Reduction In Transmitter Release Research Articles

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination.

During the development of the nervous system, there is an overproduction of neurons and synapses. Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening. We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway, at the neuromuscular junction, in the axonal development and synapse elimination process versus the synapse consolidation. The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination, in relation to other molecular pathways that we and others have found to regulate this process. In particular, we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors, coupled to downstream serine-threonine protein kinases A and C (PKA and PKC) and voltage-gated calcium channels, at different nerve endings in developmental competition. The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site, influence each other, and require careful studies to individualize the mechanisms of specific endings. We describe an activity-dependent balance (related to the extent of transmitter release) between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals. The downstream displacement of the PKA/PKC activity ratio to lower values, both in competing nerve terminals and at postsynaptic sites, plays a relevant role in controlling the elimination of supernumerary synapses. Finally, calcium entry through L- and P/Q- subtypes of voltage-gated calcium channels (both channels are present, together with the N-type channel in developing nerve terminals) contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination (the weakest in acetylcholine release and those that have already become silent). The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development. Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases.

Identifying the mechanism of action of the Kv7 channel opener, retigabine in the treatment of epilepsy.

Epilepsy is characterized by recurrent epileptic seizures caused by high levels of neuronal excitability in the brain. Voltage-sensitive K+ channels (Kv) of the Kv7 (KCNQ) family encoded by the KCNQ gene are involved in a wide range of cellular processes, i.e., KCNQ2 and KCNQ3 channels mediate M-currents to inhibit neuronal excitability and reduce transmitter release throughout the nervous system. Thus, as a positive allosteric modulator (or opener) of KCNQ channels, retigabine has been the only clinically approved anti-seizure medication that acts on the KCNQ channels. This review discusses the biochemical mechanisms about how retigabine acts on Kv7 channels, significance in neuronal pathophysiology, preclinical efficacy, and clinical stage of development. Additional efforts are being made to emphasize the possible benefits and drawbacks of retigabine compared to currently available medications for treatment-resistant epilepsy.

Activation of α-adrenoceptors depresses synaptic transmission of myelinated afferents and inhibits pathways mediating primary afferent depolarization (PAD) in the in vitro mouse spinal cord.

Somatosensory afferent transmission strength is controlled by several presynaptic mechanisms that reduce transmitter release at the spinal cord level. We focused this investigation on the role of α-adrenoceptors in modulating sensory transmission in low-threshold myelinated afferents and in pathways mediating primary afferent depolarization (PAD) of neonatal mouse spinal cord. We hypothesized that the activation of α-adrenoceptors depresses low threshold-evoked synaptic transmission and inhibits pathways mediating PAD. Extracellular field potentials (EFPs) recorded in the deep dorsal horn assessed adrenergic modulation of population monosynaptic transmission, while dorsal root potentials (DRPs) recorded at root entry zone assessed adrenergic modulation of PAD. We found that noradrenaline (NA) and the α1-adrenoceptor agonists phenylephrine and cirazoline depressed synaptic transmission (by 15, 14 and 22%, respectively). DRPs were also depressed by NA, phenylephrine and cirazoline (by 62, 30, and 64%, respectively), and by the α2-adrenoceptor agonist clonidine, although to a lower extent (20%). We conclude that NA depresses monosynaptic transmission of myelinated afferents onto deep dorsal horn neurons via α1-adrenoceptors and inhibits interneuronal pathways mediating PAD through the activation of α1- and α2-adrenoceptors. The functional significance of these modulatory actions in shaping cutaneous and muscle sensory information during motor behaviors requires further study.

