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The dental anesthesia sonophoresis device (DASD) is a novel device that is intended to reduce the discomfort associated with intraoral mucosa needle puncture. The DASD produces ultrasonic energy that provides a sonophoretic effect on the oral mucosa, generating microchannels through the lipids between the keratinized cells that make up the stratum corneum. Once the topical anesthetic has permeated the stratum corneum, it quickly diffuses through the soft tissue, desensitizing the nerve endings and reducing the perception of pain caused by needle penetration. The aim of this study is to evaluate whether topical anesthesia applied using the DASD will reduce the discomfort of the needle puncture when compared to the control device. A split-mouth model, using 50 healthy subjects with puncture site at the maxillary canine vestibule, was used for this study. Subjects received a needle puncture on both sides of the mouth. Prior to the needle puncture, there was randomized application of 5% lidocaine with the DASD and a control device. Subjects rated their discomfort after needle punctures utilizing the visual analog scale pain scoring system. There was no statistically significant difference in the pain perception using the DASD versus the control device.

Botulinum neurotoxins are used clinically for conditions characterized by hyperexcitability of peripheral nerve terminals and hypersecretory syndromes. These neurotoxins are synthesized as precursor proteins with low activity, but their effects are mediated by the active form of the neurotoxin through a multistep mechanism. Following a high-affinity interaction with a protein receptor and polysialogangliosides on the synaptic membrane, botulinum neurotoxins enter the neuron and causes a sustained inhibition of synaptic transmission. The active neurotoxin is part of a high-molecular-weight complex that protects the neurotoxin from proteolytic degradation. Although complexing proteins do not affect diffusion of therapeutic neurotoxin, they may lead to the development of neutralizing antibodies that block responsiveness to it. Nerve terminal intoxication is reversible and its duration varies for different BoNT serotypes. Although it was previously assumed that botulinum neurotoxins exert effects only on the peripheral synapses, such as the neuromuscular junction, there is now substantial evidence that these neurotoxins affect neurotransmission at distal central nervous system sites as well., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Physiological differences in low- (tonic like) and high-output (phasic like) synapses match many of the expected anatomical features of these terminals. However, investigation in the recruitment of synaptic vesicles from a reserve pool (RP) to a readily releasable pool (RRP) of synaptic vesicles within these types of nerve terminals has not been fully addressed. This study highlights physiological differences and differential modulation of the vesicles in a RP for maintaining synaptic output during evoked depression of the RRP. With the use of bafilomycin A1, a vacuolar ATPase blocker, recycling vesicles are blocked in refilling with transmitter. The tonic terminal is fatigue resistant due to a large RRP, whereas the phasic depresses rapidly upon continuous stimulation. These differences in rates of depression appear to be in the size and degree of utilization of the RRP of vesicles. The working model is that upon depression of the tonic terminal, serotonin (5-HT) has a large RP to act on in order to recruit vesicles to the RRP; whereas, the phasic terminal, 5-HT can recruit RP vesicles to the RRP prior to synaptic depression but not after depression. The vesicle pools are physiologically differentiated between phasic and tonic output terminals., (Copyright © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine stimulant drug of abuse with close structural and mechanistic similarities to methamphetamine. One of the most powerful actions associated with mephedrone is the ability to stimulate dopamine (DA) release and block its re-uptake through its interaction with the dopamine transporter (DAT). Although mephedrone does not cause toxicity to DA nerve endings, its ability to serve as a DAT blocker could provide protection against methamphetamine-induced neurotoxicity like other DAT inhibitors. To test this possibility, mice were treated with mephedrone (10, 20, or 40 mg/kg) prior to each injection of a neurotoxic regimen of methamphetamine (four injections of 2.5 or 5.0 mg/kg at 2 h intervals). The integrity of DA nerve endings of the striatum was assessed through measures of DA, DAT, and tyrosine hydroxylase levels. The moderate to severe DA toxicity associated with the different doses of methamphetamine was not prevented by any dose of mephedrone but was, in fact, significantly enhanced. The hyperthermia caused by combined treatment with mephedrone and methamphetamine was the same as seen after either drug alone. Mephedrone also enhanced the neurotoxic effects of amphetamine and 3,4-methylenedioxymethamphetamine on DA nerve endings. In contrast, nomifensine protected against methamphetamine-induced neurotoxicity. As mephedrone increases methamphetamine neurotoxicity, the present results suggest that it interacts with the DAT in a manner unlike that of other typical DAT inhibitors. The relatively innocuous effects of mephedrone alone on DA nerve endings mask a potentially dangerous interaction with drugs that are often co-abused with it, leading to heightened neurotoxicity., (© 2012 International Society for Neurochemistry.)

In experiments on frog (Rana ridibunda) neuromuscular junction the influence of cholesterol oxidation on the presynaptic vesicular cycle was investigated. Application of cholesterol oxidase (1 u. a.) during 1/2 hour led to the oxidation of - 0.007 mg cholesterol per 1 g tissue and reduced stability of lipid rafts in the nerve terminals. Using electrophysiological techniques it was shown that the cholesterol oxidation decreases the evoked neurotransmitter release. In experiments with fluorescent FM-dyes the depression of the synaptic vesicles exo-endocytosis and the dispersion of synaptic vesicles clusters were revealed. Comparative analysis of electrophysiological and optical data, as well as experiments with water soluble quencher of FM-dye indicated the possibility of some neurotransmitter release by "kiss-and-run" pathway, when short-lived fusion pore is formed. It was concluded that cholesterol oxidation inhibit evoked exocytosis, and also synaptic vesicle delivery from reserve pool to cites of exocytosis probably by break of the clusterization. Perhaps the synaptic vesicles of recycling pool release the neurotransmitter using the kiss-and-run mechanism.

This study investigated the effects and possible mechanism of ferulic acid, a naturally occurring phenolic compound, on endogenous glutamate release in the nerve terminals of the cerebral cortex in rats. Results show that ferulic acid inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effect of ferulic acid on the evoked glutamate release was prevented by chelating the extracellular Ca²⁺ ions, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Ferulic acid suppressed the depolarization-induced increase in a cytosolic-free Ca²⁺ concentration, but did not alter 4-AP-mediated depolarization. Furthermore, the effect of ferulic acid on evoked glutamate release was abolished by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca²⁺ exchange. These results show that ferulic acid inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca²⁺ entry.

