Adrenaline Facilitates Synaptic Transmission by Synchronizing Release of Acetylcholine Quanta from Motor Nerve Endings.

The long history of studies on the effect of catecholamines on synaptic transmission does not answer the main question about the mechanism of their action on quantal release in the neuromuscular junction. Currently, interest in catecholamines has increased not only because of their widespread use in the clinic for the treatment of cardiovascular and pulmonary diseases but also because of recent data on their possible use for the treatment of certain neurodegenerative diseases, muscle weakness and amyotrophic sclerosis. Nevertheless, the effects and mechanisms of catecholamines on acetylcholine release remain unclear. We investigated the action of noradrenaline and adrenaline on the spontaneous and evoked quantal secretion of acetylcholine in the neuromuscular junction of the rat soleus muscle. Noradrenaline (10 µM) did not change the spontaneous acetylcholine quantal release, the number of released quanta after nerve stimulation, or the timing of the quantal secretion. However, adrenaline at the same concentration increased spontaneous secretion by 40%, increased evoked acetylcholine quantal release by 62%, and synchronized secretion. These effects differ from those previously described by us in the synapses of the frog cutaneous pectoris muscle and mouse diaphragm. This indicates specificity in catecholamine action that depends on the functional type of muscle and the need to take the targeted type of muscle into account in clinical practice.

Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

Electroencephalogram monitoring during propofol (PRO) anesthesia typically features low-frequency oscillations, which may be involved with thalamic reticular nucleus (TRN) modulation. TRN receives noradrenergic inputs from the locus coeruleus (LC). We hypothesized that specific noradrenergic connections in the TRN may contribute to the emergence from PRO anesthesia. Intranuclei norepinephrine (NE) injections (n = 10) and designer receptors exclusively activated by designer drugs (DREADDs) (n = 10) were used to investigate the role of noradrenergic inputs from the LC to the TRN during PRO anesthesia. Whole-cell recording in acute brain slice preparations was used to identify the type of adrenoceptor that regulates noradrenergic innervation in the TRN. An intracerebral injection of NE into the TRN delays arousal in mice recovering from PRO anesthesia (means ± sd; 486.6 ± 57.32 s for the NE injection group vs. 422.4 ± 48.19 s for the control group; P = 0.0143) and increases the cortical-δ (0.1-4 Hz, 25.4 ± 2.9 for the NE injection group vs. 21.0 ± 1.7 for the control group; P = 0.0094) oscillation. An intra-TRN injection of NE also decreased the EC50 of PRO-induced unconsciousness (57.05 ± 1.78 mg/kg for the NE injection group vs. 72.44 ± 3.23 mg/kg for the control group; P = 0.0096). Moreover, the activation of LC-noradrenergic nerve terminals in the TRN using DREADDs increased the recovery time [466.1 ± 44.57 s for the clozapine N-oxide (CNO) injection group vs. 426.1 ± 38.75 s for the control group; P = 0.0033], decreased the EC50 of PRO-induced unconsciousness (64.77 ± 3.40 mg/kg for the CNO injection group vs. 74.00 ± 2.08 mg/kg for the control group; P = 0.0081), and increased the cortical-δ oscillation during PRO anesthesia (23.29 ± 2.58 for the CNO injection group vs. 19.56 ± 1.9 for the control group; P = 0.0213). In addition, whole-cell recording revealed that NE augmented the inhibitory postsynaptic currents in the TRN neurons via the α1-adrenoceptor. Our data indicated that enhanced NE signaling at the noradrenergic terminals of the LC-TRN projection delays arousal from general anesthesia, which is likely mediated by the α1-adrenoceptor activation. Our findings open a door for further understanding of the functions of various LC targets in both anesthesia and arousal.-Zhang, Y., Fu, B., Liu, C., Yu, S., Luo, T., Zhang, L., Zhou, W., Yu, T. Activation of noradrenergic terminals in the reticular thalamus delays arousal from propofol anesthesia in mice.

P2X7 receptors exert a permissive effect on the activation of presynaptic AMPA receptors in rat trigeminal caudal nucleus glutamatergic nerve terminals.