Increasing Agrin Function Antagonizes Muscle Atrophy and Motor Impairment in Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a pediatric genetic disease, characterized by motor neuron (MN) death, leading to progressive muscle weakness, respiratory failure, and, in the most severe cases, to death. Abnormalities at the neuromuscular junction (NMJ) have been reported in SMA, including neurofilament (NF) accumulation at presynaptic terminals, immature and smaller than normal endplates, reduced transmitter release, and, finally, muscle denervation. Here we have studied the role of agrin in SMAΔ7 mice, the experimental model of SMAII. We observed a 50% reduction in agrin expression levels in quadriceps of P10 SMA mice compared to age-matched WT controls. To counteract such condition, we treated SMA mice from birth onwards with therapeutic agrin biological NT-1654, an active splice variant of agrin retaining synaptogenic properties, which is also resistant to proteolytic cleavage by neurotrypsin. Mice were analyzed for behavior, muscle and NMJ histology, and survival. Motor behavior was significantly improved and survival was extended by treatment of SMA mice with NT-1654. At P10, H/E-stained sections of the quadriceps, a proximal muscle early involved in SMA, showed that NT-1654 treatment strongly prevented the size decrease of muscle fibers. Studies of NMJ morphology on whole-mount diaphragm preparations revealed that NT-1654-treated SMA mice had more mature NMJs and reduced NF accumulation, compared to vehicle-treated SMA mice. We conclude that increasing agrin function in SMA has beneficial outcomes on muscle fibers and NMJs as the agrin biological NT-1654 restores the crosstalk between muscle and MNs, delaying muscular atrophy, improving motor performance and extending survival.

The essential oil of Lippia alba and its components affect Drosophila behavior and synaptic physiology.

Lippia alba is a flowering shrub in the verbena family and its essential oil (EO) is known for its sedative, antidepressant and analgesic properties. In the Amazon region of Brazil, it is used in aquaculture to anesthetize fish during transport. Many of the specialized metabolites found in EOs presumably evolved to protect plants from herbivores, especially insects. We used Drosophila to test the behavioral and physiological actions of this EO and its components. We found that a 150 min exposure to the EO vapors resulted in immobilization of adult flies. Gas chromatography-mass spectrometry identified the major components of the EO as the monoterpenes citral (59%), carvone (7%) and limonene (7%). Fly immobilization by the EO was due to citral and carvone, with citral producing more rapid effects than carvone. We tested whether the EO affected synaptic physiology by applying it to the larval neuromuscular junction. The EO delivered at 0.012% (v/v) produced over a 50% reduction in excitatory postsynaptic potential (EPSP) amplitude within 3-4 min. When the EO components were applied at 0.4 mmol l-1, citral and carvone produced a significant reduction in EPSP amplitude, with citral producing the largest effect. Measurement of miniature EPSP amplitudes demonstrated that citral produced over a 50% reduction in transmitter release. Calcium imaging experiments showed that citral produced about 30% reduction in presynaptic Ca2+ influx, which likely resulted in the decrease in transmitter release. Thus, the EO blocks synaptic transmission, largely due to citral, and this likely contributes to its behavioral effects.

Columnar architecture improves noise robustness in a model cortical network.

Cortical columnar architecture was discovered decades ago yet there is no agreed upon explanation for its function. Indeed, some have suggested that it has no function, it is simply an epiphenomenon of developmental processes. To investigate this problem we have constructed a computer model of one square millimeter of layer 2/3 of the primary visual cortex (V1) of the cat. Model cells are connected according to data from recent paired cell studies, in particular the connection probability between pyramidal cells is inversely proportional both to the distance separating the cells and to the distance between the preferred parameters (features) of the cells. We find that these constraints, together with a columnar architecture, produce more tightly clustered populations of cells when compared to the random architecture seen in, for example, rodents. This causes the columnar network to converge more quickly and accurately on the pattern representing a particular stimulus in the presence of noise, suggesting that columnar connectivity functions to improve pattern recognition in cortical circuits. The model also suggests that synaptic failure, a phenomenon exhibited by weak synapses, may conserve metabolic resources by reducing transmitter release at these connections that do not contribute to network function.

Loss of β2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.

