Structure-activity relationship of philanthotoxins--II. Effects on the glutamate gated ion channels of the locust muscle fibre membrane.

1. PTX-4.3.3, the synthetic product of the wasp toxin, delta-philanthotoxin, blocks the open, glutamate gated, ion channels of the locust muscle. 2. This block is accompanied by the appearance of double exponential distribution of the closed times frequency, and a decrease of the inverse relationship between the open and closed states. 3. The analogues with the shortened polyamine chain, PTX-4.3 and PTX-3.3, are less potent, trifluoromethyl-PTX-4.3.3 is a more potent analogue. 4. Yet, they still behave like PTX-4.3.3 as open ion-channel blockers. 5. Another analogue, without the aromatic nucleus, dephenol-PTX-4.3.3, proved to act in a different way. 6. This less potent analogue, doesn't block the open ion channels. 7. Most likely, the not-activation induced, non-competitive antagonism, caused by dephenol-PTX-4.3.3, has a modulatory effect on glutamate receptors. 8. Methyl-PTX-4.3.3 is also an open ion-channel blocker. However, at relatively low concentrations it has an agonistic-like effect, probably of a modulatory origin as well. 9. A long-term inhibition of PTX-4.3.3 applied at low concentrations also indicates that beside an open ion-channel block, also other inhibitory processes are involved.     

delta-Philanthotoxin, a semi-irreversible blocker of ion-channels

1. The digger wasp, Philanthus triangulum, which preys on honeybees, produces a paralysing venom possessing a wide variety of activities. 2. In insects, the venom has a central as well as a peripheral effect; the latter effect consists of a presynaptic as well as a postsynaptic block of the skeletal neuromuscular transmission. 3. The presynaptic block is probably caused by an inhibition of the re-uptake of the transmitter. The postsynaptic effect probably consists of a block of open ion channels. 4. The venom contains at least four active toxins called alpha-, beta-, gamma- and delta-philanthotoxin (PTX). alpha-PTX blocks transmission in the cockroach CNS. The other three toxins block neuromuscular transmission. delta-PTX being the most active toxin in blocking glutamate evoked postsynaptic depolarizations. 5. In the junctional, as well as in the extrajunctional, muscle fibre membrane delta-PTX blocks ion channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be channels in a use-dependent manner. Once the channel has been blocked, unblocking seems to be semi-irreversible when agonist activation is low (spontaneous release of transmitter and/or leak of glutamate from the pipette). 6. The time constant of blocking is roughly estimated to be in the order of 10 msec, that of unblocking seems to be several hundreds of msec.     

Ion-channel block in insect muscle fibre membrane by the venom of the digger wasp, Philanthus triangulum F.

External miniature postsynaptic potentials were recorded from fibres of the metathoracic retractor unguis muscle of Schistocerca gregaria. At concentrations from 0.4 to 0.5 bee units/ml the venom of European Philanthus triangulum causes a decrease in amplitude and in half-decay time of miniature potentials. It is suggested that the venom, partially blocks the current by shortening the ion-channel open-life-time in the postsynaptic membrane.     

Neuromuscular block in locust skeletal muscle caused by a venom preparation made from the digger wasp Philanthus triangulum F. from Egypt.

A preparation from P. triangulum F., made by extracting abdomens and purified by Sephadex filtration, does not affect potassium ion-induced contractions of the retractor unguis muscle of S. gregaria, but the reduction of the glutamate contractions is at least as pronounced as the effect on the neurally-evoked twitch. Glutamate potentials are affected at a lower venom dose than are the neurally evoked excitatory postsynaptic potentials (EPSPs). The half-decay-time of the glutamate potentials starts to decrease just before the decrease in amplitude is initiated.In the retractor unguis muscle the resting plasma membrane is slightly depolarized at high venom concentrations, but this effect cannot explain the effects on neuromuscular transmission. It is concluded that the venom preparation of P. triangulum affects the glutamate or transmitter-induced transient permeability change, possibly by blocking the open ion-channels.     

Some receptor properties in motor neurons of an Aplysia withdrawal reflex.

Glutamate or a glutamate-related substance is the neurotransmitter used at the majority of the excitatory junctions of the neuronal network mediating the gill and siphon withdrawal reflex in Aplysia. In this report, we have studied some receptor properties of the major postsynaptic elements of the network, the motor neurons. We have examined the effect of a compound interfering with glutamate receptor, concanavalin A (con A). We found that con A treatment transforms the mainly hyperpolarizing responses to L-glutamate in motor neurons to prolonged depolarizing ones; these latter responses are sensitive to CNQX. We have also examined whether con A could enhance the CNQX sensitive excitatory postsynaptic potentials in these motor neurons. We found, by contrast, that con A did not alter the synaptic responses. The possible implications of the differential effect of con A on the glutamate responses and the synaptic responses are discussed.     