BACKGROUND: Purine receptors play roles in peripheral and central sensitization and are associated with migraine headache. We investigated the possibility that ATP plays a permissive role in the activation of AMPA receptors thus inducing Glu release from nerve terminals isolated from the rat trigeminal caudal nucleus (TCN). METHODS: Nerve endings isolated from the rat TCN were loaded with [3H]D-aspartic acid ([3H]D-ASP), layered into thermostated superfusion chambers, and perfused continuously with physiological medium, alone or with various test drugs. Radioactivity was measured to assess [3H]D-ASP release under different experimental conditions. RESULTS: Synaptosomal [3H]D-ASP spontaneous release was stimulated by ATP and to an even greater extent by the ATP analogue benzoylbenzoylATP (BzATP). The stimulation of [3H]D-ASP basal release by the purinergic agonists was prevented by the selective P2X7 receptor antagonist A438079. AMPA had no effect on basal [3H]D-ASP release, but the release observed when synaptosomes were exposed to AMPA plus a purinoceptor agonist exceeded that observed with ATP or BzATP alone. The selective AMPA receptor antagonist NBQX blocked this "excess" release. Co-exposure to AMPA and BzATP, each at a concentration with no release-stimulating effects, evoked a significant increase in [3H]D-ASP basal release, which was prevented by exposure to a selective AMPA antagonist. CONCLUSIONS: P2X7 receptors expressed on glutamatergic nerve terminals in the rat TCN can mediate Glu release directly and indirectly by facilitating the activation of presynaptic AMPA receptors. The high level of glial ATP that occurs during chronic pain states can promote widespread release of Glu as well as can increase the function of AMPA receptors. In this manner, ATP contributes to the AMPA receptor activation involved in the onset and maintenance of the central sensitization associated with chronic pain.

Age-Dependency of Levetiracetam Effects on Exocytotic GABA Release from Nerve Terminals in the Hippocampus and Cortex in Norm and After Perinatal Hypoxia.

Perinatal hypoxia can lead to multiple chronic neurological deficits, e.g., mental retardation, behavioral abnormalities, and epilepsy. Levetiracetam (LEV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an anticonvulsant drug with proven efficiency in treating patients with focal and generalized seizures. Rats were underwent hypoxia and seizures at the age of 10-12 postnatal days (pd). The ambient level and depolarization-induced exocytotic release of [3H]GABA (γ-aminobutyric acid) were analyzed in nerve terminals in the hippocampus and cortex during development at the age of pd 17-19 and pd 24-26 (infantile stage), pd 38-40 (puberty) and pd 66-73 (young adults) in norm and after perinatal hypoxia. LEV had no effects on the ambient [3H]GABA level. The latter increased during development and was further elevated after perinatal hypoxia in nerve terminals in the hippocampus during the whole period and in the cortex in young adults. Exocytotic [3H]GABA release from nerve terminals increased after perinatal hypoxia during development in the hippocampus and cortex, however this effect was preserved at all ages during blockage of GABA transporters by NO-711 in the hippocampus only. LEV realized its anticonvulsant effects at the presynaptic site through an increase in exocytotic release of GABA. LEV exerted more significant effect after perinatal hypoxia than in norm. Action of LEV was strongly age-dependent and can be registered in puberty and young adults, but the drug was inert at the infantile stage.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release.

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

Distribution of TTX-sensitive voltage-gated sodium channels in primary sensory endings of mammalian muscle spindles.

Knowledge of the molecular mechanisms underlying signaling of mechanical stimuli by muscle spindles remains incomplete. In particular, the ionic conductances that sustain tonic firing during static muscle stretch are unknown. We hypothesized that tonic firing by spindle afferents depends on sodium persistent inward current (INaP) and tested for the necessary presence of the appropriate voltage-gated sodium (NaV) channels in primary sensory endings. The NaV1.6 isoform was selected for both its capacity to produce INaP and for its presence in other mechanosensors that fire tonically. The present study shows that NaV1.6 immunoreactivity (IR) is concentrated in heminodes, presumably where tonic firing is generated, and we were surprised to find NaV1.6 IR strongly expressed also in the sensory terminals, where mechanotransduction occurs. This spatial pattern of NaV1.6 IR distribution was consistent for three mammalian species (rat, cat, and mouse), as was tonic firing by primary spindle afferents. These findings meet some of the conditions needed to establish participation of INaP in tonic firing by primary sensory endings. The study was extended to two additional NaV isoforms, selected for their sensitivity to TTX, excluding TTX-resistant NaV channels, which alone are insufficient to support firing by primary spindle endings. Positive immunoreactivity was found for NaV1.1, predominantly in sensory terminals together with NaV1.6 and for NaV1.7, mainly in preterminal axons. Differential distribution in primary sensory endings suggests specialized roles for these three NaV isoforms in the process of mechanosensory signaling by muscle spindles.NEW & NOTEWORTHY The molecular mechanisms underlying mechanosensory signaling responsible for proprioceptive functions are not completely elucidated. This study provides the first evidence that voltage-gated sodium channels (NaVs) are expressed in the spindle primary sensory ending, where NaVs are found at every site involved in transduction or encoding of muscle stretch. We propose that NaVs contribute to multiple steps in sensory signaling by muscle spindles as it does in other types of slowly adapting sensory neurons.

