Corneal acetylcholine regulates sensory nerve activity via nicotinic receptors.

PURPOSE: Sensory nerve terminals are highly distributed in the cornea, and regulate ocular surface sensation and homeostasis in response to various endogenous and exogenous stimuli. However, little is known about mediators regulating the physiological and pathophysiological activities of corneal sensory nerves. The aim of this study was to investigate the presence of cholinergic regulation in sensory nerves in the cornea. METHODS: Localization of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT) was evaluated using western blotting and immunohistochemical analysis. The synthesis and liberation of acetylcholine from the cornea were assessed using corneal segments pre-incubated with [3H]choline. The responsiveness of corneal neurons and nerves to cholinergic drugs was explored using calcium imaging with primary cultures of trigeminal ganglion neurons and extracellular recording from corneal preparations in guinea pigs. RESULTS: ChAT, but not vAChT, was highly distributed in the corneal epithelium. In corneal segments, [3H] acetylcholine was synthesized from [3H]choline, and was also released in response to electrical stimuli. In cultured corneal neurons, the population sensitive to a transient receptor potential melastatin 8 (TRPM8) agonist exhibited high probability of responding to nicotine in a calcium imaging experiment. The firing frequency of cold-sensitive corneal nerves was increased by the application of nicotine, but diminished by an α4 nicotinic acetylcholine receptor antagonist. CONCLUSIONS: The corneal epithelium can synthesize and release acetylcholine. Corneal acetylcholine can excite sensory nerves via nicotinic receptors containing the α4 subunit. Therefore, corneal acetylcholine may be one of the important regulators of corneal nerve activity arranging ocular surface condition and sensation.

Acetylcholine release from striatal cholinergic interneurons is controlled differently depending on the firing pattern.

How is the quantal size in neurotransmitter release adjusted for various firing levels? We explored the possible mechanisms that regulate acetylcholine (ACh) release from cholinergic interneurons using an ultra-mini superfusion system. After preloading [3 H]ACh in rat striatal cholinergic interneurons, the release was elicited by electrical stimulation under a condition in which presynaptic cholinergic and dopaminergic feedback was inhibited. [3 H]ACh release was reproducible at intervals of more than 10 min; shorter intervals resulted in reduced levels of ACh release. Upon persistent stimulation for 10 min, ACh release transiently increased, before gradually decreasing. Vesamicol, an inhibitor of the vesicular ACh transporter (VAChT), had no effect on the release induced by the first single pulse, but it reduced the release caused by subsequent pulses. Vesamicol also reduced the [3 H]ACh release evoked by multiple pulses, and the inhibition was enhanced by repetitive stimulation. The decreasing phase of [3 H]ACh release during persistent stimulation was accelerated by vesamicol treatment. Thus, it is likely that releasable ACh was slowly compensated for via VAChT during and after stimulation, changing the vesicular ACh content. In addition, ACh release per pulse decreased under high-frequency stimulation. The present results suggest that ACh release from striatal cholinergic interneurons may be adjusted by changes in the quantal size due to slow replenishment via VAChT, and by a reduction in release probability upon high-frequency stimulation. These two distinct processes likely enable the fine tuning of neurotransmission and neuroprotection/limitation against excessive output and have important physiological roles in the brain.

Role of Muscarinic Acetylcholine Receptors in Intestinal Epithelial Homeostasis: Insights for the Treatment of Inflammatory Bowel Disease.

Inflammatory bowel disease (IBD), which includes Crohn's disease and ulcerative colitis, is an intestinal disorder that causes prolonged inflammation of the gastrointestinal tract. Currently, the etiology of IBD is not fully understood and treatments are insufficient to completely cure the disease. In addition to absorbing essential nutrients, intestinal epithelial cells prevent the entry of foreign antigens (micro-organisms and undigested food) through mucus secretion and epithelial barrier formation. Disruption of the intestinal epithelial homeostasis exacerbates inflammation. Thus, the maintenance and reinforcement of epithelial function may have therapeutic benefits in the treatment of IBD. Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors for acetylcholine that are expressed in intestinal epithelial cells. Recent studies have revealed the role of mAChRs in the maintenance of intestinal epithelial homeostasis. The importance of non-neuronal acetylcholine in mAChR activation in epithelial cells has also been recognized. This review aimed to summarize recent advances in research on mAChRs for intestinal epithelial homeostasis and the involvement of non-neuronal acetylcholine systems, and highlight their potential as targets for IBD therapy.

