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1.  Presynaptic inhibition of the release of multiple major central nervous system neurotransmitter types by the inhaled anaesthetic isoflurane 
BJA: British Journal of Anaesthesia  2012;110(4):592-599.
Background
Presynaptic effects of general anaesthetics are not well characterized. We tested the hypothesis that isoflurane exhibits transmitter-specific effects on neurotransmitter release from neurochemically and functionally distinct isolated mammalian nerve terminals.
Methods
Nerve terminals from adult male rat brain were prelabelled with [3H]glutamate and [14C]GABA (cerebral cortex), [3H]norepinephrine (hippocampus), [14C]dopamine (striatum), or [3H]choline (precursor of [3H]acetylcholine; striatum). Release evoked by depolarizing pulses of 4-aminopyridine (4AP) or elevated KCl was quantified using a closed superfusion system.
Results
Isoflurane at clinical concentrations (<0.7 mM; ∼2 times median anaesthetic concentration) inhibited Na+ channel-dependent 4AP-evoked release of the five neurotransmitters tested in a concentration-dependent manner. Isoflurane was a more potent inhibitor [expressed as IC50 (sem)] of glutamate release [0.37 (0.03) mM; P<0.05] compared with the release of GABA [0.52 (0.03) mM], norepinephrine [0.48 (0.03) mM], dopamine [0.48 (0.03) mM], or acetylcholine [0.49 (0.02) mM]. Inhibition of Na+ channel-independent release evoked by elevated K+ was not significant at clinical concentrations of isoflurane, with the exception of dopamine release [IC50=0.59 (0.03) mM].
Conclusions
Isoflurane inhibited the release of the major central nervous system neurotransmitters with selectivity for glutamate release, consistent with both widespread inhibition and nerve terminal-specific presynaptic effects. Glutamate release was most sensitive to inhibition compared with GABA, acetylcholine, dopamine, and norepinephrine release due to presynaptic specializations in ion channel expression, regulation, and/or coupling to exocytosis. Reductions in neurotransmitter release by volatile anaesthetics could contribute to altered synaptic transmission, leading to therapeutic and toxic effects involving all major neurotransmitter systems.
doi:10.1093/bja/aes448
PMCID: PMC3600942  PMID: 23213036
acetylcholine; γ-aminobutyric acid; anaesthetics; dopamine; exocytosis; glutamate; Na+ channels; nerve terminal; neurotransmitter release; norepinephrine
2.  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.
doi:10.3389/fphar.2012.00050
PMCID: PMC3316150  PMID: 22479247
sodium channels; volatile anesthetics; presynaptic; anesthetic mechanism
3.  Nicotinic receptor-evoked hippocampal norepinephrine release is highly sensitive to inhibition by isoflurane 
BJA: British Journal of Anaesthesia  2009;102(3):355-360.
Background
Inhaled anaesthetics (IAs) produce multiple dose-dependent behavioural effects including amnesia, hypnosis, and immobility in response to painful stimuli that are mediated by distinct anatomical, cellular, and molecular mechanisms. Amnesia is produced at lower anaesthetic concentrations compared with hypnosis or immobility. Nicotinic acetylcholine receptors (nAChRs) modulate hippocampal neural network correlates of memory and are highly sensitive to IAs. Activation of hippocampal nAChRs stimulates the release of norepinephrine (NE), a neurotransmitter implicated in modulating hippocampal synaptic plasticity. We tested the hypothesis that IAs disrupt hippocampal synaptic mechanisms critical to memory by determining the effects of isoflurane on NE release from hippocampal nerve terminals.
Methods
Isolated nerve terminals prepared from adult male Sprague–Dawley rat hippocampus were radiolabelled with [3H]NE and either [14C]GABA or [14C]glutamate and superfused at 37°C. Release evoked by a 2 min pulse of 100 µM nicotine or 5 µM 4-aminopyridine was evaluated in the presence or absence of isoflurane and/or selective antagonists.
Results
Nicotine-evoked NE release from rat hippocampal nerve terminals was nAChR- and Ca2+-dependent, involved both α7 and non-α7 subunit-containing nAChRs, and was partially dependent on voltage-gated Na+ channel activation based on sensitivities to various antagonists. Isoflurane inhibited nicotine-evoked NE release (IC50=0.18 mM) more potently than depolarization-evoked NE release (IC50=0.27 mM, P=0.014), consistent with distinct presynaptic mechanisms of IA action.
Conclusions
Inhibition of hippocampal nAChR-dependent NE release by subanaesthetic concentrations of isoflurane supports a role in IA-induced amnesia.
doi:10.1093/bja/aen387
PMCID: PMC2642653  PMID: 19189985
anaesthetics volatile, isoflurane; brain, anaesthesia, molecular effects; brain, hippocampus; ions, ion channels, ligand-gated; nerve, neurotransmitters
4.  Mechanisms Involved in the Nociception Triggered by the Venom of the Armed Spider Phoneutria nigriventer 
Background
The frequency of accidental spider bites in Brazil is growing, and poisoning due to bites from the spider genus Phoneutria nigriventer is the second most frequent source of such accidents. Intense local pain is the major symptom reported after bites of P. nigriventer, although the mechanisms involved are still poorly understood. Therefore, the aim of this study was to identify the mechanisms involved in nociception triggered by the venom of Phoneutria nigriventer (PNV).