Dissecting the Influence of Two Structural Substituents on the Differential Neurotoxic Effects of Acute Methamphetamine and Mephedrone Treatment on Dopamine Nerve Endings with the Use of 4-Methylmethamphetamine and Methcathinone.

Mephedrone (MEPH) is a ß-ketoamphetamine stimulant drug of abuse that is often a constituent of illicit bath salts formulations. Although MEPH bears remarkable similarities to methamphetamine (METH) in terms of chemical structure, as well as its neurochemical and behavioral effects, it has been shown to have a reduced neurotoxic profile compared with METH. The addition of a ß-keto moiety and a 4-methyl ring substituent to METH yields MEPH, and a loss of direct neurotoxic potential. In the present study, two analogs of METH, methcathinone (MeCa) and 4-methylmethamphetamine (4MM), were assessed for their effects on mouse dopamine (DA) nerve endings to determine the relative contribution of each individual moiety to the loss of direct neurotoxicity in MEPH. Both MeCa and 4MM caused significant alterations in core body temperature as well as locomotor activity and stereotypy, but 4MM was found to elicit minimal dopaminergic toxicity only at the highest dose. By contrast, MeCa caused significant reductions in all markers of DA nerve-ending damage over a range of doses. These results lead to the conclusion that ring substitution at the 4-position profoundly reduces the neurotoxicity of METH, whereas the ß-keto group has much less influence on this property. Although the mechanism(s) by which the 4-methyl substituent reduces METH-induced neurotoxicity remains unclear, it is speculated that this effect is mediated by a loss of DA-releasing action in MEPH and 4MM at the synaptic vesicle monoamine transporter, an effect that is thought to be critical for METH-induced neurotoxicity.

Secreted herpes simplex virus-2 glycoprotein G modifies NGF-TrkA signaling to attract free nerve endings to the site of infection.

Herpes simplex virus type 1 (HSV-1) and HSV-2 are highly prevalent viruses that cause a variety of diseases, from cold sores to encephalitis. Both viruses establish latency in peripheral neurons but the molecular mechanisms facilitating the infection of neurons are not fully understood. Using surface plasmon resonance and crosslinking assays, we show that glycoprotein G (gG) from HSV-2, known to modulate immune mediators (chemokines), also interacts with neurotrophic factors, with high affinity. In our experimental model, HSV-2 secreted gG (SgG2) increases nerve growth factor (NGF)-dependent axonal growth of sympathetic neurons ex vivo, and modifies tropomyosin related kinase (Trk)A-mediated signaling. SgG2 alters TrkA recruitment to lipid rafts and decreases TrkA internalization. We could show, with microfluidic devices, that SgG2 reduced NGF-induced TrkA retrograde transport. In vivo, both HSV-2 infection and SgG2 expression in mouse hindpaw epidermis enhance axonal growth modifying the termination zone of the NGF-dependent peptidergic free nerve endings. This constitutes, to our knowledge, the discovery of the first viral protein that modulates neurotrophins, an activity that may facilitate HSV-2 infection of neurons. This dual function of the chemokine-binding protein SgG2 uncovers a novel strategy developed by HSV-2 to modulate factors from both the immune and nervous systems.

Effects of new fluorinated analogues of GABA, pregabalin bioisosters, on the ambient level and exocytotic release of [3H]GABA from rat brain nerve terminals.