Depression of glutamate-mediated synaptic transmission by benzyl alcohol.

The data obtained from this study suggest that the nonionizable anesthetic benzyl alcohol has two prominent actions on GABA- and glutamate-mediated synaptic transmission at the lobster neuromuscular junction. They are as follows: (1) depression of the excitatory end-plate potential and the postsynaptic membrane response to applied glutamate, and (2) a hyperpolarization of the postsynaptic resting membrane potential associated with a decrease in effective membrane resistance. No change in amplitude of the inhibitory end-plate potential or inhibitory reversal potential was seen. Excitatory miniature end-plate potential frequency was also unaffected. The depression of excitatory synaptic transmission appears to be due to a decreased responsiveness of the postsynaptic receptor-ionophore complex.     

Three types of miniature inhibitory potentiaes in the frog spinal cord motoneurons: the possibility of GABA and glycine co-release

Miniature inhibitory postsynaptic potentials (mlPSPs) were recorded from motoneurons of the frog isolated spinal cord after blocking action potentials and ionotropic glutamate receptors (TTX 1 mcm: CNQX 25 mcm, D-AP5 50 mcm). Three types of mlPSPs were selected by their time characteristics) fast, slow and mlPSPs with two decay time constants. We classified 8.7% of mlPSPs as dual-component, 64.5% as fast mlPSPs, and 26.8% as slow mlPSPs. The GABA(A)R blocker bicuculline (20 mcm) diminished the number of the slow and dual-component events while the number of mlPSP with a fast kinetics was increased. The GlyR antagonist strychnine (1 mcm) reduced the frequency of fast mlPSPs and increased this parameter of slow mlPSPs. These data suggest existence of three different mlPSP groups distinguished by their kinetics and sensitivity to receptor antagonists: fast events mediated by glycine, slow events mediated by GABA and dual-component mlPSPs corresponding to glycine and GABA co-release.     

Analysis of conformationally restricted alpha-ketoglutarate analogues as substrates of dehydrogenases and aminotransferases.

Five synthetic, conformationally restricted alpha-ketoglutarate analogues were tested as substrates of a variety of dehydrogenases and aminotransferases. The compounds were found not to be detectable substrates of glutamate dehydrogenase, L-leucine dehydrogenase, L-phenylalanine dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glutamine transaminase K, aspartate aminotransferase, alanine aminotransferase, and alpha-ketoglutarate dehydrogenase complex. However, two thermostable aminotransferases were identified that catalyze transamination between several L-amino acids (e.g., phenylalanine, glutamate) and the alpha-ketoglutarate analogues of interest. Transamination between L-glutamate (or L-phenylalanine) and the alpha-ketoglutarate analogues was found to be 0.13 to 1.08 micromol/h/mg at 45 degrees C. The products resulting from transamination between L-phenylalanine and the alpha-ketoglutarate analogues were separated by reverse-phase HPLC, and the newly formed amino acid analogues were analyzed by LC-MS in an ion selective mode. In each case, the ions obtained were consistent with the expected product and a representative example is provided. The possibility existed that although the alpha-ketoglutarate analogues are not substrates of the dehydrogenases and most of the aminotransferases investigated, they might be good inhibitors. Weak inhibition of aminotransferases and glutamate dehydrogenase was found with some of the alpha-ketoglutarate analogues. The newly available thermostable aminotransferases may have general utility in the synthesis of bulky L-amino acids from the corresponding alpha-keto acids.     

Basic principles of synaptic physiology illustrated by a computer model

A computer model is described that simulates many basic aspects of chemical synapse physiology. The model consists of two displays, the first being a pictorial diagram of the anatomical connections between two presynaptic neurons and one postsynaptic neuron. Either or both of the presynaptic cells can be stimulated from a control panel with variable control of the number of pulses and firing rate; the resulting presynaptic action potentials are animated. The second display plots the membrane potential of the postsynaptic cell versus time following presynaptic stimulation. The model accurately simulates temporal and spatial summation when the presynaptic cells are arranged and stimulated in parallel and simulates presynaptic inhibition when they are arranged and stimulated in series. Excitatory and inhibitory postsynaptic potentials can be demonstrated by altering the nature of the ionic conductance change occurring on the postsynaptic cell. The effects on summation of changing length constant or time constant of the postsynaptic cell can also be illustrated. The model is useful for teaching these concepts to medical, graduate, or undergraduate students and can also be used as a self-directed computer laboratory exercise. It is available for free download from the internet.     