Vinpocetine is a neuroprotective drug that exerts beneficial effects on neurological symptoms and cerebrovascular disease. 3-nitropropionic acid (3-NPA) is a toxin that irreversibly inhibits succinate dehydrogenase, the mitochondrial enzyme that acts in the electron transport chain at complex II. In previous studies in striatum-isolated nerve endings (synaptosomes), we found that vinpocetine decreased dopamine (DA) at expense of its main metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), and that 3-NPA increased DA, reactive oxygen species (ROS), DA-quinone products formation, and decreased DOPAC. Therefore, in this study, the possible effect of vinpocetine on 3-NPA-induced increase in DA, ROS, lipid peroxidation, and DA-quinone products formation in striatum synaptosomes were investigated, and compared with the effects of the antioxidant α-tocopherol. Results show that the increase in DA induced by 3-NPA was inhibited by both 25 μM vinpocetine and 50 μM α-tocopherol. Vinpocetine, as α-tocopherol, also inhibited 3-NPA-induced increase in ROS (as judged by DCF fluorescence), lipid peroxidation (as judged by TBA-RS formation), and DA-quinone products formation (as judged by the nitroblue tetrazolium reduction method). As in addition to the inhibition of complex II exerted by 3-NPA, 3-NPA increases DA-oxidation products that in turn can inhibit other sites of the respiratory chain, the drop in DA produced by vinpocetine and α-tocopherol may importantly contribute to their protective action from oxidative damage, particularly in DA-rich structures., (© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Background: Recent studies suggest that formaldehyde (FA) could be synthesized endogeneously and transient receptor potential (TRP) channel might be the sensor of FA. However, the physiological significance is still unclear., Methodology/principal Findings: The present study investigated the FA induced epithelial Cl(-) secretion by activation of TRPV-1 channel located in the nerve ending fiber. Exogenously applied FA induced an increase of I(SC) in intact rat trachea tissue but not in the primary cultured epithelial cells. Western blot and immunofluorescence analysis identified TRPV-1 expression in rat tracheal nerve ending. Capsazepine (CAZ), a TRPV-1 specific antagonist significantly blocked the I(SC) induced by FA. The TRPV-1 agonist capsaicin (Cap) induced an increase of I(SC), which was similar to the I(SC) induced by FA. L-703606, an NK-1 specific inhibitor and propranolol, an adrenalin β receptor inhibitor significantly abolished the I(SC) induced by FA or Cap. In the ion substitute analysis, FA could not induce I(SC) in the absence of extracelluar Cl(-). The I(SC) induced by FA could be blocked by the non-specific Cl(-) channel inhibitor DPC and the CFTR specific inhibitor CFTR(i-172), but not by the Ca(2+)-activated Cl(-) channel inhibitor DIDS. Furthermore, both forskolin, an agonist of adenylate cyclase (AC) and MDL-12330A, an antagonist of AC could block FA-induced I(SC)., Conclusion: Our results suggest that FA-induced epithelial I(SC) response is mediated by nerve, involving the activation of TRPV-1 and release of adrenalin as well as substance P.

This study investigates the effects and possible mechanism of coenzyme Q10 (CoQ10) on endogenous glutamate release in the cerebral cortex nerve terminals of rats. CoQ10 inhibited the release of glutamate evoked by the K+ channel blocker 4-aminopyridine (4-AP). CoQ10 reduced the depolarization-induced increase in cytosolic [Ca2+]c but did not alter the 4-AP-mediated depolarization. The effect of CoQ10 on evoked glutamate release was abolished by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) Ca2+ channels and mitogen-activated protein kinase kinase (MEK). In addition, CoQ10 decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK. Moreover, the inhibition of glutamate release by CoQ10 was strongly attenuated in mice without synapsin I. These results suggest that CoQ10 inhibits glutamate release from cortical synaptosomes in rats through the suppression of the presynaptic voltage-dependent Ca2+ entry and ERK/synapsin I signaling pathway.

Background: Acrylamide (ACR) produces cumulative neurotoxicity in exposed humans and laboratory animals through a direct inhibitory effect on presynaptic function., Objectives: In this review, we delineate how knowledge of chemistry provided an unprecedented understanding of the ACR neurotoxic mechanism. We also show how application of the hard and soft, acids and bases (HSAB) theory led to the recognition that the α,β-unsaturated carbonyl structure of ACR is a soft electrophile that preferentially forms covalent bonds with soft nucleophiles., Methods: In vivo proteomic and in chemico studies demonstrated that ACR formed covalent adducts with highly nucleophilic cysteine thiolate groups located within active sites of presynaptic proteins. Additional research showed that resulting protein inactivation disrupted nerve terminal processes and impaired neurotransmission., Discussion: ACR is a type-2 alkene, a chemical class that includes structurally related electrophilic environmental pollutants (e.g., acrolein) and endogenous mediators of cellular oxidative stress (e.g., 4-hydroxy-2-nonenal). Members of this chemical family produce toxicity via a common molecular mechanism. Although individual environmental concentrations might not be toxicologically relevant, exposure to an ambient mixture of type-2 alkene pollutants could pose a significant risk to human health. Furthermore, environmentally derived type-2 alkenes might act synergistically with endogenously generated unsaturated aldehydes to amplify cellular damage and thereby accelerate human disease/injury processes that involve oxidative stress., Conclusions: These possibilities have substantial implications for environmental risk assessment and were realized through an understanding of ACR adduct chemistry. The approach delineated here can be broadly applied because many toxicants of different chemical classes are electrophiles that produce toxicity by interacting with cellular proteins.

Background: In our earlier work, a reduction of cholesterol content increased the extracellular glutamate level in rat brain nerve terminals (synaptosomes) that was a result of the lack of transporter-mediated glutamate uptake. The aim of this study was to assess transporter-mediated release of glutamate from cholesterol-deficient synaptosomes. In stroke, cerebral hypoxia/ischemia, and traumatic brain injury, the development of neurotoxicity is provoked by enhanced extracellular glutamate, which is released from nerve cells mainly by glutamate transporter reversal - a distinctive feature of these pathological states., Methods: Laser scanning confocal microscopy, spectrofluorimetry, radiolabeled assay, and glutamate dehydrogenase assay., Results: Cholesterol acceptor methyl-β-cyclodextrin (15mM) reduced the cholesterol content in the synaptosomes by one quarter. Transporter-mediated glutamate release from synaptosomes: 1) stimulated by depolarization of the plasma membrane; 2) by means of heteroexchange with competitive transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate; 3) in low [Na(+)] medium; and 4) during dissipation of the proton gradient of synaptic vesicles by the protonophore cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP); was decreased under conditions of cholesterol deficiency by ~24, 28, 40, and 17%, respectively., Conclusions: A decrease in the level of membrane cholesterol attenuated transporter-mediated glutamate release from nerve terminals. Therefore, lowering cholesterol may be used in neuroprotection in stroke, ischemia, and traumatic brain injury which are associated with an increase in glutamate uptake reversal. This data may explain the neuroprotective effects of statins in these pathological states and provide one of the mechanisms of their neuroprotective action. However, beside these disorders, lowering cholesterol may cause harmful consequences by decreasing glutamate uptake in nerve terminals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 μM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

A study was made to determine if constitutively active adenosine receptors are present at mouse motor nerve endings. In preparations blocked by low Ca(2+)/high Mg(2+) solution, 8-cyclopentyl-1,3,dipropylxanthine (CPX, 10-100 nM), which has been reported to be both an A(1) adenosine receptor antagonist and inverse agonist, produced a dose-dependent increase in the number of acetylcholine quanta released by a nerve impulse. Adenosine deaminase, which degrades ambient adenosine into its inactive congener, inosine, failed to alter the response to 100 nM CPX. 8-Cyclopentyltheophylline (CPT, 3 μM), a competitive inhibitor at A(1) adenosine receptors, prevented the increase in acetylcholine release produced by CPX. At normal levels of acetylcholine release, neither adenosine deaminase nor CPX affected acetylcholine release at low frequencies of nerve stimulation in (+)-tubocurarine blocked preparations. The results suggest that a proportion of the acetylcholine release process is controlled by constitutively active adenosine receptors at murine motor nerve endings, providing the first evidence for constitutive activity of G-protein-coupled receptors that modulate the function of mammalian nerve endings., (Copyright © 2012 Elsevier B.V. All rights reserved.)