Recently, we have shown that new fluorinated analogues of γ-aminobutyric acid (GABA), bioisosters of pregabalin (ß-i-Bu-GABA), i.e. ß-polyfluoroalkyl-GABAs (FGABAs), with substituents: ß-CF3-ß-OH (1), ß-CF3 (2); ß-CF2CF2H (3), are able to increase the initial rate of [3H]GABA uptake by isolated rat brain nerve terminals (synaptosomes), and this effect is higher than that of pregabalin. So, synthesized FGABAs are structural but not functional analogues of GABA. Herein, we assessed the effects of synthesized FGABAs (100µM) on the ambient level and exocytotic release of [3H]GABA in nerve terminals and compared with those of pregabalin (100µM). It was shown that FGABAs 1-3 did not influence the ambient level of [3H]GABA in the synaptosomal preparations, and this parameter was also not altered by pregabalin. During blockage of GABA transporters GAT1 by specific inhibitor NO-711, FGABAs and pregabalin also did not change ambient [3H]GABA in synaptosomal preparations. Exocytotic release of [3H]GABA from synaptosomes decreased in the presence of FGABAs 1-3 and pregabalin, and the effects of FGABAs 1 &3 were more significant than those of FGABAs 2 and pregabalin. FGABAs 1-3/pregabalin-induced decrease in exocytotic release of [3H]GABA from synaptosomes was not a result of changes in the potential of the plasma membrane. Therefore, new synthesized FGABAs 1 &3 were able to decrease exocytotic release of [3H]GABA from nerve terminals more effectively in comparison to pregabalin. Absence of unspecific side effects of FGABAs 1 &3 on the membrane potential makes these compounds perspective for medical application.

Skeletal ligament healing using the recombinant human amelogenin protein.

Injuries to ligaments are common, painful and debilitating, causing joint instability and impaired protective proprioception sensation around the joint. Healing of torn ligaments usually fails to take place, and surgical replacement or reconstruction is required. Previously, we showed that in vivo application of the recombinant human amelogenin protein (rHAM(+)) resulted in enhanced healing of the tooth-supporting tissues. The aim of this study was to evaluate whether amelogenin might also enhance repair of skeletal ligaments. The rat knee medial collateral ligament (MCL) was chosen to prove the concept. Full thickness tear was created and various concentrations of rHAM(+), dissolved in propylene glycol alginate (PGA) carrier, were applied to the transected MCL. 12 weeks after transection, the mechanical properties, structure and composition of transected ligaments treated with 0.5 µg/µl rHAM(+) were similar to the normal un-transected ligaments, and were much stronger, stiffer and organized than control ligaments, treated with PGA only. Furthermore, the proprioceptive free nerve endings, in the 0.5 µg/µl rHAM(+) treated group, were parallel to the collagen fibres similar to their arrangement in normal ligament, while in the control ligaments the free nerve endings were entrapped in the scar tissue at different directions, not parallel to the axis of the force. Four days after transection, treatment with 0.5 µg/µl rHAM(+) increased the amount of cells expressing mesenchymal stem cell markers at the injured site. In conclusion application of rHAM(+) dose dependently induced mechanical, structural and sensory healing of torn skeletal ligament. Initially the process involved recruitment and proliferation of cells expressing mesenchymal stem cell markers.

Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,ß-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

Neuroactivity of detonation nanodiamonds: dose-dependent changes in transporter-mediated uptake and ambient level of excitatory/inhibitory neurotransmitters in brain nerve terminals.

BACKGROUND: Nanodiamonds are one of the most perspective nano-sized particles with superb physical and chemical properties, which are mainly composed of carbon sp(3) structures in the core with sp(2) and disorder/defect carbons on the surface. The research team recently demonstrated neuromodulatory properties of carbon nanodots with other than nanodiamonds hybridization types, i.e., sp(2) hybridized graphene islands and diamond-like sp(3) hybridized elements. RESULTS: In this study, neuroactive properties of uncoated nanodiamonds produced by detonation synthesis were assessed basing on their effects on transporter-mediated uptake and the ambient level of excitatory and inhibitory neurotransmitters, glutamate and γ-aminobutyric acid (GABA), in isolated rat brain nerve terminals. It was shown that nanodiamonds in a dose-dependent manner attenuated the initial velocity of Na(+)-dependent transporter-mediated uptake and accumulation of L-[(14)C]glutamate and [(3)H]GABA by nerve terminals and increased the ambient level of these neurotransmitters. Also, nanodiamonds caused a weak reduction in acidification of synaptic vesicles and depolarization of the plasma membrane of nerve terminals. CONCLUSIONS: Therefore, despite different types of hybridization in nanodiamonds and carbon dots, they exhibit very similar effects on glutamate and GABA transport in nerve terminals and this common feature of both nanoparticles is presumably associated with their nanoscale size. Observed neuroactive properties of pure nanodiamonds can be used in neurotheranostics for simultaneous labeling/visualization of nerve terminals and modulation of key processes of glutamate- and GABAergic neurotransmission. In comparison with carbon dots, wider medical application involving hypo/hyperthermia, external magnetic fields, and radiolabel techniques can be perspective for nanodiamonds.