Methodology/Principal Findings
Twenty microliters of PNV or PBS was injected into the mouse paw (intraplantar, i.pl.). The time spent licking the injected paw was considered indicative of the level of nociception. I.pl. injection of PNV produced spontaneous nociception, which was reduced by arachnid antivenin (ArAv), local anaesthetics, opioids, acetaminophen and dipyrone, but not indomethacin. Boiling or dialysing the venom reduced the nociception induced by the venom. PNV-induced nociception is not dependent on glutamate or histamine receptors or on mast cell degranulation, but it is mediated by the stimulation of sensory fibres that contain serotonin 4 (5-HT4) and vanilloid receptors (TRPV1). We detected a kallikrein-like kinin-generating enzyme activity in tissue treated with PNV, which also contributes to nociception. Inhibition of enzymatic activity or administration of a receptor antagonist for kinin B2 was able to inhibit the nociception induced by PNV. PNV nociception was also reduced by the blockade of tetrodotoxin-sensitive Na+ channels, acid-sensitive ion channels (ASIC) and TRPV1 receptors.
Conclusion/Significance
Results suggest that both low- and high-molecular-weight toxins of PNV produce spontaneous nociception through direct or indirect action of kinin B2, TRPV1, 5-HT4 or ASIC receptors and voltage-dependent sodium channels present in sensory neurons but not in mast cells. Understanding the mechanisms involved in nociception caused by PNV are of interest not only for better treating poisoning by P. nigriventer but also appreciating the diversity of targets triggered by PNV toxins.
Author Summary
Spiders of the Phoneutria genus live in Central and South America, where relevant envenomation cases have been reported in humans. The incidence of bite by spiders in Brazil has increased in recent years, with Phoneutria nigriventer being the second most important cause of such accidents (approximately 4,000 cases of envenomation in 2011). Pain is the primary local symptom of inoculation with Phoneutria nigriventer venom (PNV), but the mechanisms involved in pain induced by PNV are poorly understood. It is important to find effective treatments to alleviate this pain. This study examined the mechanisms involved in pain caused by PNV in a mouse model as well as the sensitivity of PNV-induced pain to clinically used analgesics. The results show that both the low- and high-molecular-weight components of PNV produce spontaneous nociception action via kinin B2, TRPV1, 5-HT4 or ASIC receptors and the voltage-gated Na+ channels present in sensory fibres. Moreover, PNV-triggered nociception could be alleviated by arachnid antivenin, local anaesthetics, opioids and atypical, but not typical, non-steroidal anti-inflammatory drugs. The elucidation of the mechanisms responsible for the nociception induced by PNV is of interest to not only better treat envenomation by P. nigriventer but also understand the diversity of targets triggered by PNV toxins.
doi:10.1371/journal.pntd.0002198
PMCID: PMC3636088  PMID: 23638210
5.  The general anaesthetic etomidate inhibits the excitability of mouse thalamocortical relay neurons by modulating multiple modes of GABAA receptor-mediated inhibition 
The European Journal of Neuroscience  2014;40(3):2487-2501.
Modulation of thalamocortical (TC) relay neuron function has been implicated in the sedative and hypnotic effects of general anaesthetics. Inhibition of TC neurons is mediated predominantly by a combination of phasic and tonic inhibition, together with a recently described ‘spillover’ mode of inhibition, generated by the dynamic recruitment of extrasynaptic γ-aminobutyric acid (GABA)A receptors (GABAARs). Previous studies demonstrated that the intravenous anaesthetic etomidate enhances tonic and phasic inhibition in TC relay neurons, but it is not known how etomidate may influence spillover inhibition. Moreover, it is unclear how etomidate influences the excitability of TC neurons. Thus, to investigate the relative contribution of synaptic (α1β2γ2) and extrasynaptic (α4β2δ) GABAARs to the thalamic effects of etomidate, we performed whole-cell recordings from mouse TC neurons lacking synaptic (α10/0) or extrasynaptic (δ0/0) GABAARs. Etomidate (3 μm) significantly inhibited action-potential discharge in a manner that was dependent on facilitation of both synaptic and extrasynaptic GABAARs, although enhanced tonic inhibition was dominant in this respect. Additionally, phasic inhibition evoked by stimulation of the nucleus reticularis exhibited a spillover component mediated by δ-GABAARs, which was significantly prolonged in the presence of etomidate. Thus, etomidate greatly enhanced the transient suppression of TC spike trains by evoked inhibitory postsynaptic potentials. Collectively, these results suggest that the deactivation of thalamus observed during etomidate-induced anaesthesia involves potentiation of tonic and phasic inhibition, and implicate amplification of spillover inhibition as a novel mechanism to regulate the gating of sensory information through the thalamus during anaesthetic states.
doi:10.1111/ejn.12601
PMCID: PMC4215602  PMID: 24773078
nucleus reticularis; phasic inhibition; spill-over inhibition; thalamus; tonic inhibition
6.  Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo 
eLife  2013;2:e00012.
Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol—consisting of pairing a postsynaptic AP with visually driven presynaptic inputs—modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course. Computational simulations could reproduce the observed subthreshold voltage changes only when presynaptic temporal jitter was included. Together this shows that STDP rules can modify output patterns of sensory neurons and the timing of single-APs plays a crucial role in sensory coding and plasticity.
DOI: http://dx.doi.org/10.7554/eLife.00012.001
eLife digest
Nerve cells, called neurons, are one of the core components of the brain and form complex networks by connecting to other neurons via long, thin ‘wire-like’ processes called axons. Axons can extend across the brain, enabling neurons to form connections—or synapses—with thousands of others. It is through these complex networks that incoming information from sensory organs, such as the eye, is propagated through the brain and encoded.
The basic unit of communication between neurons is the action potential, often called a ‘spike’, which propagates along the network of axons and, through a chemical process at synapses, communicates with the postsynaptic neurons that the axon is connected to. These action potentials excite the neuron that they arrive at, and this excitatory process can generate a new action potential that then propagates along the axon to excite additional target neurons. In the visual areas of the cortex, neurons respond with action potentials when they ‘recognize’ a particular feature in a scene—a process called tuning. How a neuron becomes tuned to certain features in the world and not to others is unclear, as are the rules that enable a neuron to change what it is tuned to. What is clear, however, is that to understand this process is to understand the basis of sensory perception.