σ-1 Receptors are expressed in the brain, and their activation has been shown to prevent neuronal death associated with glutamate toxicity. This study investigates the possible mechanism and effect of [2S-(2α,6α,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-ol (SKF10047), a σ-1 receptor agonist, on endogenous glutamate release in the nerve terminals of rat cerebral cortex. Results show that SKF10047 inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and the σ-1 receptor antagonist N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047) blocked this phenomenon. The effects of SKF10047 on the evoked glutamate release were prevented by the chelating extracellular Ca²⁺ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate did not have any effect on the action of SKF10047. SKF10047 decreased the depolarization-induced increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺](C)), but did not alter 4-AP-mediated depolarization. Furthermore, the effects of SKF10047 on evoked glutamate release were prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking the ryanodine receptors or the mitochondrial Na⁺/Ca²⁺ exchange. In addition, conventional protein kinase C (PKC) inhibitors abolished the SKF10047 effect on 4-AP-evoked glutamate release. Western blot analyses showed that SKF10047 decreased the 4-AP-induced phosphorylation of PKC and PKCα. These results show that σ-1 receptor activation inhibits glutamate release from rat cortical nerve terminals. This effect is linked to a decrease in [Ca²⁺](C) caused by Ca²⁺ entry through presynaptic voltage-dependent Ca²⁺ channels and the suppression of the PKC signaling cascade.

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.

In the present study, a possible sertraline action on cerebral pre-synaptic Na(+) channels was investigated. For this purpose, the effect of sertraline on responses induced by the Na(+) channel opener, veratridine, namely the increase in Na(+) and in neurotransmitter release in hippocampus-isolated nerve endings was investigated. Results show that sertraline in the low μM range (1.5-25 μM) progressively inhibits the rise in Na(+) and the release of pre-loaded [(3) H]Glu as well as the release of endogenous 5-HT, Glu and GABA (detected by HPLC) induced by veratridine depolarization either under external Ca(2+) -free conditions or in the presence of external Ca(2+) . In addition, under non-depolarized conditions, sertraline (25 μM) increased the external concentration of 5-HT at expense of its internal concentration, and unchanged the external and internal concentrations of the amino acid neurotransmitters and of the 5-HT main metabolite, 5-HIAA. This result is consistent with the sertraline inhibitory action of the serotonin transporter. However, sertraline is unlikely to inhibit pre-synaptic Na(+) channels permeability by increasing external 5-HT. Because 5-HT in a wide concentration range (1-1000 μM) did not change the veratridine-induced increase in Na(+) . In summary, present findings demonstrate that besides the inhibition of 5-HT reuptake, sertraline is an effective inhibitor of pre-synaptic Na(+) channels controlling neurotransmitter release., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Purpose: To determine the effects of end-to-side nerve repair performed only with fibrin glue containing nerve growth in rats., Methods: Seventy two Wistar rats were divided into six equal groups: group A was not submitted to nerve section; group B was submitted to nerve fibular section only. The others groups had the nerve fibular sectioned and then repaired in the lateral surface of an intact tibial nerve, with different procedures: group C: ETS with sutures; group D: ETS with sutures and NGF; group E: ETS with FG only; group F: ETS with FG containing NGF. The motor function was accompanied and the tibial muscle mass, the number and diameter of muscular fibers and regenerated axons were measured., Results: All the analyzed variables did not show any differences among the four operated groups (p>0.05), which were statistically superior to group B (p<0.05), but inferior to group A (p>0.05)., Conclusion: The end-to-side nerve repair presented the same recovery pattern, independent from the repair used, showing that the addition of nerve growth factor in fibrin glue was not enough for the results potentiating.

Background: Epidermal hyperinnervation in atopic dermatitis (AD) is activated directly by various external stimuli, causing enhanced itching. Nerve density is regulated by the nerve repulsion factor semaphorin 3A (Sema3A), along with nerve elongation factors., Objective: To investigate the effects of Sema3A ointment in the NC/Nga mouse model of AD., Methods: An AD-like phenotype was induced by repeated application of Dermatophagoides farinae body (Dfb) ointment to the dorsal skin of NC/Nga mice. Vaseline, heparinoid, betamethasone, tacrolimus and recombinant Sema3A ointments were applied to the lesional skin once a day for 4 days. Transepidermal water loss (TEWL) was measured before and after each treatment. We also scored the degree of dermatitis and recorded videos to observe scratching behavior. Subsequently, we collected skin samples from these mice for histological analyses., Results: Topical application of Sema3A, betamethasone and tacrolimus ointments significantly inhibited scratching behavior and improved dermatitis scores in Dfb-treated mice compared with control mice, whereas vaseline and heparinoid had no effects. A significant improvement of TEWL was observed only in Sema3A ointment-treated mice. Moreover, Sema3A ointment reduced the densities of PGP9.5- and substance P-immunoreactive nerve fibers in the epidermis and the numbers of inflammatory cells, such as CD4 immunoreactive T cells and eosinophils, and improved acanthosis in the Dfb-treated mice compared with controls., Conclusion: Sem3A ointment may have therapeutic efficacy in patients with pruritus and dermatitis of AD., (Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Mephedrone (4-methylmethcathinone) is a β-ketoamphetamine with close structural analogy to substituted amphetamines and cathinone derivatives. Abuse of mephedrone has increased dramatically in recent years and has become a significant public health problem in the United States and Europe. Unfortunately, very little information is available on the pharmacological and neurochemical actions of mephedrone. In light of the proven abuse potential of mephedrone and considering its similarity to methamphetamine and methcathinone, it is particularly important to know if mephedrone shares with these agents an ability to cause damage to dopamine nerve endings of the striatum. Accordingly, we treated mice with a binge-like regimen of mephedrone (4 × 20 or 40 mg/kg) and examined the striatum for evidence of neurotoxicity 2 or 7 days after treatment. While mephedrone caused hyperthermia and locomotor stimulation, it did not lower striatal levels of dopamine, tyrosine hydroxylase or the dopamine transporter under any of the treatment conditions used presently. Furthermore, mephedrone did not cause microglial activation in striatum nor did it increase glial fibrillary acidic protein levels. Taken together, these surprising results suggest that mephedrone, despite its numerous mechanistic overlaps with methamphetamine and the cathinone derivatives, does not cause neurotoxicity to dopamine nerve endings of the striatum., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Osthole, an active constituent isolated from Cnidium monnieri (L.) Cusson, has previously been shown to have the capacity to increase depolarization-evoked glutamate release in rat hippocampal nerve terminals. As cGMP-dependent signaling cascade has been found to modulate glutamate release at the presynaptic level, the aim of this study was to further examine the role of cGMP signaling pathway in the regulation of osthole on glutamate release in hippocampal synaptosomes. Results showed that osthole dose-dependently increased intrasynaptosomal cGMP levels. The elevation of cGMP levels by osthole was prevented by the phosphodiesterase 5 inhibitor sildenafil but was insensitive to the guanylyl cyclase inhibitor ODQ. In addition, osthole-induced facilitation of 4-aminopyridine (4-AP)-evoked glutamate release was completely prevented by the cGMP-dependent protein kinase (PKG) inhibitors, KT5823, and Rp-8-Br-PET-cGMPS. Direct activation of PKG with 8-Br-cGMP or 8-pCPT-cGMP also occluded the osthole-mediated facilitation of 4-AP-evoked glutamate release. Furthermore, sildenafil exhibited a dose-dependent facilitation of 4-AP-evoked release of glutamate and occluded the effect of osthole on the 4-AP-evoked glutamate release. Collectively, our findings suggest that osthole-mediated facilitation of glutamate release involves the activation of cGMP/PKG-dependent pathway., (Copyright © 2011 Wiley Periodicals, Inc.)