[Characterization of the dorsal raphe-periaqueductal grey DAT neurons innervating onto the extended amygdala].

It has been known that a number of tyrosine hydroxylase (TH)-positive neurons, which are regarded as dopaminergic (DA) neurons, exist in the dorsal raphe (DR). These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are thought to belong to the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. The DA transporter (DAT) neurons, which are well overlapping with DA neurons, in the DR-PAG region are also expected to be heterogeneous. However, even though the heterogeneity of DA/DATDR-PAG neurons has been suggested, the characteristics of each DA/DATDR-PAG neuron subpopulation are not well investigated. In this paper, we summarize the previous reports investigating the heterogeneity of DA/DATDR-PAG neurons and the functional importance of DA/DATDR-PAG neurons on various affective behaviors and introduce our recent findings that DATDR-PAG neurons consist of two subpopulations: TH+/vasoactive intestinal peptide (VIP)- putative DA neurons and TH-/VIP+ putative glutamatergic neurons.

Voluntary wheel-running activities ameliorate depressive-like behaviors in mouse dry eye models.

Recent clinical studies indicate that dry eye is closely associated with psychiatric disorders such as depression and anxiety. Here, we investigated whether two types of mouse dry eye models showed depressive-like behavior in forced swim and sucrose preference tests, and whether voluntary wheel-running helped ameliorate depressive states. To reproduce the dry eye models, the exorbital lacrimal glands (ELG) or exorbital and intraorbital lacrimal glands (ELG+ILG) were bilaterally excised from male C57BL/6J mice. Tear volume was persistently reduced in both models, but the ELG+ILG excision mice exhibited more severe corneal damage than the ELG excision mice. In the forced swim and sucrose preference tests, the gland excision mice showed longer immobility and shorter climbing times, and lower sucrose preference than sham-operated mice, respectively, which appeared earlier in the ELG+ILG excision mice. Wheel-running activities were significantly lower in the ELG+ILG excision mice, but not in the ELG excision mice. After short-period wheel-running, the longer immobility times and the shorter climbing times in the forced swim completely disappeared in both models. Our results suggest that dry eyes might directly cause a depressive disorder that depends on the severity and duration of the ocular surface damage, and that voluntary motor activity could help recovery from a depressive state induced by dry eye.

Evaluation of radiolabeled acetylcholine synthesis and release in rat striatum.

Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [3 H]choline and subsequent synthesis and release of [3 H]ACh were investigated in rat striatal segments. Incubation with [3 H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [3 H]choline and [3 H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [3 H]choline led predominantly to the accumulation of [3 H]ACh. Electrical stimulation and KCl depolarization selectively released [3 H]ACh but not [3 H]choline. [3 H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [3 H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [3 H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca2+ dependent and exhibited slightly different sensitivities to N- and P-type Ca2+ channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [3 H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.

Alteration of the soluble guanylate cyclase system in coronary arteries of high cholesterol diet-fed rabbits.

This study aimed to investigate how atherosclerosis affects the soluble guanylate cyclase (sGC) system in coronary arteries. Rabbits were fed a normal diet for 12 weeks (N group) or a diet containing high cholesterol (1%) for 4 weeks (S-HC group) and 12 weeks (L-HC group). Cholesterol deposition in the intima of coronary arteries was observed in the S-HC group, but the formation of an atherosclerotic plaque was not observed. In contrast, a major plaque developed in the L-HC group. The relaxant response of isolated coronary arteries to sodium nitroprusside (SNP, nitric oxide donor) was not different between the N and S-HC groups, whereas the response in the L-HC group was markedly attenuated. The relaxation induced by BAY 60-2770 (sGC activator) tended to be augmented in the S-HC group, but it was significantly impaired in the L-HC group compared to that in the N group. sGC ß1 immunostaining was equally detected in the medial layer of the arteries among the N, S-HC, and L-HC groups. In addition, a strong staining was observed in the plaque region of the L-HC group. cGMP levels in the arteries stimulated with SNP were identical in the N and S-HC groups and slightly lower in the L-HC group than the other groups. BAY 60-2770-stimulated cGMP formation tended to be increased in the S-HC and L-HC groups. These findings suggest that the sGC system was not normal in atherosclerotic coronary arteries. The redox state of sGC and the distribution pattern are likely to change with the progression of atherosclerosis.

