Inhibition Of Neurotransmitter Release Research Articles

Cannabinoid CB 1 and adenosine A 1 receptors independently inhibit hippocampal synaptic transmission

Adenosine A 1 and cannabinoid CB 1 receptors are affected by drugs widely consumed by humans, as it is the case for caffeine, an adenosine receptor antagonist, and tetrahydrocannabinol, a cannabinoid receptor agonist. These receptors are present in the hippocampus and inhibit neurotransmitter release by operating similar transduction mechanisms. We, therefore, evaluated if they cross-talk to modulate synaptic transmission in the hippocampus. To do so, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 area of rat hippocampal slices and the consequences of activation or blockade of cannabinoid CB 1 or adenosine A 1 receptors upon neuromodulation exerted by the other receptor were assessed. The cannabinoid CB 1 receptor agonist, WIN55212-2 (300 nM), slowly decreased (≅ 40%) the fEPSP slope, while the adenosine A 1 receptor agonist, cyclopenthyladenosine (CPA, 15 nM) rapidly decreased (≅ 50%) it. Blockade of cannabinoid CB 1 receptors with AM251 (1 µM) did not influence the adenosine A 1 receptor-mediated inhibition of synaptic transmission. Blockade of adenosine A 1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 50 nM) did not affect the inhibition perpetrated by cannabinoid CB 1 receptor activation. When both receptors were simultaneously activated (5 nM CPA plus 300 nM WIN55212-2) the net inhibition of synaptic transmission was about the sum of the effect of each drug applied independently. These results indicate independent synaptic transmission modulation by adenosine A 1 and cannabinoid CB 1 receptors at the hippocampus, suggesting that availability of G-proteins coupled to each receptor and availability of other signalling molecules involved in their transducing pathways, are not limiting factors for their modulatory role.

Gangliosides as High Affinity Receptors for Tetanus Neurotoxin

Tetanus neurotoxin (TeNT) is an exotoxin produced by Clostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a approximately 150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.

Presynaptic G-Protein-Coupled Receptors Dynamically Modify Vesicle Fusion, Synaptic Cleft Glutamate Concentrations, and Motor Behavior

Understanding how neuromodulators regulate behavior requires investigating their effects on functional neural systems, but also their underlying cellular mechanisms. Utilizing extensively characterized lamprey motor circuits, and the unique access to reticulospinal presynaptic terminals in the intact spinal cord that initiate these behaviors, we investigated effects of presynaptic G-protein-coupled receptors on locomotion from the systems level, to the molecular control of vesicle fusion. 5-HT inhibits neurotransmitter release via a Gbetagamma interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex that promotes kiss-and-run vesicle fusion. In the lamprey spinal cord, we demonstrate that, although presynaptic 5-HT receptors inhibit evoked neurotransmitter release from reticulospinal command neurons, their activation does not abolish locomotion but rather modulates locomotor rhythms. Liberation of presynaptic Gbetagamma causes substantial inhibition of AMPA receptor-mediated synaptic responses but leaves NMDA receptor-mediated components of neurotransmission mostly intact. Because Gbetagamma binding to the SNARE complex is displaced by Ca(2+)-synaptotagmin binding, 5-HT-mediated inhibition displays Ca(2+) sensitivity. We show that, as Ca(2+) accumulates presynaptically during physiological bouts of activity, 5-HT/Gbetagamma-mediated presynaptic inhibition is relieved, leading to a frequency-dependent increase in synaptic concentrations of glutamate. This frequency-dependent phenomenon mirrors a shift in the vesicle fusion mode and a recovery of AMPA receptor-mediated EPSCs from inhibition without a modification of NMDA receptor EPSCs. We conclude that activation of presynaptic 5-HT G-protein-coupled receptors state-dependently alters vesicle fusion properties to shift the weight of NMDA versus AMPA receptor-mediated responses at excitatory synapses. We have therefore identified a novel mechanism in which modification of vesicle fusion modes may profoundly alter locomotor behavior.

Spinal Endocannabinoids and CB 1 Receptors Mediate C-Fiber–Induced Heterosynaptic Pain Sensitization

Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.