Memory storage and formation is thought to occur at synapses. The efficiency of signal transmission between neurons can increase or decrease over time, and this process is often referred to as synaptic plasticity. But for these synaptic changes to be transmitted to target neurons, the changes must alter the number of action potentials. Although it has been shown in vitro that the efficiency of synaptic transmission—that is the strength of the synapse—can be altered by changing the order in which the pre- and postsynaptic cells are activated (referred to as ‘Spike-timing-dependent plasticity’), this has never been shown to have an effect on the number of action potentials generated in a single neuron in vivo. It is therefore unknown whether this process is functionally relevant.
Now Pawlak et al. report that spike-timing-dependent plasticity in the visual cortex of anaesthetized rats can change the spiking of neurons in the visual cortex. They used a visual stimulus (a bar flashed up for half a second) to activate a presynaptic cell, and triggered a single action potential in the postsynaptic cell a very short time later. By repeatedly activating the cells in this way, they increased the strength of the synaptic connection between the two neurons. After a small number of these pairing activations, presenting the visual stimulus alone to the presynaptic cell was enough to trigger an action potential (a suprathreshold response) in the postsynaptic neuron—even though this was not the case prior to the pairing.
This study shows that timing rules known to change the strength of synaptic connections—and proposed to underlie learning and memory—have functional relevance in vivo, and that the timing of single action potentials can change the functional status of a cortical neuron.
DOI: http://dx.doi.org/10.7554/eLife.00012.002
doi:10.7554/eLife.00012
PMCID: PMC3552422  PMID: 23359858
synaptic plasticity; STDP; visual cortex; circuits; in vivo; spiking patterns; rat
7.  General anaesthetic actions on ligand-gated ion channels 
The molecular mechanisms of general anaesthetics have remained largely obscure since their introduction into clinical practice just over 150 years ago. This review describes the actions of general anaesthetics on mammalian neurotransmitter-gated ion channels. As a result of research during the last several decades, ligand-gated ion channels have emerged as promising molecular targets for the central nervous system effects of general anaesthetics. The last 10 years have witnessed an explosion of studies of anaesthetic modulation of recombinant ligand-gated ion channels, including recent studies which utilize chimeric and mutated receptors to identify regions of ligand-gated ion channels important for the actions of general anaesthetics. Exciting future directions include structural biology and gene-targeting approaches to further the understanding of general anaesthetic molecular mechanisms.
PMCID: PMC2854026  PMID: 10487207
General anaesthesia; ligand-gated ion channels; GABA; glutamine; acetylcholine; glycine; serotonin; electrophysiology
8.  Nitric Oxide Modulation of GABAergic Synaptic Transmission in Mechanically Isolated Rat Auditory Cortical Neurons 
The auditory cortex (A1) encodes the acquired significance of sound for the perception and interpretation of sound. Nitric oxide (NO) is a gas molecule with free radical properties that functions as a transmitter molecule and can alter neural activity without direct synaptic connections. We used whole-cell recordings under voltage clamp to investigate the effect of NO on spontaneous GABAergic synaptic transmission in mechanically isolated rat auditory cortical neurons preserving functional presynaptic nerve terminals. GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in the A1 were completely blocked by bicuculline. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), reduced the GABAergic sIPSC frequency without affecting the mean current amplitude. The SNAP-induced inhibition of sIPSC frequency was mimicked by 8-bromoguanosine cyclic 3',5'-monophosphate, a membrane permeable cyclic-GMP analogue, and blocked by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a specific NO scavenger. Blockade of presynaptic K+ channels by 4-aminopyridine, a K+ channel blocker, increased the frequencies of GABAergic sIPSCs, but did not affect the inhibitory effects of SNAP. However, blocking of presynaptic Ca2+ channels by Cd2+, a general voltage-dependent Ca2+ channel blocker, decreased the frequencies of GABAergic sIPSCs, and blocked SNAP-induced reduction of sIPSC frequency. These findings suggest that NO inhibits spontaneous GABA release by activation of cGMP-dependent signaling and inhibition of presynaptic Ca2+ channels in the presynaptic nerve terminals of A1 neurons.
doi:10.4196/kjpp.2009.13.6.461
PMCID: PMC2802307  PMID: 20054493
Auditory cortex; Nitric oxide; Synaptic transmission; GABA
9.  Anaesthetic Tricaine Acts Preferentially on Neural Voltage-Gated Sodium Channels and Fails to Block Directly Evoked Muscle Contraction 
PLoS ONE  2014;9(8):e103751.
Movements in animals arise through concerted action of neurons and skeletal muscle. General anaesthetics prevent movement and cause loss of consciousness by blocking neural function. Anaesthetics of the amino amide-class are thought to act by blockade of voltage-gated sodium channels. In fish, the commonly used anaesthetic tricaine methanesulphonate, also known as 3-aminobenzoic acid ethyl ester, metacaine or MS-222, causes loss of consciousness. However, its role in blocking action potentials in distinct excitable cells is unclear, raising the possibility that tricaine could act as a neuromuscular blocking agent directly causing paralysis. Here we use evoked electrical stimulation to show that tricaine efficiently blocks neural action potentials, but does not prevent directly evoked muscle contraction. Nifedipine-sensitive L-type Cav channels affecting movement are also primarily neural, suggesting that muscle Nav channels are relatively insensitive to tricaine. These findings show that tricaine used at standard concentrations in zebrafish larvae does not paralyse muscle, thereby diminishing concern that a direct action on muscle could mask a lack of general anaesthesia.
doi:10.1371/journal.pone.0103751
PMCID: PMC4121177  PMID: 25090007
10.  Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+ Channel Function 
Anesthesiology  2009;110(3):582-590.
Background
Inhibition of voltage-gated Na+ channels (Nav) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane and desflurane) on Nav1.4, a well characterized pharmacological model for Nav effects.