Local anesthetics effectively suppress pain sensation, but most of these compounds act nonselectively, inhibiting activity of all neurons. Moreover, their actions abate slowly, preventing precise spatial and temporal control of nociception. We developed a photoisomerizable molecule, quaternary ammonium-azobenzene-quaternary ammonium (QAQ), that enables rapid and selective optical control of nociception. QAQ is membrane-impermeant and has no effect on most cells, but it infiltrates pain-sensing neurons through endogenous ion channels that are activated by noxious stimuli, primarily TRPV1. After QAQ accumulates intracellularly, it blocks voltage-gated ion channels in the trans form but not the cis form. QAQ enables reversible optical silencing of mouse nociceptive neuron firing without exogenous gene expression and can serve as a light-sensitive analgesic in rats in vivo. Because intracellular QAQ accumulation is a consequence of nociceptive ion-channel activity, QAQ-mediated photosensitization is a platform for understanding signaling mechanisms in acute and chronic pain.

GABA released from accumbal GABAergic interneurons plays an inhibitory role in the regulation of dopamine efflux through GABA(B) and GABA(A) receptors located on accumbal dopaminergic nerve endings. The cytosolic newly synthesised GABA alters vesicular GABA levels and, accordingly, the amount of GABA released from the neuron. Therefore, we hypothesised that glutamic acid decarboxylase (GAD) which generates GABA in accumbal GABAergic neurons, at least partly determines the GABA receptor subtype-mediated GABAergic tonus. To (in)validate this hypothesis, in vivo microdialysis was used to study the effects of an intra-accumbal infusion of the GAD inhibitor l-allylglycine (allylglycine) on the accumbal dopamine efflux of freely moving rats. The intra-accumbal infusion of allylglycine (50.0, 250.0 and 500.0 nmol) dose-dependently increased the accumbal dopamine levels. The co-administration of tetrodotoxin (720 pmol) suppressed the allylglycine (500.0 nmol)-induced dopamine efflux. The intra-accumbal infusion of GABA(B) receptor agonist baclofen (2.5 and 5.0 nmol) inhibited the allylglycine (500.0 nmol)-induced dopamine efflux. The baclofen's effects were counteracted by GABA(B) receptor antagonist saclofen (10.0 nmol). Neither GABA(A) receptor agonist (muscimol: 25.0 and 250.0 pmol) nor antagonist (bicuculline: 50.0 pmol) altered the allylglycine (250.0 and 500.0 nmol)-induced dopamine efflux. The present study provides in vivo neurochemical evidence that newly synthesised GABA that exerts an inhibitory tonus on the accumbal dopaminergic activity, acts at the level of GABA(B) receptors, but not GABA(A) receptors. The present study also shows that there is an allylglycine-insensitive GABA pool that release GABA exerting an inhibitory control of the accumbal dopaminergic activity, at the level of GABA(A) receptors. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose: To examine the laboratory dentin tubules occlusion and mineral precipitation capability of two potassium salts-containing desensitizing toothpastes., Methods: 40 dentin disks were obtained and divided into four groups, including artificial saliva (AS), distilled water (DW), Sensodyne Freshmint (SF) and Colgate Sensitive (CS). Dentin permeability measurement was performed after EDTA etching, initial brush, 3-day brush, 7-day brush and citric acid challenge, respectively. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to monitor the variation of mineral content before and after treatments. The ultra-morphology of dentin surface was examined using scanning electron microscopy (SEM) to evaluate tubule occlusion., Results: The two potassium salts-containing desensitizing toothpastes significantly reduced dentin permeability to less than 40% after 7-day treatment (P<0.05). The ATR-FTIR analysis showed increase of the intensity of the phosphate peak after a 7-day brushing period using both of the tested toothpastes. However, the dentin permeability significantly increased and the intensity of phosphate peak decreased after acid etching for all groups. The SEM examination revealed partially occluded dentin tubules after toothpaste treatments, but after the acid challenge the tubules were opened again.

Background: 1-Methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) is present in human and mammalian brain as a racemate (R,S) of two stereoisomers: R- and S-1MeTIQ. The racemate is a mixture of the endogenous, synthesized in the brain dextrorotary R-1MeTIQ, and the exogenous, levorotary form, S-1MeTIQ., Methods: In this study, we compared the effect of these two stereoisomers of 1MeTIQ with the racemate in the context of their influence on dopamine metabolism and in vivo dopamine release in the rat striatum. Additionally, a behavioral study was used to examine the influence of both enantiomers R- and S-1MeTIQ on locomotor activity of rats., Results: The behavioral studies showed that both R- and S-1MeTIQ enantiomers, like the racemate, produced biphasic effects: a slight decrease in the exploratory locomotor activity, and then an increase in the basal locomotor activity. Biochemical ex vivo experiments showed that S-1MeTIQ produced a much stronger inhibition of MAO-dependent dopamine oxidation than the racemate. R-1MeTIQ, in opposite to S-isoform and racemate, did not block dopamine oxidation in the structures which contain dopamine nerve endings (striatum, nucleus accumbens), and in fact did not suppress the level of its intraneuronal metabolite, DOPAC in these brain regions. However , an in vivo microdialysis study demonstrated that the racemate and both stereoisomers (R-1MeTIQ to a lesser extent) significantly elevated the concentration of dopamine in the extraneuronal space in the rat striatum. What is more, the concentration of an extraneuronal dopamine metabolite 3-methoxytyramine (3-MT), which is an indicator of its release, was considerably increased by all the compounds., Conclusions: In summary of the results, it can be stated that S-1MeTIQ, like the racemate and in contrast to R-stereoisomer, inhibits MAO-dependent dopamine oxidation in all investigated brain structures. In addition, both these substances potentiated COMT-dependent O-methylation in the brain, however, also R-1MeTIQ produced such effect but to a lesser extent. In conclusion, we suggest that R- and S-1MeTIQ enantiomers like the racemate may demonstrate neuroprotective properties in the brain via the activation of dopamine O-methylation catabolic pathway and elevation of its methylated product - 3-MT.