Cannabinoid signalling in the enteric nervous system

Cannabinoid signalling is an important mechanism of synaptic modulation in the nervous system. Endogenous cannabinoids (anandamide and 2-arachidonyl-glycerol) are synthesized and released via calcium-activated biosynthetic pathways. Exogenous cannabinoids and endocannabinoids act on CB1 and CB2 receptors. CB1 receptors are neuronal receptors which couple via G-proteins to inhibition of adenylate cyclase or to activation or inhibition of ion channels. CB2 receptors are expressed by immune cells and cannabinoids can suppress immune function. In the central nervous system, the endocannabinoids may function as retrograde signals released by the postsynaptic neuron to inhibit neurotransmitter release from presynaptic nerve terminals. Enteric neurons also express CB receptors. Exogenously applied CB receptor agonists inhibit enteric neuronal activity but it is not clear if endocannabinoids released by enteric neurons can produce similar responses in the enteric nervous system (ENS). In this issue of Neurogastroenterology and Motility, Boesmans et al. show that CB1 receptor activation on myenteric neurons maintained in primary culture can suppress neuronal activity, inhibit synaptic transmission and mitochondrial transport along axons. They also provide initial evidence that myenteric neurons (or other cell types present in the cultures) release endocannabinoids and which activate CB1 receptors constitutively. These data provide new information about targets for cannabinoid signalling in the ENS and highlight the potential importance of CB receptors as drug targets. It is necessary that future work extends these interesting findings to intact tissues and ideally to the in vivo setting.

What is the Source of the EEG?

Neurons in the human cortex generally process their information by means of electrical signals and thus enable the electrical recording of their activity, the electroencephalogram (EEG). Due to their unique orientation with their long apical dendrites perpendicular to the cortical surface, large cortical pyramidal neurons in deep cortical layers play a major role in the generation of the EEG. Specific and non-specific thalamic nuclei, as well as distant cortical areas, terminate on these apical dendrites and form myriads of excitatory and inhibitory afferents. The release of excitatory and inhibitory neurotransmitters by these fibers activates specific postsynaptic receptors and generates excitatory and inhibitory postsynaptic potentials, respectively. By electrotonic spread of postsynaptic potentials along the apical dendrites and equivalent capacitive currents, they become electrical dipoles. Positive or negative deflections are generated by both excitatory and inhibitory afferents, depending on the location of these synapses on the apical dendrites. Negative (upward) deflections are due to superficial excitatory or deep inhibitory inputs, whereas positive (downward) deflections represent deep excitatory or superficial inhibitory inputs.

Isoflurane Inhibits the Neurotransmitter Release Machinery

Despite their importance, the mechanism of action of general anesthetics is still poorly understood. Facilitation of inhibitory GABA(A) receptors plays an important role in anesthesia, but other targets have also been linked to anesthetic actions. Anesthetics are known to suppress excitatory synaptic transmission, but it has been difficult to determine whether they act on the neurotransmitter release machinery itself. By directly elevating [Ca(2+)](i) at neurotransmitter release sites without altering plasma membrane channels or receptors, we show that the commonly used inhalational general anesthetic, isoflurane, inhibits neurotransmitter release at clinically relevant concentrations, in a dose-dependent fashion in PC12 cells and hippocampal neurons. We hypothesized that a SNARE and/or SNARE-associated protein represents an important target(s) for isoflurane. Overexpression of a syntaxin 1A mutant, previously shown in Caenorhabditis elegans to block the behavioral effects of isoflurane, completely eliminated the reduction in neurotransmitter release produced by isoflurane, without affecting release itself, thereby establishing the possibility that syntaxin 1A is an intermediary in isoflurane's ability to inhibit neurotransmitter release.

Cannabinoid signaling in inhibitory autaptic hippocampal neurons

Depolarization-induced suppression of excitation and inhibition (DSE/DSI) appears to be an important form of short-term retrograde neuronal plasticity involving endocannabinoids (eCBs), the activation of presynaptic cannabinoid CB1 receptors, and the suppression of neurotransmitter release. Using murine autaptic hippocampal cultures, we have distinguished five populations of autaptic inhibitory neurons that exhibit differential cannabinoid responses, including three temporally distinct forms of DSI. One remaining population responded to cannabinoids but did not have DSI while a fifth had neither DSI nor cannabinoid responses. Of the two chief candidate eCBs, 2-AG reversibly inhibited inhibitory post synaptic currents (IPSCs) while anandamide did so irreversibly, the latter's action inconsistent with a role as a bona fide eCB mediator of DSI. The duration of depolarization necessary to elicit the two most prominent forms of DSI (effective dose (ED-50) ∼210, ∼280 ms) was far less than for autaptic DSE. However the nearly identical concentration response for 2-AG to inhibit excitatory postsynaptic currents (EPSCs) and IPSCs indicates that this difference is not due to differential cannabinoid receptor sensitivity. Interestingly, of the two populations exhibiting prominent DSI, one had a substantially faster recovery time course both after DSI and 2-AG, this despite being cultured under identical conditions. Several enzymes have been proposed to play a role in 2-AG breakdown, presumably determining the time course of DSI: fatty acid amide hydrolase (FAAH), cyclooxygenase-2 (COX-2), monoacyl glycerol lipase (MGL), and α/β-hydrolase domains 6 and 12 (ABHD6 and ABHD12). We tested the impact on DSI duration by blockers of FAAH, COX-2, MGL and ABHD6. Notably, the population with slow DSI was regulated only by MGL, whereas the fast DSI population was regulated by both MGL and COX-2. This suggests that the faster DSI time course may occur as a result of the concerted action of multiple enzymes, which may represent a more general mechanism for regulation of the duration of different forms of DSI and DSE.