Sensitization of glutamate receptor-mediated pain behaviour via nerve growth factor-dependent phosphorylation of transient receptor potential V1 under inflammatory conditions.

BACKGROUND AND PURPOSE: Glutamate and metabotropic glutamate (mGlu) receptors on primary sensory neurons are crucial in modulating pain sensitivity. However, it is unclear how inflammation affects mGlu receptor-mediated nociceptive responses. We therefore investigated the effects of mGlu1/5 receptor agonists on pain-related behaviour during persistent inflammation and their underlying mechanisms. EXPERIMENTAL APPROACH: Effects of a mGlu1/5 receptor agonist on pain-related behaviour during inflammation was assessed in mice. Intracellular calcium responses, membrane current responses, and protein expression in primary sensory neurons were examined using cultured dorsal root ganglion (DRG) neurons, dissociated from wild-type and gene knockout mice. KEY RESULTS: Persistent inflammation induced by complete Freund's adjuvant increased the duration of mGlu1/5 receptor-mediated pain behaviour, which was antagonized by inhibition of nerve growth factor (NGF)-tropomyosin receptor kinase A (TrkA) signalling. Calcium imaging revealed that NGF treatment increased the number of cultured DRG neurons responding to mGlu1/5 receptor activation. Stimulation of mGlu1/5 receptors in NGF-treated DRG neurons induced inward currents through TRPV1 channels in association with PLC but not with IP3 receptors. NGF treatment also increased the number of neurons responding to a DAG analogue via TRPV1 channel activation. Furthermore, NGF up-regulated expression of TRPV1 and A-kinase anchoring protein 5 (AKAP5), resulting in increased AKAP5-dependent TRPV1 phosphorylation. AKAP5 knockout mice did not exhibit mGlu1/5 receptor-mediated excitation in NGF-treated DRG neurons or pain response facilitation under inflammatory conditions. CONCLUSIONS AND IMPLICATIONS: NGF augments glutamate- and mGlu1/5 receptor-mediated excitation of nociceptive neurons by AKAP5-dependent phosphorylation of TRPV1 channels, potentiating hypersensitivity to glutamate in inflamed tissues.

Effect of Betanin, a Beetroot Component, on Vascular Tone in Isolated Porcine Arteries.

BACKGROUND: Beetroot has attracted much attention because of its blood pressure-lowering properties. Although beetroot contains various nutritional compounds, including inorganic nitrate, some of their physiological properties are not fully understood. In this study, we examined whether betanin, a beetroot component, has a regulatory effect on vascular tone. METHODS: Mechanical responses of isolated porcine coronary, mesenteric, and pulmonary arteries were assessed by organ chamber technique. In some cases, the vascular reactivity was observed in the presence of a physiological concentration of betanin (10 µM). RESULTS: Betanin did not induce vasorelaxation at physiological concentrations both in endothelium-intact and -denuded coronary, mesenteric, and pulmonary arteries. The endothelium-dependent agonists, bradykinin and A23187 induced vasorelaxation of endothelium-intact coronary arteries, both of which were not affected by exposure to betanin. Likewise, endothelium-independent vasorelaxation induced by sodium nitrite and sodium nitroprusside was also not affected by the presence of betanin. In addition, exposure of endothelium-intact coronary arteries to betanin did not attenuate prostaglandin F2α- and endothelin-1-induced vasocontraction. CONCLUSIONS: These findings suggest that betanin does not have a vasorelaxant activity. It is unlikely that betanin is a component directly responsible for the beetroot-induced acute blood pressure-lowering effect in a nitrate-independent manner.

