hypnotic drug widely used for relieving fear and anxiety in pediatric patients. However, mechanisms underlying the chloral hydrate-mediated analgesic action remain unexplored. Therefore, we investigated the effect of 2',2',2'-trichloroethanol (TCE), the active metabolite of chloral hydrate, on tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive sensory neurons. METHODS: The TTX-R Na+ current (INa) was recorded from acutely isolated rat trigeminal ganglion neurons using the whole-cell patch-clamp technique. RESULTS: Trichloroethanol decreased the peak amplitude of transient TTX-R INa in a concentration-dependent manner and potently inhibited persistent components of transient TTX-R INa and slow voltage-ramp-induced INa at clinically relevant concentrations. Trichloroethanol exerted multiple effects on various properties of TTX-R Na+ channels; it (1) induced a hyperpolarizing shift on the steady-state fast inactivation relationship, (2) increased use-dependent inhibition, (3) accelerated the onset of inactivation, and (4) retarded the recovery of inactivated TTX-R Na+ channels. Under current-clamp conditions, TCE increased the threshold for the generation of action potentials, as well as decreased the number of action potentials elicited by depolarizing current stimuli. CONCLUSIONS: Our findings suggest that chloral hydrate, through its active metabolite TCE, inhibits TTX-R INa and modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons. These pharmacological characteristics provide novel insights into the analgesic efficacy exerted by chloral hydrate.

Astrocyte-derived adenosine excites sleep-promoting neurons in the ventrolateral preoptic nucleus: Astrocyte-neuron interactions in the regulation of sleep.

Although ATP and/or adenosine derived from astrocytes are known to regulate sleep, the precise mechanisms underlying the somnogenic effects of ATP and adenosine remain unclear. We selectively expressed channelrhodopsin-2 (ChR2), a light-sensitive ion channel, in astrocytes within the ventrolateral preoptic nucleus (VLPO), which is an essential brain nucleus involved in sleep promotion. We then examined the effects of photostimulation of astrocytic ChR2 on neuronal excitability using whole-cell patch-clamp recordings in two functionally distinct types of VLPO neurons: sleep-promoting GABAergic projection neurons and non-sleep-promoting local GABAergic neurons. Optogenetic stimulation of VLPO astrocytes demonstrated opposite outcomes in the two types of VLPO neurons. It led to the inhibition of non-sleep-promoting neurons and excitation of sleep-promoting neurons. These responses were attenuated by blocking of either adenosine A1 receptors or tissue-nonspecific alkaline phosphatase (TNAP). In contrast, exogenous adenosine decreased the excitability of both VLPO neuron populations. Moreover, TNAP was expressed in galanin-negative VLPO neurons, but not in galanin-positive sleep-promoting projection neurons. Taken together, these results suggest that astrocyte-derived ATP is converted into adenosine by TNAP in non-sleep-promoting neurons. In turn, adenosine decreases the excitability of local GABAergic neurons, thereby increasing the excitability of sleep-promoting GABAergic projection neurons. We propose a novel mechanism involving astrocyte-neuron interactions in sleep regulation, wherein endogenous adenosine derived from astrocytes excites sleep-promoting VLPO neurons, and thus decreases neuronal excitability in arousal-related areas of the brain.

Contribution of tetrodotoxin-resistant persistent Na+ currents to the excitability of C-type dural afferent neurons in rats.