Neuritogenic Actions of Botulinum Neurotoxin A on Cultured Motor Neurons

Botulinum neurotoxins (BoNTs) are extremely potent neuromuscular poisons that act through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein cleavage to inhibit neurotransmitter release. The ability of BoNT serotype A (BoNT/A) to eliminate localized transmitter release at extremely low doses is well characterized. In the current study, we investigated the less understood characteristic of BoNT/A to induce nerve outgrowth, sometimes referred to as sprouting. This phenomenon is generally considered a secondary response to the paralytic actions of BoNT/A, and other potential factors that may initiate this sprouting have not been investigated. Alternatively, we hypothesized that BoNT/A induces sprouting through presynaptic receptor activation that is independent of its known intracellular actions on the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) synaptosomal associated protein of 25 kDa (SNAP-25). To test this, the effects of BoNT/A application on neurite outgrowth were examined using primary cultures enriched with motor neurons isolated from embryonic mouse spinal cord. In this system, BoNT/A potently stimulated neuritogenesis at concentrations as low as 0.01 nM. The neuritogenic effects of BoNT/A exposure were concentration dependent and antagonized by Triticum vulgaris lectin, a known competitive antagonist of BoNT. Similar results were observed with the isolated BoNT/A binding domain, revealing that neuritogenesis could be initiated solely by the binding actions of BoNT/A. In addition, the presence or absence of SNAP-25 cleavage by BoNT/A was not a determinant factor in BoNT/A-induced neuritogenesis. Collectively, these results suggest that binding of BoNT/A to the motor neuronal membrane activates neuritogenesis through as yet undetermined intracellular pathway(s), independent of its known action on vesicular release.

Depolarizing GABAergic synaptic input triggers endocannabinoid‐mediated retrograde synaptic signaling

Endocannabinoids released by postsynaptic neurons inhibit neurotransmitter release from presynaptic axon terminals. One typical stimulus of endocannabinoid production is an increase of calcium concentration in postsynaptic neurons. The aim of the present study was to clarify whether depolarizing GABAergic synaptic input, by increasing calcium concentration in postsynaptic neurons, can trigger endocannabinoid production. Spontaneous GABAergic inhibitory postsynaptic currents (sIPSCs) were recorded in Purkinje cells in mouse cerebellar slices with patch-clamp pipettes containing 151 mM chloride (a usual recording mode). sIPSCs were depolarizing inward currents under this condition. Combined electrophysiological and fluorometric calcium imaging experiments indicated that sIPSCs frequently triggered calcium spikes. After the calcium spikes, a short-term suppression of sIPSCs occurred. This suppression was prevented by the CB(1) cannabinoid receptor antagonist rimonabant and the diacylglycerol lipase inhibitor orlistat, but not changed by URB597, an inhibitor of anandamide degradation. It is, therefore, likely that CB(1) receptors and 2-arachidonoylglycerol were involved. For testing the physiological significance of the above observation, we carried out experiments on brains of 3- to 5-day-old mice. The gramicidin-induced perforated patch-clamp mode was used for preserving the physiological intracellular chloride concentration of the neurons. Depolarizing GABAergic sIPSCs occurred under this condition, but at a very low rate. Rimonabant did not change the frequency of these sIPSCs, arguing against the persistence of an endocannabinoid tone. The results point to a new kind of trigger of endocannabinoid production: depolarizing GABAergic synaptic input can elicit endocannabinoid production in postsynaptic neurons by activating calcium channels. The produced endocannabinoid suppresses GABA release from presynaptic axon terminals.