Chronic Tear Deficiency Sensitizes Transient Receptor Potential Vanilloid 1-Mediated Responses in Corneal Sensory Nerves.

Chronic tear deficiency enhances the excitability of corneal cold-sensitive nerves that detect ocular dryness, which can lead to discomfort in patients with dry eye disease (DED). However, changes in corneal nerve excitations through the polymodal nociceptor "transient receptor potential vanilloid 1" (TRPV1) and the potential link between this receptor and symptoms of DED remain unclear. In this study, we examined the firing properties of corneal cold-sensitive nerves expressing TRPV1 and possible contributions of chronic tear deficiency to corneal nerve excitability by TRPV1 activation. The bilateral excision of lacrimal glands in guinea pigs decreased the tear volume and increased the frequency of spontaneous eyeblinks 1-4 weeks after surgery. An analysis of the firing properties of the cold-sensitive nerves was performed by single-unit recordings of corneal preparations 4 weeks after surgery in both the sham-operated and gland-excised groups. Perfusion of the TRPV1 agonist, capsaicin (1 µM), transiently increased the firing frequency in approximately 46-48% of the cold-sensitive nerves characterized by low-background activity and high threshold (LB-HT) cold thermoreceptors in both groups. Gland excision significantly decreased the latency of capsaicin-induced firing in cold-sensitive nerves; however, its magnitude was unchanged. Calcium imaging of cultured trigeminal ganglion neurons from both groups showed that intracellular calcium elevation of corneal neurons induced by a low concentration of capsaicin (0.03 µM) was significantly larger in the gland excision group, regardless of responsiveness to cold. An immunohistochemical study of the trigeminal ganglion revealed that gland excision significantly increased the proportion of corneal neurons enclosed by glial fibrillary acidic protein (GFAP)-immunopositive satellite glial cells. Topical application of the TRPV1 antagonist, A784168 (30 µM), on the ocular surface attenuated eye-blink frequency after gland excision. Furthermore, gland excision enhanced blink behavior induced by a low concentration of capsaicin (0.1 µM). These results suggest that chronic tear deficiency sensitizes the TRPV1-mediated response in the corneal LB-HT cold thermoreceptors and cold-insensitive polymodal nociceptors, which may be linked to dry eye discomfort and hyperalgesia resulting from nociceptive stimuli in aqueous-deficient dry eyes.

Impact of cigarette smoking on nitric oxide-sensitive and nitric oxide-insensitive soluble guanylate cyclase-mediated vascular tone regulation.

Cigarette smoking induces vascular endothelial dysfunction characterized by impaired nitric oxide (NO) bioavailability. There are two types of soluble guanylate cyclase (sGC), which is a cellular target of NO: NO-sensitive reduced form (the heme moiety with a ferrous iron) and NO-insensitive oxidized (the heme moiety with a ferric iron)/heme-free form. This study investigated the influence of cigarette smoking on NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation in organ chamber experiments with isolated rat and human arteries. The rats were subcutaneously administered phosphate-buffered saline (PBS), nicotine-free cigarette smoke extract (N(-)-CSE) or nicotine-containing cigarette smoke extract (N(+)-CSE) for 4 weeks. Plasma thiobarbituric acid reactive substance (TBARS) levels were higher in the N(+)-CSE group than those in the N(-)-CSE group, and TBARS levels for these groups were higher than those for the PBS group. In the aorta and the pulmonary artery in rats administered N(-)-CSE or N(+)-CSE, acetylcholine-induced relaxation was significantly impaired compared with that in rats administered PBS; there was no significant difference in the relaxation between the N(-)-CSE and N(+)-CSE groups. However, sodium nitroprusside (NO-sensitive sGC stimulant)- and BAY 60-2770 (NO-insensitive sGC stimulant)-induced relaxations were not different among the three groups, regardless of the vessel type. In addition, in the human gastroepiploic artery, the relaxant responses to these sGC-targeting drugs were identical between nonsmokers and smokers. These findings suggest that NO-sensitive and NO-insensitive sGC-mediated vascular tone regulation functions normally even in blood vessels damaged by cigarette smoking.