Neuromuscular junctions from β2-laminin-deficient mice exhibit lower levels of calcium sensitivity. Loss of β2-laminin leads to a failure in switching from N- to P/Q-type voltage-gated calcium channel (VGCC)-mediated transmitter release that normally occurs with neuromuscular junction maturation. The motor nerve terminals from β2-laminin-deficient mice fail to up-regulate the expression of P/Q-type VGCCs clusters and down-regulate N-type VGCCs clusters, as they mature. There is decreased co-localisation of presynaptic specialisations in β2-laminin-deficient neuromuscular junctions as a consequence of lesser P/Q-type VGCC expression. These findings support the idea that β2-laminin is critical in the organisation and maintenance of active zones at the neuromuscular junction via its interaction with P/Q-type VGCCs, which aid in stabilisation of the synapse. β2-laminin is a key mediator in the differentiation and formation of the skeletal neuromuscular junction. Loss of β2-laminin results in significant structural and functional aberrations such as decreased number of active zones and reduced spontaneous release of transmitter. In vitro β2-laminin has been shown to bind directly to the pore forming subunit of P/Q-type voltage-gated calcium channels (VGCCs). Neurotransmission is initially mediated by N-type VGCCs, but by postnatal day 18 switches to P/Q-type VGCC dominance. The present study investigated the changes in neurotransmission during the switch from N- to P/Q-type VGCC-mediated transmitter release at β2-laminin-deficient junctions. Analysis of the relationship between quantal content and extracellular calcium concentrations demonstrated a decrease in the calcium sensitivity, but no change in calcium dependence at β2-laminin-deficient junctions. Electrophysiological studies on VGCC sub-types involved in transmitter release indicate N-type VGCCs remain the primary mediator of transmitter release at matured β2-laminin-deficient junctions. Immunohistochemical analyses displayed irregularly shaped and immature β2-laminin-deficient neuromuscular junctions when compared to matured wild-type junctions. β2-laminin-deficient junctions also maintained the presence of N-type VGCC clustering within the presynaptic membrane, which supported the functional findings of the present study. We conclude that β2-laminin is a key regulator in development of the NMJ, with its loss resulting in reduced transmitter release due to decreased calcium sensitivity stemming from a failure to switch from N- to P/Q-type VGCC-mediated synaptic transmission.

Serotonin, Dopamine and Noradrenaline Adjust Actions of Myelinated Afferents via Modulation of Presynaptic Inhibition in the Mouse Spinal Cord

Gain control of primary afferent neurotransmission at their intraspinal terminals occurs by several mechanisms including primary afferent depolarization (PAD). PAD produces presynaptic inhibition via a reduction in transmitter release. While it is known that descending monoaminergic pathways complexly regulate sensory processing, the extent these actions include modulation of afferent-evoked PAD remains uncertain. We investigated the effects of serotonin (5HT), dopamine (DA) and noradrenaline (NA) on afferent transmission and PAD. Responses were evoked by stimulation of myelinated hindlimb cutaneous and muscle afferents in the isolated neonatal mouse spinal cord. Monosynaptic responses were examined in the deep dorsal horn either as population excitatory synaptic responses (recorded as extracellular field potentials; EFPs) or intracellular excitatory postsynaptic currents (EPSCs). The magnitude of PAD generated intraspinally was estimated from electrotonically back-propagating dorsal root potentials (DRPs) recorded on lumbar dorsal roots. 5HT depressed the DRP by 76%. Monosynaptic actions were similarly depressed by 5HT (EFPs 54%; EPSCs 75%) but with a slower time course. This suggests that depression of monosynaptic EFPs and DRPs occurs by independent mechanisms. DA and NA had similar depressant actions on DRPs but weaker effects on EFPs. IC50 values for DRP depression were 0.6, 0.8 and 1.0 µM for 5HT, DA and NA, respectively. Depression of DRPs by monoamines was nearly-identical in both muscle and cutaneous afferent-evoked responses, supporting a global modulation of the multimodal afferents stimulated. 5HT, DA and NA produced no change in the compound antidromic potentials evoked by intraspinal microstimulation indicating that depression of the DRP is unrelated to direct changes in the excitability of intraspinal afferent fibers, but due to metabotropic receptor activation. In summary, both myelinated afferent-evoked DRPs and monosynaptic transmission in the dorsal horn are broadly reduced by descending monoamine transmitters. These actions likely integrate with modulatory actions elsewhere to reconfigure spinal circuits during motor behaviors.