BACKGROUND: Growing evidence supports the important role of persistent sodium currents (INaP) in the neuronal excitability of various central neurons. However, the role of tetrodotoxin-resistant (TTX-R) Na+ channel-mediated INaP in the neuronal excitability of nociceptive neurons remains poorly understood. METHODS: We investigated the functional role of TTX-R INaP in the excitability of C-type nociceptive dural afferent neurons, which was identified using a fluorescent dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchloride (DiI), and a whole-cell patch-clamp technique. RESULTS: TTX-R INaP were found in most DiI-positive neurons, but their density was proportional to neuronal size. Although the voltage dependence of TTX-R Na+ channels did not differ among DiI-positive neurons, the extent of the onset of slow inactivation, recovery from inactivation, and use-dependent inhibition of these channels was highly correlated with neuronal size and, to a great extent, the density of TTX-R INaP. In the presence of TTX, treatment with a specific INaP inhibitor, riluzole, substantially decreased the number of action potentials generated by depolarizing current injection, suggesting that TTX-R INaP are related to the excitability of dural afferent neurons. In animals treated chronically with inflammatory mediators, the density of TTX-R INaP was significantly increased, and it was difficult to inactivate TTX-R Na+ channels. CONCLUSIONS: TTX-R INaP apparently contributes to the differential properties of TTX-R Na+ channels and neuronal excitability. Consequently, the selective modulation of TTX-R INaP could be, at least in part, a new approach for the treatment of migraine headaches.

Astrocytes in the Ventrolateral Preoptic Area Promote Sleep.

Although ventrolateral preoptic (VLPO) nucleus is regarded as a center for sleep promotion, the exact mechanisms underlying the sleep regulation are unknown. Here, we used optogenetic tools to identify the key roles of VLPO astrocytes in sleep promotion. Optogenetic stimulation of VLPO astrocytes increased sleep duration in the active phase in naturally sleep-waking adult male rats (n = 6); it also increased the extracellular ATP concentration (n = 3) and c-Fos expression (n = 3-4) in neurons within the VLPO. In vivo microdialysis analyses revealed an increase in the activity of VLPO astrocytes and ATP levels during sleep states (n = 4). Moreover, metabolic inhibition of VLPO astrocytes reduced ATP levels (n = 4) and diminished sleep duration (n = 4). We further show that tissue-nonspecific alkaline phosphatase (TNAP), an ATP-degrading enzyme, plays a key role in mediating the somnogenic effects of ATP released from astrocytes (n = 5). An appropriate sample size for all experiments was based on statistical power calculations. Our results, taken together, indicate that astrocyte-derived ATP may be hydrolyzed into adenosine by TNAP, which may in turn act on VLPO neurons to promote sleep.SIGNIFICANCE STATEMENT Glia have recently been at the forefront of neuroscience research. Emerging evidence illustrates that astrocytes, the most abundant glial cell type, are the functional determinants for fates of neurons and other glial cells in the central nervous system. In this study, we newly identified the pivotal role of hypothalamic ventrolateral preoptic (VLPO) astrocytes in the sleep regulation, and provide novel insights into the mechanisms underlying the astrocyte-mediated sleep regulation.

Extracellular pH modulation of excitatory synaptic transmission in hippocampal CA3 neurons.

In this study, the effect of extracellular pH on glutamatergic synaptic transmission was examined in mechanically dissociated rat hippocampal CA3 pyramidal neurons using a whole-cell patch-clamp technique under voltage-clamp conditions. Native synaptic boutons were isolated without using any enzymes, using a so-called "synapse bouton preparation," and preserved for the electrical stimulation of single boutons. Both the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) were found to decrease and increase in response to modest acidic (~pH 6.5) and basic (~pH 8.5) solutions, respectively. These changes in sEPSC frequency were not affected by the addition of TTX but completely disappeared by successive addition of Cd2+. However, changes in sEPSC amplitude induced by acidic and basic extracellular solutions were not affected by the addition of neither TTX nor Cd2+. The glutamate-induced whole-cell currents were decreased and increased by acidic and basic solutions, respectively. Acidic pH also decreased the amplitude and increased the failure rate (Rf) and paired-pulse rate (PPR) of glutamatergic electrically evoked excitatory postsynaptic currents (eEPSCs), while a basic pH increased the amplitude and decreased both the Rf and PPR of eEPSCs. The kinetics of the currents were not affected by changes in pH. Acidic and basic solutions decreased and increased voltage-gated Ca2+ but not Na+ channel currents in the dentate gyrus granule cell bodies. Our results indicate that extracellular pH modulates excitatory transmission via both pre- and postsynaptic sites, with the presynaptic modulation correlated to changes in voltage-gated Ca2+ channel currents.NEW & NOTEWORTHY The effects of external pH changes on spontaneous, miniature, and evoked excitatory synaptic transmission in CA3 hippocampal synapses were examined using the isolated nerve bouton preparation, which allowed for the accurate regulation of extracellular pH at the synapses. Acidification generally reduced transmission, partly via effects on presynaptic Ca2+ channel currents, while alkalization generally enhanced transmission. Both pre- and postsynaptic sites contributed to these effects.