Responsiveness of rat aorta and pulmonary artery to cGMP generators in the presence of thiol or heme oxidant.

This study investigated the effects of thiol and heme oxidants on responsiveness to cGMP generators in isolated rat aorta and pulmonary artery using an organ chamber. The nitric oxide (NO) donor sodium nitroprusside (SNP)-induced relaxation was impaired by exposure to the thiol oxidant diamide in both the aorta and the pulmonary artery, whereas the soluble guanylate cyclase (sGC) stimulator BAY 41-2272- or the sGC activator BAY 60-2770-induced relaxation was not affected. The impairment by diamide of SNP-induced aortic and pulmonary arterial relaxation was completely restored by post-treatment with the thiol reductant dithiothreitol. However, regardless of the vessel type, the relaxant response to SNP or BAY 41-2272 was impaired by exposure to the heme oxidant ODQ, whereas the response to BAY 60-2770 was enhanced. The ODQ-induced effects were reversed partially by post-treatment with the heme reductant dithionite. These findings indicate that thiol oxidation attenuates only the vascular responsiveness to NO donors and that heme oxidation attenuates the responsiveness to NO donors and sGC stimulators but augments that to sGC activators. Therefore, under oxidative stress, the order of usability of the vasodilators is suggested to be: NO donors < sGC stimulators < sGC activators.

Chronological Change of Vascular Reactivity to cGMP Generators in the Balloon-Injured Rat Carotid Artery.

BACKGROUND/AIMS: Soluble guanylate cyclase (sGC) exists as reduced, oxidized, and heme-free forms. Currently, it is unclear whether endovascular mechanical stenosis has an impact on vascular tone control by drugs targeting sGC, namely cGMP generators. METHODS: Pharmacological responses to acidified sodium nitrite (reduced sGC stimulant) and BAY 60-2770 (oxidized/heme-free sGC stimulant) were studied in balloon-injured rat carotid arteries at several time points. In addition, sGC expression was detected by immunohistochemistry. RESULTS: At 1 day after injury, acidified sodium nitrite-induced relaxation was attenuated in the injured artery, whereas BAY 60-2770-induced relaxation was augmented. Similar attenuation of response to acidified sodium nitrite was seen at 7 and 14 days after injury. On the other hand, the augmentation of response to BAY 60-2770 disappeared at 7 and 14 days after injury. At 1 day after injury, the immunohistochemical expression pattern of sGC in the smooth muscle layer of the injured artery was not different from that of the uninjured artery. However, in the injured artery, the intensity of sGC staining was weak at 7 and 14 days after injury. CONCLUSION: Balloon injury alters vascular responsiveness to cGMP generators, which seems to be associated with the form and/or expression of sGC.

Novel regulatory systems for acetylcholine release in rat striatum and anti-Alzheimer's disease drugs.

Regulation of neurotransmitter release in the central nervous system is complex. Here, we investigated regulatory mechanisms for acetylcholine (ACh) release from cholinergic neurons by performing superfusion experiments with rat striatal segments after labelling the cellular ACh pool with [3 H]choline. Electrical stimulation-evoked pronounced [3 H]ACh release from cholinergic neurons. The estimated quantity of [3 H]ACh release per pulse of electrical stimulation was reduced by an increase in stimulus frequency, showing an inverse correlation between release probability of ACh and neuronal excitation. ACh release was also negatively regulated by pre-synaptic muscarinic ACh receptors (mAChRs). The autoinhibition induced by released ACh was predominantly suppressed by the M2 -selective antagonist AF-DX 116, partially inhibited by M3 -selective darifenacin, and minimally by M4 -selective PD 102807. Other subtype-selective antagonists had no effect at subtype-selective concentrations. ACh esterase (AChE) inhibitors (diisopropylfluorophosphate, donepezil and galantamine) at concentrations that mostly inhibit esterase activity reduced [3 H]ACh release, and the reduction was abolished by treatment with atropine. This implies that pre-synaptic autoreceptors are activated more after blockade of ACh hydrolysis, leading to autoinhibition of ACh release and consequent reduction in synaptic ACh concentrations. [3 H]efflux was also enhanced by ACh uptake inhibitors (100 µM hemicholinium-3 and physostigmine), regardless of ACh hydrolysis. This study shows that synaptic ACh concentrations in striatal cholinergic neurons are regulated in a complex manner by many factors such as release probability, pre-synaptic M2 /M3 /M4 mAChRs, AChE and post-synaptic ACh uptake, and provides important information about cholinergic neurotransmission for future exploration of therapeutic strategies for Alzheimer's and other central nervous system diseases. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/openscience-badges/.