Paclitaxel alters the evoked release of calcitonin gene-related peptide from rat sensory neurons in culture

Peripheral neuropathy (PN) is a debilitating and dose-limiting side effect of treatment with the chemotherapeutic agent, paclitaxel. Understanding the effects of paclitaxel on sensory neuronal function and the signaling pathways which mediate these paclitaxel-induced changes in function are critical for the development of therapies to prevent or alleviate the PN. The effects of long-term administration of paclitaxel on the function of sensory neurons grown in culture, using the release of the neuropeptide calcitonin gene-related peptide (CGRP) as an endpoint of sensory neuronal function, were examined. Dorsal root ganglion cultures were treated with low (10nM) and high (300nM) concentrations of paclitaxel for 1, 3, or 5days. Following paclitaxel treatment, the release of CGRP was determined using capsaicin, a TRPV1 agonist; allyl isothiocyanate (AITC), a TRPA1 agonist; or high extracellular potassium. The effects of paclitaxel on the release of CGRP were stimulant-, concentration-, and time-dependent. When neurons were stimulated with capsaicin or AITC, a low concentration of paclitaxel (10nM) augmented transmitter release, whereas a high concentration (300nM) reduced transmitter release in a time-dependent manner; however, when high extracellular potassium was used as the evoking stimulus, all concentrations of paclitaxel augmented CGRP release from sensory neurons. These results suggest that paclitaxel alters the function of sensory neurons in vitro, and suggest that the mechanisms by which paclitaxel alters neuronal function may include functional changes in TRP channel activity. The described in vitro model will facilitate future studies to identify the signaling pathways by which paclitaxel alters neuronal sensitivity.