An alien divalent ion reveals a major role for Ca²âº buffering in controlling slow transmitter release.

Ca(2+)-dependent transmitter release occurs in a fast and in a slow phase, but the differential roles of Ca(2+) buffers and Ca(2+) sensors in shaping release kinetics are still controversial. Replacing extracellular Ca(2+) by Sr(2+) causes decreased fast release but enhanced slow release at many synapses. Here, we established presynaptic Sr(2+) uncaging and made quantitative Sr(2+)- and Ca(2+)-imaging experiments at the mouse calyx of Held synapse, to reveal the interplay between Ca(2+) sensors and Ca(2+) buffers in the control of fast and slow release. We show that Sr(2+) activates the fast, Synaptotagmin-2 (Syt2) sensor for vesicle fusion with sixfold lower affinity but unchanged high cooperativity. Surprisingly, Sr(2+) also activates the slow sensor that remains in Syt2 knock-out synapses with a lower efficiency, and Sr(2+) was less efficient than Ca(2+) in the limit of low concentrations in wild-type synapses. Quantitative imaging experiments show that the buffering capacity of the nerve terminal is markedly lower for Sr(2+) than for Ca(2+) (~5-fold). This, together with an enhanced Sr(2+) permeation through presynaptic Ca(2+) channels (~2-fold), admits a drastically higher spatially averaged Sr(2+) transient compared with Ca(2+). Together, despite the lower affinity of Sr(2+) at the fast and slow sensors, the massively higher amplitudes of spatially averaged Sr(2+) transients explain the enhanced late release. This also allows us to conclude that Ca(2+) buffering normally controls late release and prevents the activation of the fast release sensor by residual Ca(2+).

Central vagal afferent endings mediate reduction of food intake by melanocortin-3/4 receptor agonist.

Injection of the melanocortin-3/4 receptor agonist melanotan-II (MTII) into the nucleus of the solitary tract (NTS) produces rapid and sustained reduction of food intake. Melanocortin-4 receptors (MC4Rs) are expressed by vagal afferent endings in the NTS, but it is not known whether these endings participate in MTII-induced reduction of food intake. In experiments described here, we evaluated the contribution of central vagal afferent endings in MTII-induced reduction of food intake. Examination of rat hindbrain sections revealed that neuronal processes expressing immunoreactivity for the endogenous MC4R agonist α-melanoctyte-stimulating hormone course parallel and wrap around anterogradely labeled vagal afferent endings in the NTS and thus are aptly positioned to activate vagal afferent MC4Rs. Furthermore, MTII and endogenous MC4R agonists increased protein kinase A (PKA)-catalyzed phosphorylation of synapsin I in vagal afferent endings, an effect known to increase synaptic strength by enhancing neurotransmitter release in other neural systems. Hindbrain injection of a PKA inhibitor, KT5720, significantly attenuated MTII-induced reduction of food intake and the increase in synapsin I phosphorylation. Finally, unilateral nodose ganglion removal, resulting in degeneration of vagal afferent endings in the ipsilateral NTS, abolished MTII-induced synapsin I phosphorylation ipsilateral to nodose ganglion removal. Moreover, reduction of food intake following MTII injection into the NTS ipsilateral to nodose ganglion removal was significantly attenuated, whereas the response to MTII was not diminished when injected into the contralateral NTS. Altogether, our results suggest that reduction of food intake following hindbrain MC4R activation is mediated by central vagal afferent endings.

3,4-Methylenedioxypyrovalerone prevents while methylone enhances methamphetamine-induced damage to dopamine nerve endings: ß-ketoamphetamine modulation of neurotoxicity by the dopamine transporter.