Persistent activation of histamine H1 receptors in the hippocampal CA1 region enhances NMDA receptor-mediated synaptic excitation and long-term potentiation in astrocyte- and D-serine-dependent manner.

Behavioral studies using pharmacological tools have implicated histamine H1 receptors in cognitive function via their interactions with N-methyl-D-aspartate receptors (NMDARs) in the hippocampus. However, little is known about the neurophysiological mechanism that underlies the interaction between H1 receptors and NMDARs. To explore how H1 receptor activation affects hippocampal excitatory neurotransmission and synaptic plasticity, this study aimed to examine the effect of H1 receptor ligands on both NMDAR-mediated synaptic currents and long-term potentiation (LTP) at synapses between Schaffer collaterals and CA1 pyramidal neurons using acute mouse hippocampal slices. We found that the H1 receptor antagonist/inverse agonists, pyrilamine (0.1 µM) and cetirizine (10 µM), decreased the NMDAR-mediated component of stimulation-induced excitatory postsynaptic currents (EPSCs) recorded from CA1 pyramidal neurons without affecting the AMPA receptor-mediated component of EPSCs and its paired pulse ratio. Pretreatment of slices with either the glial metabolism inhibitor, fluoroacetate (5 mM), or D-serine (100 µM) diminished the pyrilamine- or cetirizine-induced attenuation of the NMDAR-mediated EPSCs. Furthermore, the LTP of field excitatory postsynaptic potentials induced following high frequency stimulation of Schaffer collaterals was attenuated with application of pyrilamine or cetirizine. Pretreatment with D-serine again attenuated the pyrilamine-induced suppression of LTP. Our data suggest that H1 receptors in the CA1 can undergo persistent activation induced by their constitutive receptor activity and/or tonic release of endogenous histamine, resulting in facilitation of the NMDAR activity in a manner dependent of astrocytes and the release of D-serine. This led to the enhancement of NMDA-component EPSC and LTP at the Schaffer collateral-CA1 pyramidal neuron synapses.

Augmentation of Endogenous Acetylcholine Uptake and Cholinergic Facilitation of Hippocampal Long-Term Potentiation by Acetylcholinesterase Inhibition.

Hippocampal cholinergic activity enhances long-term potentiation (LTP) of synaptic transmission in intrahippocampal circuits and regulates cognitive function. We recently demonstrated intracellular distribution of functional M1-muscarinic acetylcholine receptors (mAChRs) and neuronal uptake of acetylcholine (ACh) in the central nervous system. Here we examined whether endogenous ACh acts on intracellular M1-mAChRs following its uptake and causes cholinergic facilitation of hippocampal LTP. ACh esterase (AChE) activities and [3H]ACh uptake was measured in rat hippocampal segments. LTP of evoked field excitatory postsynaptic potentials at CA1 synapses was induced by high frequency stimulation in hippocampal slices. Pretreatment with diisopropylfluorophosphate (DFP) irreversibly inhibited AChE, augmented ACh uptake, and significantly enhanced the LTP. This cholinergic facilitation was inhibited by pirenzepine, a membrane-permeable M1 antagonist, while only the early stage of cholinergic facilitation was inhibited by a membrane-impermeable M1 antagonist, muscarinic toxin 7. Tetraethylammonium (TEA) inhibited ACh uptake in hippocampal segments and selectively suppressed late stage cholinergic facilitation without changing the early stage. In contrast, LTP in DFP-untreated slices was not affected by the muscarinic antagonists and TEA. Carbachol (CCh; an AChE-resistant muscarinic agonist) competed with ACh for its uptake and produced cholinergic facilitation of LTP in DFP-untreated slices. The late stage of CCh-induced facilitation was also selectively inhibited by TEA. Our results suggest that when AChE is inactivated by inhibitors, LTP in hippocampal slices is significantly enhanced by endogenous ACh and that cholinergic facilitation is caused by direct activation of cell-surface M1-mAChRs and subsequent activation of intracellular M1-mAChRs after ACh uptake.