Golgi meet Cajal: coupling and feedback at retinal photoreceptors

Photoreceptors not only transduce and relay light signals, they also receive a dazzling array of synaptic input from the retinal network including electrical and chemical synaptic signals from other photoreceptors, lateral feedback from horizontal cells, and long range feedback from interplexiform cells. In this issue of The Journal of PhysiologyGao et al. (2013) quantitatively tackle the interplay between some of these circuits. Gap junction coupling in the retina is pervasive and perplexing. It starts at the first cell layer, the photoreceptors, and is observed in every cell type as visual signals travel through this network. In a tissue designed for acuity, photoreceptor coupling could blur images and dampen signals. Instead, evidence indicates that coupling reduces noise and leads to an increase in both gain and dynamic range: an engineering marvel (Attwell et al. 1987). Coupling can be either homotypic (rod–rod, cone–cone) or heterotypic (rod–cone). Rods are high sensitivity, low resolution light detectors where strong homotypic coupling has clear advantages in improving dim light communication. Furthermore, heterotypic coupling allows rods to pass signals through the cone circuitry with a shift in sensitivity that expands the dynamic range of rod vision (Bloomfield & Dacheux, 2001). Gao et al. (2013) have now presented detailed measurements of this heterotypic coupling in salamander retina. Studies over several decades, often emanating from this laboratory, have made this animal a model system. In addition to the previously described strong heterotypic coupling of a subset (∼20%) of salamander rods, there is also coupling of variable strength with all cone subtypes. The coupling is linear and symmetrical. The rod–cone coupling is less than rod homotypic coupling, ranging from approximately half in the strongly coupled rod subset to about 10% in principal double cones. Surprisingly, the coupling is not under circadian control, unlike fish or rodents, although it is altered by background illumination. But in addition to sign-conserved coupling, Gao et al. (2013) explore the unidirectional ‘sign-inverting’ synapse from rods to cones (Attwell et al. 1983). They found that this feedback, generated by B-type horizontal cells, affects all but accessory cones (these receive A-type horizontal cell feedback). Surprisingly, the horizontal input to cones induces a chloride current, mediated by a glutamate transporter. A similar glutamate-gated channel has been described in bipolar cells, where it is an autoreceptor that suppresses transmitter release (Veruki et al. 2006). At the photoreceptor feedback synapse the mechanism may be more complex because it has to compete with glutamate release from photoreceptors and the potential stimulation of metabotropic receptors. The synaptic calcium channel that induces photoreceptor glutamate release is also under the direct control of horizontal cells. A further complication is that the chloride reversal potential sits slightly below the cone's dark membrane potential, instead of the more negative potential in bipolar cell terminals. Thus, while horizontal cell depolarization linked to surround responses hyperpolarizes and/or reduces transmitter release from cones, this B-type horizontal cell pathway may depolarize cones. The horizontal cell input would occur at light offset and oppose the effect of rod–cone coupling. As the authors suggest, horizontal cell lateral pathways are diverse and involve activation of multiple mechanisms. There is no shortage of candidates, including feedback by pH, GABA and ephaptic synapses (Thoreson & Mangel, 2012). And if unexpectedly glutamate is the transmitter, what role does this leave for GABA found in B-type horizontal cells? The report of Gao et al. (2013) fills in many of the gaps, but (Gao et al. 2013) the saga of horizontal cell feedback continues and you know that with the introduction of a new set of characters the story will not end soon.

High Frequency Burst Firing of Granule Cells Ensures Transmission at the Parallel Fiber to Purkinje Cell Synapse at the Cost of Temporal Coding

Cerebellar granule cells (GrCs) convey information from mossy fibers (MFs) to Purkinje cells (PCs) via their parallel fibers (PFs). MF to GrC signaling allows transmission of frequencies up to 1 kHz and GrCs themselves can also fire bursts of action potentials with instantaneous frequencies up to 1 kHz. So far, in the scientific literature no evidence has been shown that these high-frequency bursts also exist in awake, behaving animals. More so, it remains to be shown whether such high-frequency bursts can transmit temporally coded information from MFs to PCs and/or whether these patterns of activity contribute to the spatiotemporal filtering properties of the GrC layer. Here, we show that, upon sensory stimulation in both un-anesthetized rabbits and mice, GrCs can show bursts that consist of tens of spikes at instantaneous frequencies over 800 Hz. In vitro recordings from individual GrC-PC pairs following high-frequency stimulation revealed an overall low initial release probability of ~0.17. Nevertheless, high-frequency burst activity induced a short-lived facilitation to ensure signaling within the first few spikes, which was rapidly followed by a reduction in transmitter release. The facilitation rate among individual GrC-PC pairs was heterogeneously distributed and could be classified as either “reluctant” or “responsive” according to their release characteristics. Despite the variety of efficacy at individual connections, grouped activity in GrCs resulted in a linear relationship between PC response and PF burst duration at frequencies up to 300 Hz allowing rate coding to persist at the network level. Together, these findings support the hypothesis that the cerebellar granular layer acts as a spatiotemporal filter between MF input and PC output (D’Angelo and De Zeeuw, 2009).