Methods
Na+ currents (INa) from rat Nav1.4 α-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording.
Results
Halogenated inhaled anesthetics reversibly inhibited Nav1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equi-anesthetic concentrations, peak INa was inhibited with a rank order of desflurane > halothane ≈ enflurane > isoflurane ≈ sevoflurane from a physiological holding potential (−80 mV). This suggests that the contribution of Na+ channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (−120 mV), peak INa was inhibited with a rank order of potency for tonic inhibition of peak INa of halothane > isoflurane ≈ sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane.
Conclusions
Five halogenated inhaled anesthetics all inhibit a voltage-gated Na+ channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na+ channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.
doi:10.1097/ALN.0b013e318197941e
PMCID: PMC2699670  PMID: 19225394
11.  Regional differences in the effects of isoflurane on neurotransmitter release 
Neuropharmacology  2011;61(4):699-706.
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 [3H]glutamate and [14C]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 Ca2+ channel-mediated presynaptic effects. Isoflurane completely inhibited total 4AP-evoked glutamate release (IC50=0.42 ± 0.03 mM) more potently than GABA release (IC50=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 Ca2+ entry. Isoflurane inhibited Na+ channel-mediated (tetrodotoxin-sensitive) 4AP-evoked glutamate release (IC50=0.30 ± 0.03 mM) more potently than GABA release (IC50=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.
doi:10.1016/j.neuropharm.2011.05.013
PMCID: PMC3130078  PMID: 21651920
Na+ channels; glutamate; GABA; nerve terminal; tetrodotoxin; rat
12.  Dominance of P/Q-type calcium channels in depolarization-induced presynaptic fm dye release in cultured hippocampal neurons☆ 
Neuroscience  2013;253(100):330-340.
Highlights
•We analyzed depolarization-induced synaptic FM dye release in hippocampal neurons.•We pharmacologically isolated the contribution of voltage-gated Ca2+ channels.•85% of synapses utilize N- and P/Q-type channels, 15% only P/Q-type channels.•In both groups of synapses release kinetics are determined by P/Q-type channels.•We propose a more direct coupling of P/Q-type channels to synaptic release.
Neurotransmitter release probability is related by high power to the local concentration of calcium in presynaptic terminals, which in turn is controlled by voltage-gated calcium channels. P/Q- and N-type channels trigger synaptic transmission in the majority of neurons of the central nervous system. However, whether and under which conditions both channel types act cooperatively or independently is still insufficiently understood. Previous studies suggested either a dominance of N- or P/Q-type channels, or a synergistic action of both channels, depending on the experimental paradigms. Thus, to provide insight into the properties of neurotransmitter release in cultured mouse hippocampal neurons, we used quantitative analysis of FM dye release from presynaptic boutons induced by high potassium membrane depolarization. Increasing extracellular potassium concentrations revealed a sigmoid dependence of FM dye release to the stimulation strength. Individual and combined application of the P/Q- and N-type channel-specific blockers ω-agatoxin-IVA and ω-conotoxin-GVIA, respectively, allowed us to specifically isolate the contribution of both channel types to release triggered with 40 mM KCl. Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels. Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels. Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.
doi:10.1016/j.neuroscience.2013.08.052
PMCID: PMC3824072  PMID: 24012836
A, release amplitude; Aga, ω-agatoxin-IVA; CaV, voltage-gated Ca2+ channel; CTx, ω-conotoxin GVIA; DIV, days in vitro; [K+], extracellular potassium concentration; Rf, fractional release; τ, release time constant; voltage-gated Ca2+ channels; synapse function; N-type; P/Q-type; neurotransmitter release; calcium channel physiology
13.  Presynaptic CaV3.2 channels regulate excitatory neurotransmission in nociceptive dorsal horn neurons 
The Journal of Neuroscience  2012;32(27):9374-9382.
It is generally accepted that presynaptic transmitter release is mainly regulated by subtypes of neuronal high-voltage-activated (HVA) Ca2+ channels. Here for the first time, we examined the role of T-type Ca2+ channels (T-channels) in synaptic transmission in the dorsal horn (DH) of the spinal cord using patch-clamp recordings from acute spinal cord preparations from both rat and mouse. We found that selective pharmacological antagonism of T-channels inhibited spontaneous synaptic release of glutamate in superficial laminae I-II of the DH, while GABA release was spared. We found similar effect in identified nociceptive projection neurons of lamina I of the DH, but not in inhibitory DH interneurons. In comparison, antagonism of T-channels did not affect excitatory transmission in deeper non-nociceptive DH laminae. Furthermore, we used isoform-specific agents, knockout mice and immunohistochemistry to specifically implicate presynaptic CaV3.2 channels. We also used an animal model of painful diabetic neuropathy to demonstrate that blocking T-channels in superficial DH neurons suppressed spontaneous excitatory synaptic transmission in diabetic rats in greater degree than in healthy age-matched animals. These studies provide previously unknown information regarding the role of presynaptic T-channels in nociceptive signaling in the spinal cord.