Methylone, 3,4-methylenedioxypyrovalerone (MDPV), and mephedrone are psychoactive ingredients of 'bath salts' and their abuse represents a growing public health care concern. These drugs are cathinone derivatives and are classified chemically as ß-ketoamphetamines. Because of their close structural similarity to the amphetamines, methylone, MDPV, and mephedrone share most of their pharmacological, neurochemical, and behavioral properties. One point of divergence in their actions is the ability to cause damage to the CNS. Unlike methamphetamine, the ß-ketoamphetamines do not damage dopamine (DA) nerve endings. However, mephedrone has been shown to significantly accentuate methamphetamine neurotoxicity. Bath salt formulations contain numerous different psychoactive ingredients, and individuals who abuse bath salts also coabuse other illicit drugs. Therefore, we have evaluated the effects of methylone, MDPV, mephedrone, and methamphetamine on DA nerve endings. The ß-ketoamphetamines alone or in all possible two-drug combinations do not result in damage to DA nerve endings but do cause hyperthermia. MDPV completely protects against the neurotoxic effects of methamphetamine while methylone accentuates it. Neither MDPV nor methylone attenuates the hyperthermic effects of methamphetamine. The potent neuroprotective effects of MDPV extend to amphetamine-, 3,4-methylenedioxymethamphetamine-, and MPTP-induced neurotoxicity. These results indicate that ß-ketoamphetamine drugs that are non-substrate blockers of the DA transporter (i.e., MDPV) protect against methamphetamine neurotoxicity, whereas those that are substrates for uptake by the DA transporter and which cause DA release (i.e., methylone, mephedrone) accentuate neurotoxicity. METH (a) enters DA nerve endings via the DAT, causes leakage of DA into the cytoplasm and then into the synapse via DAT-mediated reverse transport. Methylone (METHY) and mephedrone (MEPH; b), like METH, are substrates for the DAT but release DA from cytoplasmic pools selectively. When METH is combined with METHY or MEPH (c), DA efflux and neurotoxicity are enhanced. MDPV (d), which is a non-substrate blocker of the DAT, prevents METH uptake and efflux of DA. Therefore, bath salts that are substrates for the DAT and release DA (METHY, MEPH) accentuate METH neurotoxicity, whereas those that are non-substrate blockers of the DAT (MDPV) are neuroprotective.

Rapid optical control of nociception with an ion-channel photoswitch.

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.

Comparative biochemical responses and antioxidant activities of the rabbit urinary bladder to whole grapes versus resveratrol.

The objective of this study is to compare the antioxidant activity of a whole-grape suspension with the antioxidant activity or pure resveratrol on the effect of hydrogen peroxide (H2O2) on malondialdehyde (MDA) generation, choline acetyltransferase (ChAT) activity, calcium ATPase activity, and sarcoendoplasmic reticular ATPase (SERCA) of the male rabbit urinary bladder. MDA was used as a model for the effect of H2O2 on lipid peroxidation. ChAT, SERCA, and calcium ATPase were evaluated based on their importance in urinary bladder physiology and pathology. Four male rabbit bladders were used. Each bladder was separated into muscle and mucosa, frozen under liquid nitrogen and stored at -80 °C for biochemical evaluation. The effect of H2O2 on the enzymes listed above was determined in the presence and absence of either resveratrol or a whole-grape suspension. (1) Resveratrol was significantly more effective than the grape suspension at protecting the bladder muscle and mucosa against peroxidation as quantitated by MDA formation. (2) The grape suspension was significantly more effective at protecting ChAT activity against oxidative stress of the muscle than resveratrol. (3) Neither the grape suspension nor resveratrol were particularly effective at protecting the bladder muscle or mucosa calcium ATPase or SERCA against oxidative stress. (4) ChAT was significantly more sensitive to oxidative stress than either calcium ATPase or SERCA. These data support the idea that the grape suspension protects the mitochondria and nerve terminals to a significantly greater degree than resveratrol which suggests that the activities of the grape suspension are due to the combination of active components found in the grape suspension and not just resveratrol alone.

Activity-dependent neurotrophin signaling underlies developmental switch of Ca2+ channel subtypes mediating neurotransmitter release.