γ-Aminobutryic Acid

γ-Aminobutryic Acid

Increased glutamate and decreased glycine release in the rostral ventromedial medulla during induction of a pre-clinical model of chronic widespread muscle pain

Two injections of acidic saline into the gastrocnemius muscle produce long-lasting hyperalgesia that is initiated and maintained by changes in the rostroventromedial medulla (RVM). Potential underlying mechanisms could be increased release of excitatory neurotransmitters and/or reduced release of inhibitory neurotransmitters, in the RVM. We tested this hypothesis by measuring concentrations of aspartate, glutamate and glycine in response to the first and second injection of acidic saline and compared to intramuscular injections of normal saline using microdialysis with HPLC analysis. We show a significant increase in aspartate and glutamate during the second acidic saline injection compared to normal saline injections or the first injection of acidic saline. There were also long-lasting decreases in glycine concentrations in the RVM in response to both the first and second injection of acidic saline. It is possible that disinhibition after the first injection leads to long-lasting neuronal changes that allow a greater release of excitatory neurotransmitters after the second injection. We hypothesize that increased release of excitatory neurotransmitters in the RVM drives the release of excitatory neurotransmitters in the spinal cord, central sensitization and the consequent hyperalgesia.

Distinctive roles of different β‐amyloid 42 aggregates in modulation of synaptic functions

To determine how endogenously secreted beta-amyloid 42 (Abeta42) aggregates regulate synaptic functions, we examined effects of Abeta42 at the neuromuscular junction of Drosophila larvae. Voltage-clamp recordings of synaptic transmission and optical analysis of vesicle recycling at presynaptic terminals show that expression of Abeta42 in neurons leads to a reduction of neurotransmitter release. However, expression of Abeta42 in postsynaptic muscle cells enhanced neurotransmitter release. Both effects are neutralized by Abeta antibody, suggesting a role for secreted Abeta42 peptides. Application of exogenously prepared Abeta42 oligomers leads to a reduction in synaptic responses, whereas mixed Abeta42 aggregates with mainly fibrils elicit an opposite effect by increasing synaptic transmission. Further analysis of long-term depression (LTD) confirms differential effects of different Abeta42 aggregates. Taken together, our data suggest that Abeta42 is secreted from neurons primarily as oligomers that inhibit neurotransmitter release and exert no effect on LTD. Whereas larger-sized aggregates, possibly fibrils, are major components secreted from muscle cells, which enhance synaptic transmission and LTD. Thus, different types of cells may secrete distinct forms of Abeta42 aggregates, leading to different modulation of synaptic functions.

Suppressive Effect of Orthovanadate on Hepatic Stellate Cell Activation and Liver Fibrosis in Rats

Orthovanadate (OV), an inhibitor of protein tyrosine phosphatases, affects various biological processes in a cell-type-specific manner. In this study, we investigated the effect of OV on hepatic stellate cells (HSCs). When primary rat HSCs were cultured in the presence of 10% serum, they spontaneously lost characteristic stellate morphology, proliferated, and were transformed into an activated state with the formation of abundant stress fibers and increased expression of both alpha-smooth muscle actin and collagen type I mRNA. OV treatment inhibited proliferation and activation of HSCs and partially reversed the phenotype of activated HSCs. Among the signaling molecules investigated, phosphorylation of the Src protein at tyrosine 416 was the most striking in OV-treated HSCs. Treatment of cells with Src family inhibitors partially abrogated the effects of OV. Furthermore, transfection of v-Src into activated HSCs induced a stellate morphology similar to that in the quiescent state. We then examined whether OV could effectively suppress HSC activation in vivo after liver injury induced by either carbon tetrachloride or dimethylnitrosamine. OV significantly reduced the appearance of alpha-smooth muscle actin-positive cells and decreased collagen deposition, concomitant with an improvement in liver function. Our study showed for the first time that OV was able to suppress the activation of HSCs, possibly through the modulation of Src activity, and attenuated fibrosis after chronic liver injury.