doi:10.1523/JNEUROSCI.0068-12.2012
PMCID: PMC3398424  PMID: 22764245
glutamate; dorsal horn; presynaptic mechanisms; low-threshold calcium channel; nociception
14.  Calcium-sensing receptor activation depresses synaptic transmission 
At excitatory synapses, decreases in cleft [Ca] arising from activity-dependent transmembrane Ca flux reduce the probability of subsequent transmitter release. Intense neural activity, induced by physiological and pathological stimuli, disturb the external microenvironment reducing extracellular [Ca] ([Ca]o) and thus may impair neurotransmission. Increases in [Ca]o activate the extracellular calcium sensing receptor (CaSR) which in turn inhibits non-selective cation channels (NSCC) at the majority of cortical nerve terminals. This pathway may modulate synaptic transmission by attenuating the impact of decreases in [Ca]o on synaptic transmission. Using patch-clamp recording from isolated cortical terminals, cortical neuronal pairs and isolated neuronal soma we examined the modulation of synaptic transmission by CaSR. Excitatory postsynaptic currents were increased on average by 88% in reduced affinity CaSR-mutant (CaSR−/−) neurons compared to wild-type. Variance-mean analysis indicates that the enhanced synaptic transmission was due largely to an increase in average probability of release (0.27 vs 0.46 for wild-type vs CaSR−/− pairs) with little change in quantal size (23 ± 4 pA vs 22 ± 4 pA) or number of release sites (11 vs 13). In addition, the CaSR agonist spermidine reduced synaptic transmission and increased paired-pulse depression at physiological [Ca]o. Spermidine did not affect quantal size, consistent with a presynaptic mechanism of action, nor did it affect voltage-activated Ca channel currents. In summary, reduced CaSR function enhanced synaptic transmission and CaSR stimulation had the opposite effect. Thus CaSR provides a mechanism that may compensate for the fall in release probability that accompanies decreases in [Ca]o .
doi:10.1523/JNEUROSCI.4134-08.2008
PMCID: PMC2761683  PMID: 19005071
calcium; GPCR; Depression; Synaptic plasticity; synaptic transmission; Synaptic vesicle release; Synaptosome; Extracellular; Receptor
15.  Are anaesthetics toxic to the brain? 
It has been assumed that anaesthetics have minimal or no persistent effects after emergence from anaesthesia. However, general anaesthetics act on multiple ion channels, receptors, and cell signalling systems in the central nervous system to produce anaesthesia, so it should come as no surprise that they also have non-anaesthetic actions that range from beneficial to detrimental. Accumulating evidence is forcing the anaesthesia community to question the safety of general anaesthesia at the extremes of age. Preclinical data suggest that inhaled anaesthetics can have profound and long-lasting effects during key neurodevelopmental periods in neonatal animals by increasing neuronal cell death (apoptosis) and reducing neurogenesis. Clinical data remain conflicting on the significance of these laboratory data to the paediatric population. At the opposite extreme in age, elderly patients are recognized to be at an increased risk of postoperative cognitive dysfunction (POCD) with a well-recognized decline in cognitive function after surgery. The underlying mechanisms and the contribution of anaesthesia in particular to POCD remain unclear. Laboratory models suggest anaesthetic interactions with neurodegenerative mechanisms, such as those linked to the onset and progression of Alzheimer's disease, but their clinical relevance remains inconclusive. Prospective randomized clinical trials are underway to address the clinical significance of these findings, but there are major challenges in designing, executing, and interpreting such trials. It is unlikely that definitive clinical studies absolving general anaesthetics of neurotoxicity will become available in the near future, requiring clinicians to use careful judgement when using these profound neurodepressants in vulnerable patients.
doi:10.1093/bja/aer122
PMCID: PMC3159425  PMID: 21616941
anaesthesia, general; Alzheimer's disease; neurobehavioural manifestations; postoperative complications
16.  Xenon inhibits excitatory but not inhibitory transmission in rat spinal cord dorsal horn neurons 
Molecular Pain  2010;6:25.
Background
The molecular targets for the promising gaseous anaesthetic xenon are still under investigation. Most studies identify N-methyl-D-aspartate (NMDA) receptors as the primary molecular target for xenon, but the role of α-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionic acid (AMPA) receptors is less clear. In this study we evaluated the effect of xenon on excitatory and inhibitory synaptic transmission in the superficial dorsal horn of the spinal cord using in vitro patch-clamp recordings from rat spinal cord slices. We further evaluated the effects of xenon on innocuous and noxious stimuli using in vivo patch-clamp method.
Results
In vitro, xenon decreased the amplitude and area under the curve of currents induced by exogenous NMDA and AMPA and inhibited dorsal root stimulation-evoked excitatory postsynaptic currents. Xenon decreased the amplitude, but not the frequency, of miniature excitatory postsynaptic currents. There was no discernible effect on miniature or evoked inhibitory postsynaptic currents or on the current induced by inhibitory neurotransmitters. In vivo, xenon inhibited responses to tactile and painful stimuli even in the presence of NMDA receptor antagonist.
Conclusions
Xenon inhibits glutamatergic excitatory transmission in the superficial dorsal horn via a postsynaptic mechanism. There is no substantial effect on inhibitory synaptic transmission at the concentration we used. The blunting of excitation in the dorsal horn lamina II neurons could underlie the analgesic effect of xenon.
doi:10.1186/1744-8069-6-25
PMCID: PMC2873505  PMID: 20444263
17.  Slob, a Slowpoke channel–binding protein, modulates synaptic transmission 
The Journal of General Physiology  2011;137(2):225-238.
Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel–binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission.
doi:10.1085/jgp.201010439
PMCID: PMC3032372  PMID: 21282401
18.  Free-energy Landscapes of Ion-channel Gating Are Malleable: changes in the number of bound ligands are accompanied by changes in the location of the transition state in acetylcholine-receptor channels† 
Biochemistry  2003;42(50):14977-14987.
Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing (‘gating’) a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed ⇌ open equilibrium constant because the ion-impermeable → ion-permeable transition of the ion pathway is accompanied by a low → high affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di-) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonist-binding sites and propagates towards the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed ⇌ open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating.