Inhibitory Effect of Amitriptyline on the Impulse Activity of Cold Thermoreceptor Terminals of Intact and Tear-Deficient Guinea Pig Corneas.

PURPOSE: Chronic dryness of the ocular surface evokes sensitization of corneal cold-sensitive neurons through an increase of sodium currents and a decrease of potassium currents, leading to the unpleasant dryness and pain sensations typical of dry eye disease. Here, we explored the effects of amitriptyline, a voltage-gated Na+ channel blocker used for the treatment of depression and chronic pain, on nerve terminal impulse (NTI) activity of cold-sensitive nerve terminals recorded in intact and tear-deficient guinea pig corneas. METHODS: Main lachrymal gland was surgically removed in anesthetized guinea pigs to induce chronic tear deficiency. Four to 6 weeks afterward, animals were sacrificed and both corneas placed in a perfusion chamber superfused at 34°C. Thermal stimuli were induced by changing the solution temperature from 34°C to 20°C (cooling ramp) and from 34°C to 50°C (heating ramp). Spontaneous and stimulus-evoked NTIs of cold-sensitive nerve terminals were recorded before, during, and after perfusion with solutions containing amitriptyline at different concentrations (3-30 µM). RESULTS: Perfusion with amitriptyline inhibited irreversibly and in a concentration-dependent manner the spontaneous NTI activity of cold thermoreceptors of intact corneas. This effect was less evident in tear-deficient corneas. In addition, amitriptyline (10 µM) attenuated the maximal response to cooling ramps without changing cold threshold in intact but not in tear-deficient corneas. Only cold thermoreceptors with low cooling threshold values were sensitive to amitriptyline. CONCLUSION: Amitriptyline effectively reduces the activity of cold thermoreceptors, although its efficacy is different in intact and tear-deficient corneas, which might be due to the changes induced by ocular dryness in the expression of the various voltage-gated Na+ channels responsible of the action potential generation and propagation.

Regulation of synaptic acetylcholine concentrations by acetylcholine transport in rat striatal cholinergic transmission.

In addition to hydrolysis by acetylcholine esterase (AChE), acetylcholine (ACh) is also directly taken up into brain tissues. In this study, we examined whether the uptake of ACh is involved in the regulation of synaptic ACh concentrations. Superfusion experiments with rat striatal segments pre-incubated with [3 H]choline were performed using an ultra-mini superfusion vessel, which was developed to minimize superfusate retention within the vessel. Hemicholinium-3 (HC-3) at concentrations less than 1 µM, selectively inhibited the uptake of [3 H]choline by the high affinity-choline transporter 1 and had no effect on basal and electrically evoked [3 H]efflux in superfusion experiments. In contrast, HC-3 at higher concentrations, as well as tetraethylammonium (>10 µM), which inhibited the uptake of both [3 H]choline and [3 H]ACh, increased basal [3 H]overflow and potentiated electrically evoked [3 H]efflux. These effects of HC-3 and tetraethylammonium were also observed under conditions where tissue AChE was irreversibly inactivated by diisopropylfluorophosphate. Specifically, the potentiation of evoked [3 H]efflux was significantly higher in AChE-inactivated preparations and was attenuated by atropine. On the other hand, striatal segments pre-incubated with [3 H]ACh failed to increase [3 H]overflow in response to electrical stimulation. These results show that synaptic ACh concentrations are significantly regulated by the postsynaptic uptake of ACh, as well as by AChE hydrolysis and modulation of ACh release mediated through presynaptic muscarinic ACh receptors. In addition, these data suggest that the recycling of ACh-derived choline may be minor in cholinergic terminals. This study reveals a new mechanism of cholinergic transmission in the central nervous system.

TRPA1 Channels Modify TRPV1-Mediated Current Responses in Dorsal Root Ganglion Neurons.

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.