Effect of carbamazepine on tetrodotoxin-resistant Na+ channels in trigeminal ganglion neurons innervating to the dura.

Migraine is a neurological disorder characterized by recurrent and disabling severe headaches. Although several anticonvulsant drugs that block voltage-dependent Na+ channels are widely used for migraine, far less is known about the therapeutic actions of carbamazepine on migraine. In the present study, therefore, we characterized the effects of carbamazepine on tetrodotoxin-resistant (TTX-R) Na+ channels in acutely isolated rat dural afferent neurons, which were identified by the fluorescent dye DiI. The TTX-R Na+ currents were measured in medium-sized DiIpositive neurons using the whole-cell patch clamp technique in the voltage-clamp mode. While carbamazepine had little effect on the peak amplitude of transient Na+ currents, it strongly inhibited steady-state currents of transient as well as persistent Na+ currents in a concentration-dependent manner. Carbamazepine had only minor effects on the voltage-activation relationship, the voltage-inactivation relationship, and the use-dependent inhibition of TTX-R Na+ channels. However, carbamazepine changed the inactivation kinetics of TTX-R Na+ channels, significantly accelerating the development of inactivation and delaying the recovery from inactivation. In the current-clamp mode, carbamazepine decreased the number of action potentials without changing the action potential threshold. Given that the sensitization of dural afferent neurons by inflammatory mediators triggers acute migraine headaches and that inflammatory mediators potentiate TTX-R Na+ currents, the present results suggest that carbamazepine may be useful for the treatment of migraine headaches.

Effects of acidic pH on voltage-gated ion channels in rat trigeminal mesencephalic nucleus neurons.

The effects of acidic pH on several voltage-dependent ion channels, such as voltage-dependent K+ and Ca2+ channels, and hyperpolarization-gated and cyclic nucleotide-activated cation (HCN) channels, were examined using a whole-cell patch clamp technique on mechanically isolated rat mesencephalic trigeminal nucleus neurons. The application of a pH 6.5 solution had no effect on the peak amplitude of voltage-dependent K+ currents. A pH 6.0 solution slightly, but significantly inhibited the peak amplitude of voltage-dependent K+ currents. The pH 6.0 also shifted both the current-voltage and conductance-voltage relationships to the depolarization range. The application of a pH 6.5 solution scarcely affected the peak amplitude of membrane currents mediated by HCN channels, which were profoundly inhibited by the general HCN channel blocker Cs+ (1 mM). However, the pH 6.0 solution slightly, but significantly inhibited the peak amplitude of HCN-mediated currents. Although the pH 6.0 solution showed complex modulation of the current-voltage and conductance-voltage relationships, the midpoint voltages for the activation of HCN channels were not changed by acidic pH. On the other hand, voltage-dependent Ca2+ channels were significantly inhibited by an acidic pH. The application of an acidic pH solution significantly shifted the current-voltage and conductance-voltage relationships to the depolarization range. The modulation of several voltage-dependent ion channels by an acidic pH might affect the excitability of mesencephalic trigeminal nucleus neurons, and thus physiological functions mediated by the mesencephalic trigeminal nucleus could be affected in acidic pH conditions.

Phenotypic polarization of activated astrocytes: the critical role of lipocalin-2 in the classical inflammatory activation of astrocytes.

Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4-STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Chronic sleep deprivation-induced proteome changes in astrocytes of the rat hypothalamus.

Sleep deprivation (SD) can influence cognition, memory, and sleep/wake homeostasis and can cause impairments in many physiological processes. Because the homeostatic control of the sleep/wake cycle is closely associated with the hypothalamus, the current study was undertaken to examine proteomic changes occurring in hypothalamic astrocytes following chronic partial SD. After chronic partial SD for 7 days, astrocytes were prepared from rat hypothalamus using a Percoll gradient method, and their proteome profiles were determined by LC-MS/MS. Comparisons of the proteome profiles of hypothalamic astrocytes revealed that chronic partial SD increased (≥1.5-fold) 89 proteins and decreased (≤0.7-fold) 50 proteins; these changes in protein expression were validated by western blot or immunohistochemistry. DAVID and IPA analyses of these proteins suggested that SD may influence gliotransmission and astrocyte activation. PPP2R1A, RTN4, VAMP-2, LGI-1, and SLC17A7 were identified and validated as the main targets of SD in astrocytes. Our results suggest that SD may modulate gliotransmission in the hypothalamus, thereby disturbing sleep/wake homeostasis and increasing susceptibility to neurological disease; however, further studies are required to confirm whether the proteome changes are specific to SD.

Push-in Head Restraining Apparatus for Intracranial Self Stimulation Tasks in Rats.

Head restraining is an experimental technique that firmly secures the animal's head to a fixation apparatus for the precise control and sensing of behaviors. However, procedural and surgical difficulties and limitations have been obstructing the use of the technique in neurophysiological and behavioral experiments. Here, we propose a novel design of the head-restraining apparatus which is easy to develop and convenient for practical use. Head restraining procedure can be completed by sliding the head mounter, which is molded by dental cement during implantation surgery, into the port, which serves as matching guide rails for the mounter, of the fixation bar. So neither skull-attached plates nor screws for fixation are needed. We performed intracranial self stimulation experiment in rats using the newly designed device. Rats were habituated to acclimatize the head-restraint environment and trained to discriminate two spatially distinguished cues using a customized push-pull lever as an operandum. Direct electrical stimulation into the medial forebrain bundle served as reward. We confirmed that head restraining was stable throughout experiments and rats were able to learn to manipulate the lever after successful habituation. Our experimental framework might help precise control or sensing of behavior under head fixed rats using direct electrical brain stimulation as a reward.

Synaptic effects of xenon on NMDA receptor-mediated response in rat spinal neuron.

To investigate the precise mechanism of xenon (Xe), pharmacologically isolated AMPA/KA and NMDA receptor-mediated spontaneous (s) and evoked (e) excitatory postsynaptic currents (s/eEPSCAMPA/KA and s/eEPSCNMDA) were recorded from mechanically isolated single spinal sacral dorsal commissural nucleus (SDCN) neurons attached with glutamatergic nerve endings (boutons) using conventional whole-cell patch-clamp technique. We analysed kinetic properties of both s/eEPSCAMPA/KA and s/eEPSCNMDA by focal single- and/or paired-pulse electrical stimulation to compare them. The s/eEPSCNMDA showed smaller amplitude, slower rise time, and slower 1/e decay time constant (τDecay) than those of s/eEPSCAMPA/KA. We previously examined how Xe modulates s/eEPSCAMPA/KA, therefore, examined the effects on s/eEPSCNMDA in the present study. Xe decreased the frequency and amplitude of sEPSCNMDA, and decreased the amplitude but increased the failure rate and paired-pulse ratio of eEPSCNMDA without affecting their τDecay. It was concluded that Xe might suppress NMDA receptor-mediated synaptic transmission via both presynaptic and postsynaptic mechanisms.

α(2A) adrenoceptor-mediated presynaptic inhibition of GABAergic transmission in rat tuberomammillary nucleus neurons.