Effects and Mechanisms of Action of Nitric Oxide on Transmitter Release in Mouse Motor Nerve Terminals

We examined the mechanisms of NO action on transmitter release in neuromuscular preparations of the mouse diaphragm muscle using a standard microelectrode technique. A donor of NO (SNAP, 100 μM) and a substrate for NO synthesis (L-arginine, 100 μM) reduced the evoked transmitter release from motor nerve endings. At the same time, SNAP did not change the frequency and amplitude of miniature end-plate potentials, while an inhibitor of NO synthase, L-NAME, exerted no effect on evoked and spontaneous transmitter release. Inhibition of soluble guanylate cyclase by ODQ (2.5 μM) abolished the effect of NO on the evoked transmitter release, while the elevation of cGMP-level by its membranepenetrating analog 8BrcGMP did not prevent such effect. The elevation of intracellular concentration of cAMP by 100 μM of its analog 8(4CPT) cAMP or inhibition of phosphodiesterase (PDE) by the action of 100 μM IBMX eliminated NO effects on transmitter release. It is concluded that NO activates soluble guanylate cyclase and intensifies the cGMP synthesis. Activation of the PDE II via an increase in the cGMP level with consequent reduction of the level of intracellular cAMP and decrease in the activity of PKA reduced transmitter release from mouse motor nerve ending.

Activation of metabotropic glutamate receptors improves the accuracy of coincidence detection by presynaptic mechanisms in the nucleus laminaris of the chick

Interaural time difference (ITD) is a major cue for localizing a sound source and is processed in the nucleus laminaris (NL) in birds. Coincidence detection (CD) is a crucial step for processing ITD and critically depends on the size and time course of excitatory postsynaptic potentials (EPSPs). Here, we investigated a role of metabotropic glutamate receptors (mGluRs) in the regulation of EPSP amplitude and CD in the NL of chicks. A non-specific agonist of mGluRs ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) reduced the amplitude and extent of depression of excitatory postsynaptic currents (EPSCs) during a stimulus train, while the paired pulse ratio and coefficient of variation of EPSC amplitude were increased. In contrast, the amplitudes of spontaneous EPSCs were not affected, but the frequency was reduced. Thus, the effects of t-ACPD were presynaptic and reduced the release of neurotransmitter from terminals in the NL. Expression of group II mGluRs was graded along the tonotopic axis and was stronger towards the low frequency region in the NL. Both group II (DCG-IV) and group III (l-AP4) specific agonists reduced EPSC amplitude by presynaptic mechanisms, and the reduction was larger in the low frequency region; however, we could not find any effects of group I-specific agonists on EPSCs. The reduced EPSP amplitude in DCG-IV improved CD. A specific antagonist of group II mGluRs (LY341495) increased the amplitude of both EPSCs and EPSPs and enhanced the depression during a stimulus train, indicating constitutive activation of mGluRs in the NL. These observations indicate that mGluRs may work as autoreceptors and regulate EPSP size to improve CD in the NL.

G-protein–mediated inhibition of the Trp channel TRPM1 requires the Gβγ dimer

ON bipolar cells are critical for the function of the ON pathway in the visual system. They express a metabotropic glutamate receptor (mGluR6) that, when activated, couples to the G(o) class of G protein. The channel that is primarily responsible for the synaptic response has been recently identified as the transient receptor potential cation channel subfamily M member 1 (TRPM1); TRPM1 is negatively coupled to the mGluR6/Go cascade such that activation of the cascade results in closure of the channel. Light indirectly opens TRPM1 by reducing transmitter release from presynaptic photoreceptors, resulting in a decrease in mGluR6 activation. Conversely, in the dark, binding of synaptic glutamate to mGluR6 inhibits TRPM1 current. Closure of TRPM1 by G-protein activation in the dark is a critical step in the process of ON bipolar cell signal transduction, but the precise pathway linking these two events is not understood. To address this question, we measured TRPM1 activity in retinal bipolar cells, in human ependymal melanocytes (HEMs) that endogenously express TRPM1, and in HEK293 cells transfected with TRPM1. Dialysis of the Gβγ subunit dimer, but not Gα(o), closed TRPM1 channels in every cell type that we tested. In addition, activation of an endogenous G-protein-coupled receptor pathway in HEK293 cells that releases Gβγ without activating Go protein also closed TRPM1 channels. These results suggest a model in which the Gβγ dimer that is released as a result of the dissociation from Gα(o) upon activation of mGluR6 closes the TRPM1 channel, perhaps via a direct interaction.