The muscle acetylcholine receptor channel (AChR)1 is the neurotransmitter-gated ion channel that mediates neuromuscular synaptic transmission in vertebrates (1). Although the structure of this large pentameric transmembrane protein (∼470 residues per subunit) is not known with atomic resolution, a wealth of structural information exists, mainly from mutational studies, affinity labeling, chemical modification of specific residues, electron microscopy, and crystallography (reviewed in ref. 2). As is the case of any other allosteric protein, the dynamic behavior of this receptor-channel can be understood in the framework of thermodynamic cycles, with conformational changes and ligand-binding events as the elementary steps (3-5). Thus, the AChR can adopt a variety of different conformations that can interconvert (closed, open, and desensitized ‘states’), and each conformation has a distinct ligand-binding affinity (low affinity in the closed state and high affinity in the open and desensitized states) and a particular ‘catalytic efficiency’ (ion-impermeable in the closed and desensitized states, and ion-permeable in the open state). To meet the physiological requirement of a small closed ⇌ open (‘gating’) equilibrium constant for the unliganded receptor, and a large gating equilibrium constant for the ACh-diliganded receptor, the affinity of the AChR for ACh must be higher in the open than in the closed conformation (4-6). This follows from the notion that the equilibrium constants governing the different reaction steps (ligand binding and gating) of these cyclic reaction schemes are constrained by the principle of detailed balance.
Hence, irrespective of whether the receptor is diliganded, monoliganded or unliganded, two changes must take place in going from the closed state (low ligand affinity and ion-impermeable) to the open state (high ligand affinity and ion-permeable): a) the pore becomes permeable to ions, and b) the transmitter-binding sites, some 50 Å away from the pore domain (7), increase their affinity for the ligand (with the reverse changes taking place during closing). The apparent lack of stable intermediates between the closed and open conformations, inferred from kinetic modeling of the diliganded-gating reaction (8), suggests that these two changes occur as a result of a one-step, global conformational change. The question, then, arises as to whether this concerted conformational change proceeds synchronously (i.e., every residue of the protein moves ‘in unison’) or asynchronously (i.e., following a sequence of events; ref. 9) and, if the latter were the case, whether multiple, few, or just one sequence of events is actually traversed by the channel to ‘connect’ the end states.
Analysis of the correlation between rate and equilibrium constants of gating in diliganded AChRs has allowed us to address some of these issues by probing the structure of the transition state (8, 10-12), that is, the intermediate species between the end states of a one-step reaction that can be most easily studied. Interpretation of these results in the framework of the classical rate-equilibrium free-energy relationships of physical organic chemistry (13, 14), revealed that AChR diliganded gating is a highly asynchronous reaction, and suggested that the transition-state ensemble is quite homogeneous, as if the crossing of the energy barrier were confined to a narrow pass at the top of the energy landscape. In the opening direction, the conformational rearrangement that leads to the low-to-high affinity change at the extracellular binding sites precedes the conformational rearrangement of the pore that renders the channel ion-permeable. This propagated global conformational change, which we have referred to as a ‘conformational wave’ (11), must reverse during channel closing so that closing starts at the pore and propagates all the way to the binding sites.
It is not at all obvious why the diliganded-gating conformational change starts at the binding sites when the channel opens, nor even why the conformational change propagates at all through the receptor, instead of taking place synchronously throughout the protein. Is there any correlation between the location of the domain that binds agonist and the location of the initiation site for the opening conformational change? Could the latter have started from the intracellular end of the pore, for example, and have propagated to the (extracellular) transmitter-binding sites? What difference does it make to be liganded or unliganded as far as the mechanism of the gating conformational change is concerned? To address these issues, I set out to explore the mechanism of gating in unliganded AChRs by probing the structure of the corresponding transition state using kinetic measurements, site-directed mutagenesis, and the concepts of rate-equilibrium free-energy relationships and Φ-value analysis.
Briefly, a Φ-value can be assigned to any position in the protein by estimating the slope of a ‘Brönsted plot’2 [log (gating rate constant) versus log (gating equilibrium constant)] where each point corresponds to a different amino-acid substitution at that given position. More coarsegrained Φ-values can also be obtained by using different agonists or different transmembrane potentials, for example, as a means of altering the rate and equilibrium constants of gating. Very often, rate-equilibrium plots are linear, and 0 < Φ < 1. A value of Φ = 0 suggests that the position in question (in the case of a mutation series) experiences a closed-state-like environment at the transition state whereas a value of Φ = 1 suggests an open-state-like environment. A fractional Φ-value suggests an environment that is intermediate between those experienced in the closed and open states (16).
Earlier results indicated that the Φ-values obtained by varying the transmembrane potential are different in diliganded and unliganded AChRs. These Φ-values, which are a measure of the closed-state-like versus open-state-like character of the channel’s voltage-sensing elements at the transition state, are 0.070 ± 0.060 in diliganded receptors (17), and 1.025 ± 0.053 in unliganded AChRs (11, 18). The present study reveals that residues at the transmitter-binding sites (Figure 1), the extracellular loop that links the second (M2) and third (M3) transmembrane segments (M2-M3 linker), and the upper and lower half of M2, which during diliganded gating have Φ-values of ∼1 (ref. 11), ∼0.7 (ref. 10), ∼0.35 (refs 8, 11, 12), and ∼0 (ref. 12), respectively, have also Φ-values very close to 1 during unliganded gating. This generalized shift in Φ-values suggests that the diliganded → unliganded perturbation deforms the energy landscape of gating in such a way that the ‘new’ transition state occurs very close to the open state, to such an extent that all tested positions experience an open-state-like environment at the transition state of unliganded gating. Thus, the transition state occurs so ‘late’ (i.e., so close to the open state) that its inferred structure does not provide any clues as to the intermediate stages of this reaction.
Hence, the mechanism of unliganded gating remains obscure. The change in the position of the transition state along a reaction coordinate, as a result of perturbations to the energy landscape, is a very well known phenomenon in organic chemistry (e.g., refs 20-26), and protein folding (e.g., refs 27-34). In this paper, I show that this phenomenon can also take place in the case of allosteric transitions and, therefore, that the structure of the transition state of a global conformational change need not be fixed; rather, it can change depending on the experimental conditions.
doi:10.1021/bi0354334
PMCID: PMC1463891  PMID: 14674774
19.  Differential Occurrence of Reluctant Openings in G-Protein–Inhibited N- and P/Q-Type Calcium Channels 
The Journal of General Physiology  2000;115(2):175-192.
Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel “slope” analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the “willing–reluctant” model, in which binding of G-protein βγ subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca2+ entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.
PMCID: PMC2217198  PMID: 10653895
α1A; α1B; channel modulation; heterologous expression; short-term synaptic plasticity
20.  The GAD-given Right of Dentate Gyrus Granule Cells to Become GABAergic 
Epilepsy Currents  2002;2(5):143-145.
Janus, the ancient Roman God of Gates and Doors had two faces: one looked into the past, and the other, into the future. Do neurons possess a Janus face when it comes to neurotransmitters, or a given neuron is to be forever solely γ-aminobutyric acid (GABA) ergic, glutamatergic, dopaminergic, peptidergic, or YOURPREFERREDTRANSMITTERergic? The answer is that the terminals of many neurons are homes to even more than two neurotransmitters. All this in spite of the “one neuron–one transmitter” usual misinterpretation of Sir Henry Hallett Dale's postulate, originally meant to indicate that a metabolic process taking place in the cell body can influence all processes of the same neuron. A large variety of neurons in the CNS, many of them GABAergic, produce and release chemicals that satisfy some of the criteria used to define neurotransmitters. The usual scenario for a dual-transmitter terminal is that the fast-acting transmitter such as GABA or glutamate is stored in regular synaptic vesicles, whereas a neuropeptide is stored in dense core vesicles 1. The vesicular zinc found in many glutamatergic terminals also may be considered to be a second neurotransmitter, based on its vesicular packaging with the aid of a specific vesicular transporter, and its postsynaptic actions through high-affinity binding sites and permeation through certain channels 2. Whenever a “fast” and a “slow” neurotransmitter are present in the same presynaptic terminal, it is customary to assume that their release can be differentially regulated 1. There is little convincing experimental support for this phenomenon in the mammalian CNS. The coexistence of two “fast” neurotransmitters in the same terminal is less frequent, but not unheard of. In neonatal sympathetic neurons cocultured with cardiac myocytes, norepinephrine and acetylcholine coexist and have opposite actions on the cardiac muscle cells 3. Very recently we learned that brain-derived neurotrophic factor acting at the low-affinity neurotrophin receptor p75NTR, perhaps as part of a programmed developmental switch, can convert the phenotype of the sympathetic neuron from noradrenergic to cholinergic 4. Other examples of two fast neurotransmitters released from the same neuron include GABA and glycine in interneurons of the spinal cord 5 and glutamate and dopamine in ventral midbrain dopamine neurons 6. Of all CNS neurons, the granule cells of the dentate gyrus appear to be the champions of neurotransmitter colocalization: glutamate, enkephalin, dynorphin, zinc, and finally GABA 2, 7, 8, 9. With this many transmitters in a single neuron, there are probably different ways in which they can be released. Dynorphin and other opioid peptides can be released directly from the dendrites to inhibit excitatory transmission 8. A similar mechanism may take place for GABA, as described in cortical GABAergic neurons 10.
doi:10.1046/j.1535-7597.2002.00053.x
PMCID: PMC321043  PMID: 15309121
21.  Anaesthetic Impairment of Immune Function Is Mediated via GABAA Receptors 
PLoS ONE  2011;6(2):e17152.
Background
GABAA receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs [1]. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear [2]. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die [3]–[6]. As many anaesthetics act via GABAA receptors [7], the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients.
Principal Findings
We demonstrate, using RT-PCR, that monocytes express GABAA receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABAA receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABAA receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin.
Significance
Our results show that functional GABAA receptors are present on monocytes with properties similar to CNS GABAA receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABAA receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABAA receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.
doi:10.1371/journal.pone.0017152
PMCID: PMC3044756  PMID: 21390329
22.  Dynamic Causal Models and Physiological Inference: A Validation Study Using Isoflurane Anaesthesia in Rodents 
PLoS ONE  2011;6(8):e22790.
Generative models of neuroimaging and electrophysiological data present new opportunities for accessing hidden or latent brain states. Dynamic causal modeling (DCM) uses Bayesian model inversion and selection to infer the synaptic mechanisms underlying empirically observed brain responses. DCM for electrophysiological data, in particular, aims to estimate the relative strength of synaptic transmission at different cell types and via specific neurotransmitters. Here, we report a DCM validation study concerning inference on excitatory and inhibitory synaptic transmission, using different doses of a volatile anaesthetic agent (isoflurane) to parametrically modify excitatory and inhibitory synaptic processing while recording local field potentials (LFPs) from primary auditory cortex (A1) and the posterior auditory field (PAF) in the auditory belt region in rodents. We test whether DCM can infer, from the LFP measurements, the expected drug-induced changes in synaptic transmission mediated via fast ionotropic receptors; i.e., excitatory (glutamatergic) AMPA and inhibitory GABAA receptors. Cross- and auto-spectra from the two regions were used to optimise three DCMs based on biologically plausible neural mass models and specific network architectures. Consistent with known extrinsic connectivity patterns in sensory hierarchies, we found that a model comprising forward connections from A1 to PAF and backward connections from PAF to A1 outperformed a model with forward connections from PAF to A1 and backward connections from A1 to PAF and a model with reciprocal lateral connections. The parameter estimates from the most plausible model indicated that the amplitude of fast glutamatergic excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) behaved as predicted by previous neurophysiological studies. Specifically, with increasing levels of anaesthesia, glutamatergic EPSPs decreased linearly, whereas fast GABAergic IPSPs displayed a nonlinear (saturating) increase. The consistency of our model-based in vivo results with experimental in vitro results lends further validity to the capacity of DCM to infer on synaptic processes using macroscopic neurophysiological data.
doi:10.1371/journal.pone.0022790
PMCID: PMC3149050  PMID: 21829652
23.  AMPA receptor-mediated presynaptic inhibition at cerebellar GABAergic synapses: a characterization of molecular mechanisms 
The European journal of neuroscience  2004;19(9):2464-2474.