Histaminergic neurons within the tuberomammillary nucleus (TMN) play an important role in the regulation of sleep-wakefulness. Here, we report the adrenergic modulation of GABAergic transmission in rat TMN histaminergic neurons using a conventional whole-cell patch clamp technique. Norepinephrine (NE) reversibly decreased the amplitude of action potential-dependent GABAergic inhibitory post-synaptic currents (IPSCs) and increased the paired pulse ratio. The NE-induced inhibition of GABAergic IPSCs was mimicked by clonidine, a selective α2 adrenoceptor agonist. However, cirazoline and isoproterenol, nonselective α1 and ß adrenoceptor agonists, respectively, had no effect on GABAergic IPSCs. The NE-induced inhibition of GABAergic IPSCs was significantly blocked by BRL44408, a selective α2A adrenoceptor antagonist, but not imiloxan or JP1302, a selective α2B and α2C adrenoceptor antagonists. The extent of NE-induced inhibition of GABAergic IPSCs was inversely proportional to the extracellular Ca(2+) concentration. Pharmacological agents affecting the activities of adenylyl cyclase or G-protein-coupled inwardly rectifying K(+) channels did not affect the NE-induced inhibition of GABAergic IPSCs. However, NE had no effect on the frequency and amplitude of GABAergic miniature IPSCs. These results suggest that NE acts on presynaptic α2A adrenoceptor to inhibit action potential-dependent GABA release via the inhibition of Ca(2+) influx from the extracellular space to GABAergic nerve terminals, and that this α2A adrenoceptor-mediated modulation of GABAergic transmission may be involved in regulating the excitability of TMN histaminergic neurons.

Presynaptic glycine receptors increase GABAergic neurotransmission in rat periaqueductal gray neurons.

The periaqueductal gray (PAG) is involved in the central regulation of nociceptive transmission by affecting the descending inhibitory pathway. In the present study, we have addressed the functional role of presynaptic glycine receptors in spontaneous glutamatergic transmission. Spontaneous EPSCs (sEPSCs) were recorded in mechanically dissociated rat PAG neurons using a conventional whole-cell patch recording technique under voltage-clamp conditions. The application of glycine (100 µM) significantly increased the frequency of sEPSCs, without affecting the amplitude of sEPSCs. The glycine-induced increase in sEPSC frequency was blocked by 1 µM strychnine, a specific glycine receptor antagonist. The results suggest that glycine acts on presynaptic glycine receptors to increase the probability of glutamate release from excitatory nerve terminals. The glycine-induced increase in sEPSC frequency completely disappeared either in the presence of tetrodotoxin or Cd(2+), voltage-gated Na(+), or Ca(2+) channel blockers, suggesting that the activation of presynaptic glycine receptors might depolarize excitatory nerve terminals. The present results suggest that presynaptic glycine receptors can regulate the excitability of PAG neurons by enhancing glutamatergic transmission and therefore play an important role in the regulation of various physiological functions mediated by the PAG.

Ibuprofen modulates tetrodotoxin-resistant persistent Na+ currents at acidic pH in rat trigeminal ganglion neurons.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to relieve various symptoms such as headache, arthralgia, and dental pain. While the primary mechanism of NSAID-based pain relief is the inhibition of cyclooxygenase-2, several NSAIDs also modulate other molecular targets related to nociceptive transmission such as voltage-gated Na+ channels. In the present study, we examined the effects of NSAIDs on persistent Na+ current (INaP) mediated by tetrodotoxin-resistant (TTX-R) Na+ channels in small-to medium-sized trigeminal ganglion neurons using a whole-cell patch-clamp technique. At clinically relevant concentrations, all propionic acid derivatives tested (ibuprofen, naproxen, fenoprofen, and flurbiprofen) preferentially inhibited the TTX-R INaP. The inhibition was more potent at acidic extracellular pH (pH 6.5) than at normal pH (pH 7.4). Other NSAIDs, such as ketorolac, piroxicam, and aspirin, had a negligible effect on the TTX-R INaP. Ibuprofen both accelerated the onset of inactivation and retarded the recovery from inactivation of TTX-R Na+ channels at acidic extracellular pH. However, all NSAIDs tested in this study had minor effects on voltage-gated K+ currents, as well as hyperpolarization-activated and cyclic nucleotide-gated cation currents, at both acidic and normal extracellular pH. Under current-clamp conditions, ibuprofen decreased the number of action potentials elicited by depolarizing current stimuli at acidic (pH 6.5) extracellular pH. Considering that extracellular pH falls as low as 5.5 in inflamed tissues, TTX-R INaP inhibition could be a mechanism by which ibuprofen and propionic acid derivative NSAIDs modulate inflammatory pain.