At the nerve terminal, neurotransmitter release is triggered by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCCs). During postnatal development, VGCC subtypes in the nerve terminal switch at many synapses. In immature rodent cerebella, N-type and P/Q-type VGCCs mediate GABAergic neurotransmission from Purkinje cells (PCs) to deep nuclear cells, but as animals mature, neurotransmission becomes entirely P/Q-type dependent. We reproduced this developmental switch in rat cerebellar slice culture to address the underlying mechanism. Chronic block of cerebellar neuronal activity with tetrodotoxin (TTX) in slice culture, or in vivo, reversed the switch, leaving neurotransmission predominantly N-type channel-dependent. Brain-derived neurotrophic factor or neurotrophin-4 rescued this TTX effect, whereas pharmacological blockade of neurotrophin receptors mimicked the TTX effect. In PC somata, unlike in presynaptic terminals, TTX had no effect on the proportion of Ca(2+) channel subtype currents. We conclude that neuronal activity activates the neurotrophin-TrkB signaling pathway, thereby causing the N-to-P/Q channel switch in presynaptic terminals.

Co-administration of betulinic acid and methamphetamine causes toxicity to dopaminergic and serotonergic nerve terminals in the striatum of late adolescent rats.

Psychostimulant methamphetamine (METH) is toxic to striatal dopaminergic and serotonergic nerve terminals in adult, but not in the adolescent, brain. Betulinic acid (BA) and its derivatives are promising anti-HIV agents with some toxic properties. Many METH users, particularly young men, are HIV-positive; therefore, they might be treated with BA or its derivative for HIV infection. It is not known whether BA, or any of its derivatives, are neurotoxic in combination with METH in the adolescent brain. The present study investigated the effects of BA and binge METH in the striatum of late adolescent rats. BA or METH alone did not decrease the levels of dopaminergic or serotonergic markers in the striatum whereas BA and METH together decreased these markers in a BA dose-dependent manner. BA+METH also caused decreases in the levels of mitochondrial complex I in the same manner; BA alone only slightly decreased the levels of this enzyme in striatal synaptosomes. BA or METH alone increased cytochrome c. METH alone decreased parkin, increased complex II and striatal BA levels. These results suggest that METH in combination with BA can be neurotoxic to striatal dopaminergic and serotonergic nerve terminals in the late adolescent brain via mitochondrial dysfunction and parkin deficit. We report a synergistic neurotoxicity of betulinic acid (BA) and methamphetamine (METH) to monoaminergic terminals in the striatum of male late adolescent rats. BA contribution to the neurotoxicity is decreasing mitochondrial complex I whereas METH contribution is decreasing parkin and increasing brain concentration of BA. We propose that clinical use of BA in young male METH users can be neurotoxic.

Correlation between the effects of local and intracerebroventricular infusions of galanin on 5-HT release studied by microdialysis, and distribution of galanin and galanin receptors in prefrontal cortex, ventral hippocampus, amygdala, hypothalamus, and striatum of awake rats.

The neuropeptide galanin is implicated in regulation of affective behavior, including modulation of 5-HT signaling. Here, we investigated, by use of microdialysis in freely moving rats, the effects of intracerebral (i.c.) and intracerebroventricular (i.c.v.) infusions of galanin on basal extracellular 5-HT levels in medial prefrontal cortex (mPFC), CA1 area of ventral hippocampus (vHPC), central amygdaloid nucleus (CeA), ventromedial hypothalamic nucleus ventrolateral part (VMHvl), and ventromedial caudate putamen (CPu). These results were compared with a parallel immunohistochemical analysis of the distribution of galanin, 5-HT, and noradrenaline (NA) nerve terminals, and with data on galanin receptors. Galanin i.c.v. significantly decreased the 5-HT levels in mPFC to 79% and in vHPC to 72%. Local infusions of galanin caused a long-lasting decrease in 5-HT levels in vHPC to 88%, and a moderate decrease in CeA, whereas the 5-HT levels in mPFC significantly increased to 121%. These effects of i.c. galanin correlated well with the density of 5-HT and galanin nerve terminals and galanin receptors autoradiography in mPFC, vHPC, and CeA. No effects of i.c. or i.c.v. galanin on 5-HT levels were observed in CPu or VMHvl, in agreement with the low numbers of galanin-positive terminals and low/moderate galanin receptor density. Galanin was often found to coexist in NA, but could never be detected in 5-HT terminals. Together the results show a neuroanatomical correlation between the effects of galanin infusions on 5-HT release and distribution of galanin and its receptors, and that i.c.v. and i.c. administration can give opposite effects on 5-HT release.