Mu-opioid and corticotropin-releasing-factor receptors show largely postsynaptic co-expression, and separate presynaptic distributions, in the mouse central amygdala and bed nucleus of the stria terminalis

The anxiolytic effects of opiates active at the mu-opioid receptor (μ-OR) may be ascribed, in part, to suppression of neurons that are responsive to the stress-associated peptide, corticotropin releasing factor (CRF), in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). The corticotropin releasing factor receptor (CRFr) and μ-OR are expressed in both the CeA and BNST, but their subcellular relationship to each other is not known in either region. To address this question, we used dual electron microscopic immunolabeling of μ-OR and CRFr in the mouse lateral CeA and anterolateral BNST. Immunolabeling for each receptor was detected in the same as well as in separate somatic, dendritic and axonal profiles of neurons in each region. CRFr had a plasmalemmal or cytoplasmic distribution in many dendrites, including those co-expressing μ-OR. The co-expression of CRFr and μ-OR also was seen near excitatory-type synapses on dendritic spines. In both the CeA and BNST, over 50% of the CRFr-labeled dendritic profiles (dendrites and spines) contained immunoreactivity for the μ-OR. However, less than 25% of the dendritic profiles containing the μ-OR were labeled for CRFr in either region, suggesting that opiate activation of the μ-OR affects many neurons in addition to those responsive to CRF. The dendritic profiles containing CRFr and/or μ-OR received asymmetric, excitatory-type synapses from unlabeled or CRFr-labeled axon terminals. In contrast, the μ-OR was identified in terminals forming symmetric, inhibitory-type synapses. Thus, in both the CeA and BNST, μ-OR and CRFr have strategic locations for mediation of CRF and opioid effects on the postsynaptic excitability of single neurons, and on the respective presynaptic release of excitatory and inhibitory neurotransmitters. The commonalities in the synaptic location of both receptors in the CeA and BNST suggest that this is a fundamental cellular association of relevance to both drug addiction and stress-induced disorders.

Involvement of S-Nitrosylation of Actin in Inhibition of Neurotransmitter Release by Nitric Oxide

BackgroundThe role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. S-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of S-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying S-nitrosylated proteins.ResultsHere we show that actin was a major protein S-nitrosylated in the spinal cord by the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP). Interestingly, actin was S-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid S-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase.ConclusionThe present study demonstrates that actin is a major S-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by S-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.

Deletion of N-type Ca2+ Channel Cav2.2 Results in Hyperaggressive Behaviors in Mice

Voltage-dependent N-type Ca(2+) channels play important roles in the regulation of diverse neuronal functions in the brain, but little is known about its role in social aggressive behaviors. Mice lacking the alpha1B subunit (Ca(v)2.2) of N-type Ca(2+) channels showed markedly enhanced aggressive behaviors to an intruder mouse in the resident-intruder test. The dorsal raphe nucleus (DRN), which contains serotonin neurons, is known to be involved in aggression in animals. We thus examined the DRN neurons in the Ca(v)2.2-deficient (Ca(v)2.2(-/-)) mice. Microinjection of omega-conotoxin GVIA, an N-type Ca(2+) channel-specific blocker, into the DRN of wild type mice resulted in escalated aggression, mimicking the phenotypes of Ca(v)2.2(-/-). Electrophysiological analysis showed increased firing activity of serotonin neurons with a reduced inhibitory neurotransmission in the Ca(v)2.2(-/-) DRN. Ca(v)2.2(-/-) mice showed an elevated level of arginine vasopressin, an aggression-related hormone, in the cerebrospinal fluid. In addition, Ca(v)2.2(-/-) mice showed an increase of serotonin in the hypothalamus. These results suggest that N-type Ca(2+) channels at the DRN have a key role in the control of aggression.

Modulation of Excitatory Synaptic Transmission by Δ9-Tetrahydrocannabinol Switches from Agonist to Antagonist Depending on Firing Rate

Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive ingredient in marijuana, acts as a partial agonist on presynaptic cannabinoid type 1 (CB1) receptors to inhibit neurotransmitter release. Here, we report that THC inhibits excitatory neurotransmission between cultured rat hippocampal neurons in a manner highly sensitive to stimulus rate. THC (1 microM) inhibited excitatory postsynaptic currents (EPSCs) and whole-cell I(Ca) evoked at 0.1 Hz but at 0.5 Hz THC had little effect. The cannabinoid receptor full agonists [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate salt] (Win55212-2) (100 nM) and 2-arachidonylglycerol (1 microM) inhibited EPSCs independent of stimulation at 0.1 or 0.5 Hz. THC occupied CB1 receptors at 0.5 Hz, but the receptors failed to couple to presynaptic Ca(2+) channels. Consequently, 1 microM THC blocked the inhibition of EPSC amplitude by Win55212-2 when EPSCs were evoked at 0.5 Hz. A depolarizing prepulse to 0 mV reversed THC inhibition of I(Ca), but reversal of the inhibition produced by Win55212-2 required a pulse to +80 mV, suggesting that the voltage-dependent reversal of Gbetagamma inhibition of voltage-gated Ca(2+) channels accounts for the frequency-dependence of cannabinoid action. THC blocked depolarization-induced suppression of EPSCs evoked at 0.5 Hz, indicating that it inhibited retrograde endocannabinoid signaling in a frequency-dependent manner. Thus, THC displayed a state-dependent switching from agonist to antagonist that may account for its complex actions in vivo.