A major subtype of glutamate receptors, AMPA receptors (AMPARs), are generally thought to mediate excitation at mammalian central synapses via the ionotropic action of ligand-gated channel opening. It has recently emerged, however, that synaptic activation of AMPARs by glutamate released from the climbing fibre input elicits not only postsynaptic excitation but also presynaptic inhibition of GABAergic transmission onto Purkinje cells in the cerebellar cortex. Although presynaptic inhibition is critical for information processing at central synapses, the molecular mechanisms by which AMPARs take part in such actions are not known. This study therefore aimed at further examining the properties of AMPAR-mediated presynaptic inhibition at GABAergic synapses in the rat cerebellum. Our data provide evidence that the climbing fibre-induced inhibition of GABA release from interneurons depends on AMPAR-mediated activation of GTP-binding proteins coupled with down-regulation of presynaptic voltage-dependent Ca2+ channels. A Gi/o-protein inhibitor, N-ethylmaleimide, selectively abolished the AMPAR-mediated presynaptic inhibition at cerebellar GABAergic synapses but did not affect AMPAR-mediated excitatory actions on Purkinje cells. Furthermore, both Gi/o-coupled receptor agonists, baclofen and DCG-IV, and the P/Q-type calcium channel blocker ω-agatoxin IVA markedly occluded the AMPAR-mediated inhibition of GABAergic transmission. Conversely, AMPAR activation inhibited action potential-triggered Ca2+ influx into individual axonal boutons of cerebellar GABAergic interneurons. By suppressing the inhibitory inputs to Purkinje cells, the AMPAR-mediated presynaptic inhibition could thus provide a feed-forward mechanism for the information flow from the cerebellar cortex.
doi:10.1111/j.0953-816X.2004.03347.x
PMCID: PMC3387903  PMID: 15128400
AMPA-type glutamate receptor; cerebellum; GABAergic inhibitory synapse; presynaptic inhibition; rat
24.  Interplay between low threshold voltage-gated K+ channels and synaptic inhibition in neurons of the chicken nucleus laminaris along its frequency axis 
Central auditory neurons that localize sound in horizontal space have specialized intrinsic and synaptic cellular mechanisms to tightly control the threshold and timing for action potential generation. However, the critical interplay between intrinsic voltage-gated conductances and extrinsic synaptic conductances in determining neuronal output are not well understood. In chicken, neurons in the nucleus laminaris (NL) encode sound location using interaural time difference (ITD) as a cue. Along the tonotopic axis of NL, there exist robust differences among low, middle, and high frequency (LF, MF, and HF, respectively) neurons in a variety of neuronal properties such as low threshold voltage-gated K+ (LTK) channels and depolarizing inhibition. This establishes NL as an ideal model to examine the interactions between LTK currents and synaptic inhibition across the tonotopic axis. Using whole-cell patch clamp recordings prepared from chicken embryos (E17–E18), we found that LTK currents were larger in MF and HF neurons than in LF neurons. Kinetic analysis revealed that LTK currents in MF neurons activated at lower voltages than in LF and HF neurons, whereas the inactivation of the currents was similar across the tonotopic axis. Surprisingly, blockade of LTK currents using dendrotoxin-I (DTX) tended to broaden the duration and increase the amplitude of the depolarizing inhibitory postsynaptic potentials (IPSPs) in NL neurons without dependence on coding frequency regions. Analyses of the effects of DTX on inhibitory postsynaptic currents led us to interpret this unexpected observation as a result of primarily postsynaptic effects of LTK currents on MF and HF neurons, and combined presynaptic and postsynaptic effects in LF neurons. Furthermore, DTX transferred subthreshold IPSPs to spikes. Taken together, the results suggest a critical role for LTK currents in regulating inhibitory synaptic strength in ITD-coding neurons at various frequencies.
doi:10.3389/fncir.2014.00051
PMCID: PMC4033047  PMID: 24904297
GABAergic inhibition; voltage-gated low-threshold potassium current; IPSC; IPSP; tonotopy; whole-cell patch; interaural time difference
25.  Cyclic-nucleotide-gated channels mediate synaptic feedback by nitric oxide 
Nature  1997;390(6661):10.1038/37803.
Cyclic-nucleotide-gated (CNG) channels in outer segments of vertebrate photoreceptors generate electrical signals in response to changes in cyclic GMP concentration during phototransduction1. CNG channels also allow the influx of Ca2+, which is essential for photoreceptor adaptation2. In cone photoreceptors, cGMP triggers an increase in membrane capacitance indicative of exocytosis, suggesting that CNG channels are also involved in synaptic function3. Here we examine whether CNG channels reside in cone terminals and whether they regulate neurotransmitter release, specifically in response to nitric oxide (NO), a retrograde transmitter that increases cGMP synthesis and potentiates synaptic transmission in the brain4–6. Using intact retina, we show that endogenous NO modulates synapses between cones and horizontal cells. In experiments on isolated cones, we show directly that CNG channels occur in clusters and are indirectly activated by S-nitrosocysteine (SNC), an NO donor. Furthermore, both SNC and pCPT–cGMP, a membrane-permeant analogue of cGMP, trigger the release of transmitter from the cone terminals. The NO-induced transmitter release is suppressed by guanylate cyclase inhibitors and prevented by direct activation of CNG channels, indicating that their activation is required for NO to elicit release. These results expand our view of CNG channel function to include the regulation of synaptic transmission and mediation of the presynaptic effects of NO.
doi:10.1038/37803
PMCID: PMC3858101  PMID: 9414163

Results 1-25 (1105569)