Enhanced production of angiotensin II and excessive release of norepinephrine in the ischemic heart are major causes of arrhythmias and sudden cardiac death. Mast cell-dependent mechanisms are pivotal in the local formation of angiotensin II and modulation of norepinephrine release in cardiac pathophysiology. Cardiac mast cells increase in number in myocardial ischemia and are located in close proximity to sympathetic neurons expressing angiotensin AT1- and histamine H3-receptors. Once activated, cardiac mast cells release a host of potent pro-inflammatory and pro-fibrotic cytokines, chemokines, preformed mediators (e.g., histamine) and proteases (e.g., renin). In myocardial ischemia, angiotensin II (formed locally from mast cell-derived renin) and histamine (also released from local mast cells) respectively activate AT1- and H3-receptors on sympathetic nerve endings. Stimulation of angiotensin AT1-receptors is arrhythmogenic whereas H3-receptor activation is cardioprotective. It is likely that in ischemia/reperfusion the balance may be tipped toward the deleterious effects of mast cell renin, as demonstrated in mast cell-deficient mice, lacking mast cell renin and histamine in the heart. In these mice, no ventricular fibrillation occurs at reperfusion following ischemia, as opposed to wild-type hearts which all fibrillate. Preventing mast cell degranulation in the heart and inhibiting the activation of a local renin-angiotensin system, hence abolishing its detrimental effects on cardiac rhythmicity, appears to be more significant than the loss of histamine-induced cardioprotection. This suggests that therapeutic targets in the treatment of myocardial ischemia, and potentially congestive heart failure and hypertension, should include prevention of mast cell degranulation, mast cell renin inhibition, local ACE inhibition, ANG II antagonism and H3-receptor activation.

4-Aminopyridine (4-AP) is a convulsing agent that in vivo preferentially releases Glu, the most important excitatory amino acid neurotransmitter in the brain. Here the ionic dependence of 4-AP-induced Glu release and the effects of several of the most common antiepileptic drugs (AEDs) and of the new potential AED, vinpocetine on 4-AP-induced Glu release were characterized in hippocampus isolated nerve endings pre-loaded with labelled Glu ([3H]Glu). 4-AP-induced [3H]Glu release was composed by a tetrodotoxin (TTX) sensitive and external Ca2+ dependent fraction and a TTX insensitive fraction that was sensitive to the excitatory amino acid transporter inhibitor, TBOA. The AEDs: carbamazepine, phenytoin, lamotrigine and oxcarbazepine at the highest dose tested only reduced [3H]Glu release to 4-AP between 50-60%, and topiramate was ineffective. Vinpocetine at a much lower concentration than the above AEDs, abolished [3H]Glu release to 4-AP. We conclude that the decrease in [3H]Glu release linked to the direct blockade of presynaptic Na+ channels, may importantly contribute to the anticonvulsant actions of all the drugs tested here (except topiramate); and that the significantly greater vinpocetine effect in magnitude and potency on [3H]Glu release when excitability is exacerbated like during seizures, may involve the increase additionally exerted by vinpocetine in some K+ channels permeability., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [³H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca²⁺-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 μM), but not of 4-AP (1 mM), involved also external Ca²⁺-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca²⁺-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca²⁺ channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca²⁺-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na⁺/Ca²⁺ exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca²⁺ originated through mitochondrial Na⁺/Ca²⁺ exchangers. Ca²⁺-induced Ca²⁺ release (CICR) mediated by inositoltrisphosphate receptors (InsP₃Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP₃R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets., (© 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.)

Stimulus evoked neurotransmitter release requires that Na(+) channel-dependent nerve terminal depolarization be transduced into synaptic vesicle exocytosis. Inhaled anesthetics block presynaptic Na(+) channels and selectively inhibit glutamate over GABA release from isolated nerve terminals, indicating mechanistic differences between excitatory and inhibitory transmitter release. We compared the effects of isoflurane on depolarization-evoked [(3)H]glutamate and [(14)C]GABA release from isolated nerve terminals prepared from four regions of rat CNS evoked by 4-aminopyridine (4AP), veratridine (VTD), or elevated K(+). These mechanistically distinct secretegogues distinguished between Na(+) channel- and/or Ca(2+) channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC(50) = 0.42 ± 0.03 mM) more potently than GABA release (IC(50) = 0.56 ± 0.02 mM) from cerebral cortex (1.3-fold greater potency), hippocampus and striatum, but inhibited glutamate and GABA release from spinal cord terminals equipotently. Na(+) channel-specific VTD-evoked glutamate release from cortex was also significantly more sensitive to inhibition by isoflurane than was GABA release. Na(+) channel-independent K(+)-evoked release was insensitive to isoflurane at clinical concentrations in all four regions, consistent with a target upstream of Ca(2+) entry. Isoflurane inhibited Na(+) channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC(50) = 0.30 ± 0.03 mM) more potently than GABA release (IC(50) = 0.67 ± 0.04 mM) from cortex (2.2-fold greater potency). The magnitude of inhibition of Na(+) channel-mediated 4AP-evoked release by a single clinical concentration of isoflurane (0.35 mM) varied by region and transmitter: Inhibition of glutamate release from spinal cord was greater than from the three brain regions and greater than GABA release for each CNS region. These findings indicate that isoflurane selectively inhibits glutamate release compared to GABA release via Na(+) channel-mediated transduction in the four CNS regions tested, and that differences in presynaptic Na(+) channel involvement determine differences in anesthetic pharmacology., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

There is abundant evidence suggesting the relevance of glutamate to depression and antidepressant mechanisms. Curcumin, a major active compound of Curcuma longa, has been reported to have the biological function of antidepressant. The aim of the present study was to investigate the effect of curcumin on endogenous glutamate release in nerve terminals of rat prefrontal cortex and the underlying mechanisms. The results showed that curcumin inhibited the release of glutamate that was evoked by exposing synaptosomes to the K(+) channel blocker 4-aminopyridine (4-AP). This phenomenon was blocked by the chelating the extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-β-benzyl-oxyaspartate (DL-TBOA). Further experiments demonstrated that curcumin decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting membrane potential or 4-AP-mediated depolarization. Furthermore, the inhibitory effect of curcumin on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. These results suggest that curcumin inhibits evoked glutamate release from rat prefrontocortical synaptosomes by the suppression of presynaptic Ca(v)2.2 and Ca(v)2.1 channels. Additionally, we also found that the inhibitory effect of curcumin on 4-AP-evoked glutamate release was completely abolished by the clinically effective antidepressant fluoxetine. This suggests that curcumin and fluoxetine use a common intracellular mechanism to inhibit glutamate release from rat prefrontal cortex nerve terminals., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The present study investigated the effect and possible mechanism of luteolin, a food-derived flavonoid, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Luteolin inhibited the release of glutamate evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent. The effect of luteolin on the evoked glutamate release was prevented by the chelation of the extracellular Ca(2+) ions and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Luteolin decreased the 4-AP-induced increase in [Ca(2+)](C), whereas it did not alter 4-AP-mediated depolarization. Furthermore, the effect of luteolin on evoked glutamate release was abolished by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking the ryanodine receptors or the mitochondrial Na(+)/Ca(2+) exchange. In addition, the inhibitory effect of luteolin on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase (MEK) inhibitors. Western blot analyses showed that luteolin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK. Thus, it was concluded that luteolin inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca(2+) entry and MEK/ERK signaling cascade.