Modulation of Group III Metabotropic Glutamate Receptors by Hydrogen Ions

Protons act as neuromodulators and produce significant effects on synaptic transmission. The molecular basis of neuromodulation by extracellular protons is partially explained by their effects on certain neurotransmitter receptors and ion channels. The metabotropic glutamate receptors (mGluRs) are a family of eight receptor subtypes that are widely expressed throughout the mammalian CNS. In this study, the effects of physiologically relevant changes in extracellular pH were examined in mammalian cells expressing the mGluR subtypes: human mGluR1a, mGluR4a, mGluR5d or mGluR8b. The signal transduction coupling properties of mGluR4a and mGluR8b were switched from the adenylate cyclase (G_i) pathway to the phospholipase C (G_q) pathway by coexpression of a promiscuous G protein. Fluorometric imaging plate reader was used to measure changes in cytoplasmic calcium concentrations in response to agonist. Extracellular acidification from pH 8.0 to pH 6.5 progressively diminished mGluR4 responsiveness to the agonists L-glutamate and (2S,1′S,2′R)-2-(carboxycyclopropyl)glycine (L-CCG-I), and this inhibition was characterized by insurmountable antagonism. By comparison, extracellular acidification did not significantly alter mGluR8 responses to agonists. Furthermore, agonist activation of mGluR1a and mGluR5d was virtually unaffected by changes in pH. Because mGluR4 is expressed presynaptically and its activation inhibits the release of neurotransmitters such as glutamate and GABA, we propose that the net effect of proton inhibition of mGluR4 would be to reverse or prevent that suppression of neurotransmitter release. As such, local decreases in pH could have significant effects on the regulation of transmitter release and synaptic tone via modulation of mGluR4.

The Effects of Volatile Aromatic Anesthetics on Voltage-Gated Na+ Channels Expressed in Xenopus Oocytes

Many inhaled anesthetics inhibit voltage-gated sodium channels at clinically relevant concentrations, and suppression of neurotransmitter release by these anesthetics results, at least partly, from decreased presynaptic sodium channel activity. Volatile aromatic anesthetics can inhibit N-methyl-D-aspartate (NMDA) receptor function and enhance gamma-amino butyric acid A receptor function, but these effects depend strongly on the chemical properties of the aromatic compounds. In the present study we tested whether diverse aromatic anesthetics consistently inhibit sodium channel function. We studied the effect of eight aromatic anesthetics on Na(v)1.2 sodium channels with beta(1) subunits, using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. All aromatic anesthetics inhibited I(Na) (sodium currents) at a holding potential which produce half-maximal current (V(1/2)) (partial depolarization); inhibition was modest with 1,3,5-trifluorobenzene (8% +/- 2%), pentafluorobenzene (13% +/- 2%), and hexafluorobenzene (13% +/- 2%), but greater with benzene (37% +/- 2%), fluorobenzene (39% +/- 2%), 1,2-difluorobenzene (48% +/- 2%), 1,4-difluorobenzene (31 +/- 3%), and 1,2,4-trifluorobenzene (33% +/- 1%). Such dichotomous effects were noted by others for NMDA and gamma-aminobutyric acid A receptors. Parallel, but much smaller inhibition, was found for I(Na) at a holding potential which produced near maximal current (-90 mV) (V(H-90)), and hexafluorobenzene caused small (6% +/- 1%) enhancement of this current. These changes in sodium channel function were correlated with effectiveness for inhibiting NMDA receptors, with lipid solubility of the compounds, with molecular volume, and with cation-pi interactions. Aromatic compounds vary in their actions on the kinetics of sodium channel gating and this may underlie their variable inhibition. The range of inhibition produced by minimum alveolar anesthetic concentration concentrations of inhaled anesthetics indicates that sodium channel inhibition may underlie the action of some of these anesthetics, but not others.