Sodium Channels as Targets for Volatile Anesthetics

The molecular mechanisms of modern inhaled anesthetics are still poorly understood although they are widely used in clinical settings. Considerable evidence supports effects on membrane proteins including ligand- and voltage-gated ion channels of excitable cells. Na+ channels are crucial to action potential initiation and propagation, and represent potential targets for volatile anesthetic effects on central nervous system depression. Inhibition of presynaptic Na+ channels leads to reduced neurotransmitter release at the synapse and could therefore contribute to the mechanisms by which volatile anesthetics produce their characteristic end points: amnesia, unconsciousness, and immobility. Early studies on crayfish and squid giant axon showed inhibition of Na+ currents by volatile anesthetics at high concentrations. Subsequent studies using native neuronal preparations and heterologous expression systems with various mammalian Na+ channel isoforms implicated inhibition of presynaptic Na+ channels in anesthetic actions at clinical concentrations. Volatile anesthetics reduce peak Na+ current (INa) and shift the voltage of half-maximal steady-state inactivation (h∞) toward more negative potentials, thus stabilizing the fast-inactivated state. Furthermore recovery from fast-inactivation is slowed, together with enhanced use-dependent block during pulse train protocols. These effects can depress presynaptic excitability, depolarization and Ca2+ entry, and ultimately reduce transmitter release. This reduction in transmitter release is more potent for glutamatergic compared to GABAergic terminals. Involvement of Na+ channel inhibition in mediating the immobility caused by volatile anesthetics has been demonstrated in animal studies, in which intrathecal infusion of the Na+ channel blocker tetrodotoxin increases volatile anesthetic potency, whereas infusion of the Na+ channels agonist veratridine reduces anesthetic potency. These studies indicate that inhibition of presynaptic Na+ channels by volatile anesthetics is involved in mediating some of their effects.

Effects of a bicyclic thymoleptic drug (LU 3-010) on neuroeffector function in rat isolated portal veins.

Abstract The effects of the thymoleptic drug, LU 3–010, on adrenergic transmitter release and reuptake and on effector cell function in rat isolated portal veins have been investigated. The contractile response to exogenous noradrenaline was potentiated by LU 3–010 and its concentration-effect curves after LU 3–010 10−7 g/ml were displaced to the left to a similar extent as had been found in chronically denervated preparations, indicating efficient blockade of the membrane pump mechanism. LU 3–010 10−7g/ml slightly potentiated the neurogenic response to electrical field stimulation, whereas 10−6 g/ml or more reduced the neural effector response in a concentration- dependent manner. The output of radioactive material per nerve impulse after incubation with 3H-NA was also studied. At 10−7 and 3 × 10−7 g/ml of LU 3–010 no significant change in transmitter overflow was observed. At 10−6 g/ml there was a decrease in the “fractional release”, suggesting that LU 3–010 concentrations of 10−6 g/ml or higher reduce transmitter release. Evidence is also given for a direct inhibitory effect of LU 3–010 on the smooth muscle.

GluK1 inhibits calcium dependent and independent transmitter release at associational/commissural synapses in area CA3 of the hippocampus