Acrylamide (ACR) intoxication is associated with selective nerve terminal damage in the central and peripheral nervous systems. As a soft electrophile, ACR could form adducts with nucleophilic sulfhydryl groups on cysteine residues of kelch-like erythroid cell-derived protein with CNS homology-associated protein 1 (Keap1) leading to dissociation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activation of the antioxidant-responsive element (ARE) and subsequent upregulated gene expression of phase II detoxification enzymes and anitoxidant proteins might provide protection in neuronal regions with transcriptional capabilities (e.g., cell body). In contrast, non-transcriptional cell regions (axons, nerve terminals) might be vulnerable to electrophile-induced damage. To test this possibility, immunoblot analysis was used to measure protein products of Nrf2-activated ARE genes in nerve terminals and in cytosolic/nuclear factions of neuronal cell bodies isolated from rats intoxicated at two different ACR dose-rates; i.e., 50mg/kg/d×10 days, 21mg/kg/d×38 days. To detect possible differences in cell-specific induction, the cytoprotective response to ACR intoxication was determined in hepatic cells. Results show that control brain and hepatic cell fractions exhibited distinct subcellular distributions of Nrf2, Keap1 and several ARE protein products. ACR intoxication, however, did not alter the levels of these proteins in synaptosomal, brain cytoplasm or liver cell fractions. These data indicate that ACR was an insufficient electrophilic signal for ARE induction in all subcellular fractions tested. Because a cytoprotective response was not induced in any fraction, nerve terminal vulnerability to ACR cannot be ascribed to the absence of transcription-based defense mechanisms in this neuronal region., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The present study was aimed at investigating the effect and the possible mechanism of idebenone on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Idebenone inhibited the release of glutamate that was evoked by exposing synaptosomes to the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration dependent. Inhibition of glutamate release by idebenone was prevented by chelating extracellular Ca(2+), or by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to DL-threo-beta-benzyl-oxyaspartate, a glutamate transporter inhibitor. Idebenone decreased the depolarization-induced increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](C)),whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The inhibitory effect of idebenone on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. Furthermore, the idebenone effect on 4-AP-evoked Ca(2+) influx and glutamate release was completely abolished by the protein kinase A (PKA) inhibitors, H89 and KT5720. On the basis of these results, it was concluded that idebenone inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in [Ca(2+)](C) contributed by Ca(2+) entry through presynaptic voltage-dependent Ca(2+) channels and to the suppression of PKA signaling cascade.

Methylmalonic acidemia and propionic acidemia are organic acidemias biochemically characterized by predominant tissue accumulation of methylmalonic acid (MMA) and propionic acid (PA), respectively. Affected patients present predominantly neurological symptoms, whose pathogenesis is not yet fully established. In the present study we investigated the in vitro effects of MMA and PA on important parameters of lipid and protein oxidative damage and on the production of reactive species in synaptosomes from cerebrum of developing rats. Synaptosomes correspond to nerve terminals that have been used to investigate toxic properties of compounds on neuronal cells. The in vivo effects of intrastriatal injection of MMA and PA on the same parameters and on enzymatic antioxidant defenses, were also studied. MMA-induced in vitro and in vivo lipid peroxidation and protein oxidative damage. Furthermore, the lipid oxidative damage was attenuated or prevented, pending on the doses utilized, by the free radical scavengers α-tocopherol, melatonin and by the NMDA glutamate receptor antagonist MK-801, implying the involvement of reactive species and glutamate receptor activation in these effects. In addition, 2',7'-dichlorofluorescein diacetate oxidation was significantly increased in synaptosomes by MMA, reinforcing that reactive species generation is elicited by this organic acid. We also verified that glutathione peroxidase activity was inhibited by intrastriatal MMA injection. In contrast, PA did not induce any significant effect on all parameters examined in vitro and in vivo, implying a selective action for MMA. The present data demonstrate that oxidative stress is induced by MMA in vitro in nerve terminals and in vivo in striatum, suggesting the participation of neuronal cells in MMA-elicited oxidative damage.

Chronic unproductive coughing and dyspnea are symptoms that severely diminish the quality of life in a substantial proportion of the population. There are presently few if any drugs that effectively treat these symptoms. Rational drug targets for cough and dyspnea have emerged over the recent years based on developments in our understanding of the innervation of the respiratory tract. These drug targets can be subcategorized into those that target the vagal afferent nerve endings, and those that target neural activity within the CNS. This review focuses on targets presumed to be in the peripheral terminals of afferent nerves within the airways. Conceptually, the activity of peripheral afferent nerves involved with unwanted urge-to-cough or dyspnea sensations can be inhibited by limiting the intensity of the stimulus, inhibiting the amplitude of the stimulus-induced generator potential, or inhibiting the transduction between the generator potential and action potential discharge and conduction. These mechanisms reveal many therapeutic strategies for anti-tussive and anti-dyspnea drug development with peripheral sites of action., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Central glutamate neurotransmission has been postulated to play a role in pathophysiology of depression and in the mechanism of antidepressants. The present study was undertaken to elucidate the effect and the possible mechanism of bupropion, an atypical antidepressant, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Result showed that bupropion exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of bupropion on the evoked glutamate release was prevented by the chelating the intrasynaptosomal Ca(2+) ions, and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Bupropion decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of bupropion on evoked glutamate release was abolished by the N-, P- and Q-type Ca(2+) channel blocker, but not by the ryanodine receptor blocker, or the mitochondrial Na(+)/Ca(2+) exchanger blocker. In addition, the inhibitory effect of bupropion on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Western blot analyses showed that bupropion significantly decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and this effect also was blocked by MEK inhibitor. These results are the first to suggest that, in rat cerebrocortical nerve terminals, bupropion suppresses voltage-dependent Ca(2+) channel and MEK/ERK activity and in so doing inhibits evoked glutamate release. This finding may provide important information regarding the beneficial effects of bupropion in the brain., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Adenosine, dopamine and endocannabinoids strictly modulate the release of one another in the dorsolateral striatum thereby controlling synaptic plasticity. As a second level of interaction, they regulate the action of one another via receptor heteromer formation. Here we investigated a putative third level of interaction, i.e. the possible control by cannabinoids of synaptic dopamine and adenosine reuptake. We found that a large number of endo- and exogenous cannabinoid ligands inhibit the uptake of [(3)H]adenosine and [(3)H]dopamine in rat sriatal nerve terminals. Maximal effects were often comparable to those of the dopamine transporter inhibitor, GBR12783 and the equilibrative nucleoside transporter inhibitor, dipyridamole. Cannabinoid ligands were generally more potent to inhibit the uptake of adenosine than that of dopamine. The inhibitory effect was: (1) unrelated to the pharmacological profile(s) of the ligands at the cannabinoid CB(1), CB(2), GPR55 and at the vanilloid TRPV(1) receptors; (2) not prevented by the cannabinoid CB(1) receptor antagonist/inverse agonist, LY320135; and (3) maintained in the cannabinoid CB(1) receptor knockout mice. In the same experiments, only O-2050, cannabidiol, and WIN55212-3 inhibited the simultaneously measured DL-TBOA-sensitive uptake of [(14)C]glutamate. In summary, many cannabinoid ligands are able to inhibit the synaptic uptake of adenosine and dopamine. These effects are not mediated by cannabinoid CB(1) receptors, and should be an additional mechanism to consider when interpreting synaptic effects of cannabinoids., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Synaptic release of neurotransmitters is evoked by activity-dependent Ca(2+) entry into the nerve terminal. However, here it is shown that robust synaptic neuropeptide release from Drosophila motoneurons is evoked in the absence of extracellular Ca(2+) by octopamine, the arthropod homolog to norepinephrine. Genetic and pharmacology experiments demonstrate that this surprising peptidergic transmission requires cAMP-dependent protein kinase, with only a minor contribution of exchange protein activated by cAMP (epac). Octopamine-evoked neuropeptide release also requires endoplasmic reticulum Ca(2+) mobilization by the ryanodine receptor and the inositol trisphosphate receptor. Hence, rather than relying exclusively on activity-dependent Ca(2+) entry into the nerve terminal, a behaviorally important neuromodulator uses synergistic cAMP-dependent protein kinase and endoplasmic reticulum Ca(2+) signaling to induce synaptic neuropeptide release.