Menthol excites dural afferent neurons by inhibiting leak K+ conductance in rats.

Menthol-a natural organic compound-is widely used for relieving various pain conditions including migraine. However, a high dose of menthol reportedly decreases pain thresholds and enhances pain responses. Accordingly, in the present study, we addressed the effect of menthol on the excitability of acutely isolated dural afferent neurons, which were identified with a fluorescent dye, using the whole-cell patch-clamp technique. Under a voltage-clamped condition, menthol altered the holding current levels in a concentration-dependent manner. The menthol-induced current (IMenthol) remained unaffected by the addition of selective transient receptor potential melastatin 8 antagonists. Moreover, the reversal potential of IMenthol was similar to the equilibrium potential of K+. IMenthol was accompanied by an increase in input resistance, thereby suggesting that menthol decreases the leak K+ conductance. Under a current-clamped condition, menthol caused depolarization of the membrane potential and decreased the threshold for the generation of action potential. While the IMenthol was substantially inhibited by 10 µM XE-991, a selective KV7 blocker, the M-current mediated by KV7 was not detected in the nociceptive neurons tested in the present study. Moreover, IMenthol decreased under acidic extracellular pH conditions or in the presence of 3 µM A-1899, a selective K2P3.1 and K2P9.1 blocker. The present results suggest that menthol inhibits leak K+ channels, possibly acid-sensitive two-pore domain K+ channels, thereby increasing the excitability of nociceptive sensory neurons. The resultant increase in neuron excitability may partially be responsible for the pronociceptive effect mediated by high menthol doses.

TRPA1-like channels enhance glycinergic transmission in medullary dorsal horn neurons.

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca(2+), suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca(2+) influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na(+) -free external solution or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca(2+), to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.

Propranolol modulation of tetrodotoxin-resistant Na+ channels in dural afferent neurons.

Propranolol, a representative adrenergic ß-receptor antagonist, is widely used to prevent migraine attacks. Although propranolol is well known to inhibit tetrodotoxin-resistant (TTX-R) Na+ channels in cardiac myocytes, it is unclear whether the drug modulates these channels expressed in dural afferent neurons. In this study, we examined the effects of propranolol on TTX-R Na+ channels in medium-sized dural afferent neurons identified by the fluorescent dye DiI. The TTX-R Na+ currents (INa) were recorded from acutely isolated DiI-positive neurons using a whole-cell patch clamp technique under voltage-clamp conditions. Propranolol inhibited the noninactivating steady-state component more potently than the peak component of transient TTX-R INa. Propranolol also potently inhibited the slow voltage ramp-induced TTX-R INa in a concentration-dependent manner, suggesting that it preferentially inhibited the noninactivating or persistent INa in DiI-positive neurons. Propranolol had little effect on voltage dependence, but it increased the extent of the use-dependent inhibition of TTX-R Na+ channels. Propranolol also accelerated the onset of inactivation and retarded recovery from inactivation in these channels. Under current-clamp conditions, propranolol decreased the number of action potentials elicited by depolarizing current stimuli. In conclusion, the propranolol-mediated preferential inhibition of persistent INa and modulation of the inactivation kinetics of TTX-R Na+ channels might represent additional mechanisms for migraine prophylaxis.