CA3 pyramidal cells receive three main excitatory inputs: the first one is the mossy fiber input, synapsing mainly on the proximal apical dendrites. Second, entorhinal cortex cells form excitatory connections with CA3 pyramidal cells via the perforant path in the stratum lacunosum moleculare. The third input involves the ipsi-and contralateral connections, termed the associational/commissural (A/C) pathway terminating in the stratum radiatum of CA3, thus forming a feedback loop within this region. Since this excitatory recurrent synapse makes the CA3 region extremely prone to seizure development, understanding the regulation of synaptic strength of this connection is of crucial interest. Several studies suggest that kainate receptors (KAR) play a role in the regulation of synaptic strength. Our aim was to characterize the influence of KAR on A/C synaptic transmission: application of ATPA, a selective agonist of the GluK1 KAR, depressed the amplitude fEPSP without affecting the size of the fiber volley. Blockade of GABA receptors had no influence on this effect, arguing against the influence of interneuronal KARs. Pharmacological and genetic deletion studies could show that this effect was selectively due to GluK1 receptor activation. Several lines of evidence, such as PPF changes, coefficient of variance-analysis and glutamate uncaging experiments strongly argue for a presynaptic locus of suppression. This is accompanied by an ATPA-mediated reduction in Ca(2+) influx at excitatory synaptic terminals, which is most likely mediated by a G-Protein dependent mechanism, as suggested by application of pertussis toxin. Finally, analysis of miniature EPSCs in the presence and absence of extracellular Ca(2+) suggest that presynaptic KAR can also reduce transmitter release downstream and therefore independent of Ca(2+) influx.

Voltage‐dependent κ‐opioid modulation of action potential waveform‐elicited calcium currents in neurohypophysial terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca(2+) channels results in less calcium influx into the terminals and presumably a reduction in transmitter release. In the neurohypophysis (NH), Ca(2+) channel kinetics, and the associated Ca(2+) influx, is primarily controlled by membrane voltage and can be modulated, in a voltage-dependent manner, by G-protein subunits interacting with voltage-gated calcium channels (VGCCs). In this series of experiments we test whether the kappa- and micro-opioid inhibition of Ca(2+) currents in NH terminals is voltage-dependent. Voltage-dependent relief of G-protein inhibition of VGCC can be achieved with either a depolarizing square pre-pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the kappa- and micro-receptors in neurohypophysial terminals. The kappa-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane delimited pathway. In contrast, micro-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, relief of kappa-opioid inhibition during a physiologic action potential (AP) burst stimulation indicates the possibility of activity-dependent modulation in vivo. Differences in the facilitation of Ca(2+) channels due to specific G-protein modulation during a burst of APs may contribute to the fine-tuning of Ca(2+)-dependent neuropeptide release in other CNS terminals, as well.

V-ATPase Membrane Sector Associates with Synaptobrevin to Modulate Neurotransmitter Release

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.

Voltage-Dependent Kappa Opioid Modulation of Calcium Currents Elicited by Action Potential Waveforms in Neurohypophysial Terminals

Release of neurotransmitter is activated by the influx of calcium. Inhibition of Ca2+ channels results in less calcium influx into the terminal and, presumably, a reduction in transmitter release. In the nerve terminals of the neurohypophysis, voltage-gated calcium channels (VGCC) are primarily controlled by membrane voltage and their activity can be modulated, in a voltage-dependent manner, by their interaction with G-protein subunits. Endogenous opioids also affect (inhibit) these calcium channels, upon binding to μ- and κ-receptors at the terminals.Voltage-dependent relief of G-protein inhibition of VGCC is achieved with either a depolarizing square pre-pulse or by action potential waveforms. Both protocols were tested in the presence and absence of opioid agonists targeting the μ- and κ-receptors. The κ-opioid VGCC inhibition is relieved by such pre-pulses, suggesting that this receptor is involved in a voltage-dependent membrane-delimited G-protein pathway. In contrast, μ-opioid inhibition of VGCC is not relieved by such pre-pulses, indicating a voltage-independent diffusible second-messenger signaling pathway. Furthermore, κ-opioid inhibition is also relieved during stimulation with action potential bursts with physiological characteristics. This indicates the possibility of activity-dependent modulation in vivo.Differences in the facilitation of Ca2+ channels due to specific G-protein modulation during a burst of action potentials may contribute to the fine-tuning of Ca2+-dependent neuropeptide release in other central nervous system synapses, as well. [Supported by NIH Grant NS29470].