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.

The pathogenesis of Parkinson's disease is thought to involve a self-sustaining cycle of neuroinflammation and neurodegeneration. In order to develop novel anti-inflammatory therapies to break this cycle, it is crucial that the temporal relationship between neurodegeneration and neuroinflammation is characterised in pre-clinical models to maximise their predictive validity. Thus, this study aimed to investigate the progression of neuroinflammation relative to nigrostriatal neurodegeneration in the two most commonly-used rat models of Parkinson's disease. Male Sprague-Dawley rats were lesioned by terminal or axonal administration of 6-hydroxydopamine, and were sacrificed for quantitative immunohistochemistry (to assess nigrostriatal integrity (anti-tyrosine hydroxylase), microgliosis (anti-OX42) and astrocytosis (anti-GFAP)) at 6 h 24 h 72 h or 2 weeks post-lesion. Following terminal lesion, dopaminergic deafferentation of the striatum was evident from 6 h post-lesion and was accompanied by microglial and astroglial activation. Dopamine neuron loss from the substantia nigra did not occur until 2 weeks after terminal lesion, and this was preceded by microglial, but not astroglial, activation. Following axonal lesion, retraction of nigrostriatal terminals from the striatum was not observed until the 72 h time-point, and this was associated with a slight astrocytosis, but not microgliosis. Degeneration of dopaminergic neurons from the substantia nigra was also evident from 72 h after axonal lesion, and was accompanied by nigral microgliosis and astrocytosis by 2 weeks. This study highlights the temporal relationship between neurodegeneration and neuroinflammation in models of Parkinson's disease, and should facilitate use of these models in the development of anti-inflammatory therapies for the human condition., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Many studies have demonstrated that premenopausal women are at increased risk for various pain disorders. Pain-sensing neurons, termed "nociceptors," in the trigeminal ganglia (TG) and dorsal root ganglia (DRG) express receptors for inflammatory mediators and noxious physical stimuli and transmit signals for central processing of pain sensation. Estrogen receptors (ERs) are also expressed on nociceptors in the TG and DRG, and there is ample literature to suggest that activation of ERs can influence pain mechanisms. However, the mechanism for ER modulation of nociceptor activity is incompletely understood. The aim of this study was to characterize the effect of 17β-estradiol (17β-E(2)) on signaling of the inflammatory mediator bradykinin (BK) in primary cultures of rat sensory neurons and a behavioral model of thermal allodynia in rats. Here, we show that exposure to 17β-E(2) rapidly (within 15 min) enhanced responses to BK in vitro and in vivo. The 17β-E(2)-mediated enhancement of BK signaling was not blocked by the transcription inhibitor anisomycin and was mediated by a membrane-associated ER. The effect of 17β-E(2) to enhance BK responses required activation of β1-containing, RGD-binding integrins. These data show that 17β-E(2) rapidly enhances inflammatory mediator responses both in vitro and in vivo and suggest that 17β-E(2) acting at primary sensory pain neurons may participate in regulating the sensitivity of women to painful stimuli.

Background: Topical photodynamic therapy is a good treatment option for extensively photodamaged skin with multiple actinic keratoses. Pain is one of the major adverse effects during and after the treatment., Objective: We sought to determine the pain intensity and its influencing factors during the extensive photodynamic treatment of complete cosmetic units., Methods: In total, 104 patients with 411 treated fields were enrolled in this retrospective monocentric study. All patients had multiple actinic keratoses on the face, scalp, or back of hands and received an extensive treatment of the complete photodamaged area in our dermatologic outpatient department between February and May 2009. Pain was rated using a visual analog scale directly and 8 hours after photodynamic therapy., Results: Multifactorial analysis of the data shows that pain intensity is dependent on sex of the patient (P = .030) and location of the treated field (P < .001). Visual analog scale scores were independent of the age and skin type of the patient. No significant difference in pain between the use of 5-amino-4-oxo-pentanoate (methylaminolevulinate) and 5-aminolevulinic acid was noticed. During treatment, mean visual analog scale scores +/- SEM of the different locations were 2.5 +/- 0.36 (hand), 3.6 +/- 0.35 (occiput), 5.2 +/- 0.19 (forehead), 5.9 +/- 0.20 (cheeks), and 7.4 +/- 0.48 (lips). Eight hours after treatment the mean pain +/- SEM in all locations was reduced significantly (P < .001) from 5.2 +/- 0.14 to 3.0 +/- 0.14. The rate of therapy interruptions showed a significant correlation (P = .044) with the location of the treated field. In all, 71% of the patients showed a good and 29% a poor clinical outcome., Limitations: Because of the retrospective study design not all factors that may influence pain (eg, protoporphyrin IX fluorescence) were recorded., Conclusion: These results show that pain intensity is dependent on the location of the treated field. Pain intensity is higher in male patients. After 8 hours pain decreases significantly., (Copyright 2009 American Academy of Dermatology, Inc. Published by Mosby, Inc. All rights reserved.)

The purpose of this study was to examine the effect and mechanism of astaxanthin, a natural carotenoid, on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Results showed that astaxanthin exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate. The effect of astaxanthin on the evoked glutamate release was prevented by chelating the intrasynaptosomal Ca(2+) ions and by the vesicular transporter inhibitor, but was insensitive to the glutamate transporter inhibitor. Astaxanthin decreased depolarization-induced increase in [Ca(2+)](C), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of astaxanthin on evoked glutamate release was abolished by the N-, P- and Q-type Ca(2+) channel blockers, but not by the ryanodine receptor blocker or the mitochondrial Na(+)/Ca(2+) exchanger blocker. In addition, the inhibitory effect of astaxanthin on evoked glutamate release was prevented by the mitogen-activated protein kinase (MAPK) inhibitors PD98059 and U0126. Western blot analyses showed that astaxanthin significantly decreased the 4-AP-induced phosphorylation of MAPK, and this effect was blocked by PD98059. On the basis of these results, it was concluded that astaxanthin inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca(2+) entry and MAPK signaling cascade.

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

Polyethylene glycol (PEG) has been widely used as a solvent among other applications. An ideal solvent is one that does not interfere with an in vitro biological system, unless it is a bioactive agent. Herein, a facilitatory neurotransmission effect was exhibited by PEG (20 microM) in mammalian (67 +/- 12.5%, n = 4) and avian (74 +/- 6.8%, n = 6) neuromuscular preparations. In curarized preparations, PEG did not reverse the neurotransmission blockade induced by D-tubocurarine (D-Tc, 5.8 microM, n = 6) as promoted by neostigmine (12 microM, n = 4). A possible presynaptic action of PEG was ruled out, because quantal acetylcholine (ACh) content was similar to the control Tyrode-incubated mammalian preparation. PEG showed improved sarcolemmal sensitivity, both under direct (sarcolemma) and indirect stimulation (motor axon), because it was able to release calcium from the sarcoplasmic reticulum, even when 30 microM dantrolene (n = 5) was previously applied. Neurotransmission decreased at a higher PEG concentration (100 microM, n = -6) in the depolarized membrane, but it did not alter normal muscle fiber morphology. In addition, it partially recovered twitch tension amplitude (55 +/- 5.7%) after washing the preparations. More than a simple solvent, we suggest that PEG 400 is able to act on the sarcolemmal membrane, probably at the triad level, which is in line with its well-known ability as drug carrier.

Aberrant accumulation of amyloid beta (Abeta) oligomers may underlie the cognitive failure of Alzheimer's disease (AD). All species of Abeta peptides are produced physiologically during normal brain activity. Therefore, elucidation of mechanisms that interconnect excitatory glutamatergic neurotransmission, synaptic amyloid precursor protein (APP) processing and production of its metabolite, Abeta, may reveal synapse-specific strategies for suppressing the pathological accumulation of Abeta oligomers and fibrils that characterize AD. To study synaptic APP processing, we used isolated intact nerve terminals (cortical synaptoneurosomes) from TgCRND8 mice, which express a human APP with familial AD mutations. Potassium chloride depolarization caused sustained release from synaptoneurosomes of Abeta(42) as well as Abeta(40), and appeared to coactivate alpha-, beta- and gamma-secretases, which are known to generate a family of released peptides, including Abeta(40) and Abeta(42). Stimulation of postsynaptic group I metabotropic glutamate receptor (mGluRs) with DHPG (3,5-dihydroxyphenylglycine) induced a rapid accumulation of APP C-terminal fragments (CTFs) in the synaptoneurosomes, a family of membrane-bound intermediates generated from APP metabolized by alpha- and beta-secretases. Following stimulation with the group II mGluR agonist DCG-IV, levels of APP CTFs in the synaptoneurosomes initially increased but then returned to baseline by 10 min after stimulation. This APP CTF degradation phase was accompanied by sustained accumulation of Abeta(42) in the releasate, which was blocked by the group II mGluR antagonist LY341495. These data suggest that group II mGluR may trigger synaptic activation of all three secretases and that suppression of group II mGluR signaling may be a therapeutic strategy for selectively reducing synaptic generation of Abeta(42).

5R-L-Erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) is an essential cofactor for tyrosine hydroxylase (TH). Recently, a type of dopa-responsive dystonia (DRD) (DYT5, Segawa's disease) was revealed to be caused by dominant mutations of the gene encoding GTP cyclohydrolase I (GCHI), which is the rate-limiting enzyme of BH(4) biosynthesis. In order to probe the role of BH(4) in vivo, we established BH(4)-depleted mice by disrupting the 6-pyruvoyltetrahydropterin synthase (PTS) gene (Pts(-/-)) and rescued them by introducing human PTS cDNA under the control of the human dopamine β-hydroxylase (DBH) promoter (Pts(-/-)-DPS). The Pts(-/-)-DPS mice developed hyperphenylalaninemia. Interestingly, tyrosine hydroxylase protein was dramatically reduced in the dopaminergic nerve terminals of these mice, and they developed abnormal posture and motor disturbance. We propose that the biochemical and pathologic changes of Pts(-/-)-DPS mice are caused by mechanisms common to human DRD, and understanding these mechanisms could give us insight into other movement disorders.

Botulinum neurotoxin A (BoNT/A), the most toxic, naturally occurring protein, cleaves synapse-associated protein of 25 kDa and inhibits acetylcholine release from motor nerve endings (MNEs). This leads to paralysis of skeletal muscles. Our study demonstrates that capsaicin protects mouse neuromuscular junctions from the neuroparalytic effects of BoNT/A. Bilateral injection of BoNT/A near the innervation of the Extensor digitorum longus (EDL) muscle of adult Swiss-Webster mice inhibited the toe spread reflex (TSR). However, when capsaicin was coinjected bilaterally, or injected 4 or 8 h before injecting BoNT/A, the TSR remained normal. In animals that were pretreated with capsazepine, capsaicin failed to protect against the neuroparalytic effects of BoNT/A. In vivo analyses demonstrated that capsaicin protected muscle functions and electromygraphic activity from the incapacitating effects of BoNT/A. The twitch response to nerve stimulation was greater for EDL preparations isolated from mice injected with capsaicin before BoNT/A. Capsaicin pretreatment also prevented the inhibitory effects of BoNT/A on end-plate currents. Furthermore, pretreatment of Neuro 2a cells with capsaicin significantly preserved labeling of synaptic vesicles by FM 1-43. This protective effect of capsaicin was observed only in the presence of extracellular Ca(2+) and was inhibited by capsazepine. Immunohistochemistry demonstrated that MNEs express transient receptor potential protein of the vanilloid subfamily, TRPV1, the capsaicin receptor. Capsaicin pretreatment, in vitro, reduced nerve stimulation or KCl-induced uptake of BoNT/A into motor nerve endings and cholinergic Neuro 2a cells. These data demonstrate that capsaicin interacts with TRPV1 receptors on MNEs to reduce BoNT/A uptake via a Ca(2+)-dependent mechanism.

Snake presynaptic neurotoxins with phospholipase A2 (PLA2) activity cause degeneration of the neuromuscular junction. They induce depletion of synaptic vesicles and increase the membrane permeability to Ca(2+) which fluxes from the outside into the nerve terminal. Moreover, several toxins were shown to enter the nerve terminals of cultured neurons, where they may display their PLA2 activity on internal membranes. The relative contribution of these different actions in nerve terminal degeneration remains to be established. To gather information on this point, we have compared the effects of beta-bungarotoxin, taipoxin, notexin and textilotoxin with those of alpha-latrotoxin on the basis of the notion that this latter toxin is well known to cause massive Ca(2+) influx and exocytosis of synaptic vesicles. All the parameters analysed here, including calcium imaging, are very similar for the two classes of neurotoxins. This indicates that Ca(2+) overloading plays a major role in the degeneration of nerve terminals induced by the snake presynaptic neurotoxins.