Multivesicular release at climbing fiber-Purkinje cell synapses.

Synapses driven by action potentials are thought to release transmitter in an all-or-none fashion; either one synaptic vesicle undergoes exocytosis, or there is no release. We have estimated the glutamate concentration transient at climbing fiber synapses on Purkinje cells by measuring the inhibition of excitatory postsynaptic currents (EPSCs) produced by a low-affinity competitive antagonist of AMPA receptors, gamma-DGG. The results, together with simulations using a kinetic model of the AMPA receptor, suggest that the peak glutamate concentration at this synapse is dependent on release probability but is not affected by pooling of transmitter released from neighboring synapses. We propose that the mechanism responsible for the elevated glutamate concentration at this synapse is the simultaneous release of multiple vesicles per site.     

β-philanthotoxin,a novel glutamatergic antagonist for insect neuromuscular transmission

1. The molecular structure of β-philanthotoxin (β-PTX), a toxin from the insect paralysing venom of the wasp Philanthus triangulum, has been elucidated using NMR and mass spectrometry. β-PTX has a polyamine character: C₅H₁₁·NH·CO·(CH₂)₄·NH·(CH₂)₃·NH₂. This structure has been confirmed by synthesis.2. In the locust muscle fibre β-PTX blocks iontophoretically evoked glutamate potentials in a non-activation induced manner. β-PTX also blocks the nicotinic transmission in the insect CNS, however, at much higher toxin concentrations.3. β-PTX reduces the frequency of postsynaptic channel opening and reduces the duration of open times.4. These results suggest an effect of β-PTX on kinetics of glutamate activated ion channels, different from acting as an open channel blocker.     

Noise analysis of the glutamate-activated current in photoreceptors.

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.     

Blockade of NMDA-receptors or calcium-channels attenuates the ischaemia-evoked efflux of glutamate and phosphoethanolamine and depression of neuronal activity in rat organotypic hippocampal slice cultures

We have investigated the effects of various insults on extracellular glutamate and phosphoethanolamine levels as well as electrical activity alterations in the early period following these insults in organotypic hippocampal slice cultures. Cultures prepared from 7-day-old rats were maintained in vitro for 7-14 days and then metabolic inhibition was induced: cultures were briefly exposed to potassium cyanide to induce chemical anoxia, 2-deoxyglucose with glucose removal to produce hypoglycaemia, or a combination of both to simulate ischaemia. Chemical anoxia induced a small increase in glutamate and a reversible decrease in evoked field potentials and these were greatly potentiated following simulated ischaemia: high, biphasic glutamate efflux and irreversible field potential abolition as well as increase in phosphoethanolamine levels were observed. We have characterised the effects of treatments using NMDA-receptor antagonists and the L-type calcium channel blocker diltiazem. Anoxia-induced glutamate accumulation was prevented by MK-801 and diltiazem D-AP5. Following simulated ischaemia, diltiazem totally prevented glutamate and phosphoethanolamine accumulations, whereas MK-801 did not block the first phase of glutamate accumulation and D-AP5 prevented none. We demonstrated that glutamate and phosphoethanolamine ischaemic-evoked efflux as well as the recovery of electrical activity in organotypic hippocampal slice cultures are sensitive to both NMDA-receptor and calcium-channel blockade. This model thus represents a useful in vitro system for the study of ischaemic neurodegeneration paralleling results reported using in vivo models.     

Two classes of channel-specific toxins from funnel web spider venom

1. The paralytic effects and neuromuscular actions of Agelenopsis aperta venom on insects were analyzed biochemically and electrophysiologically. 2. Paralysis caused by Agelenopsis venom is correlated with two effects on neuromuscular transmission: postsynaptic inhibition and presynaptic excitation. These effects are explained by the actions of two classes of toxins purified by RPLC, the alpha- and mu-agatoxins. 3. The alpha-agatoxins are low molecular weight, acylpolyamines which cause rapid, reversible paralysis correlated with use-dependent postsynaptic block of EPSPs and ionophoretic glutamate potentials. The mu-agatoxins are cysteine-rich polypeptides which cause irreversible paralysis and repetitive action potentials originating in presynaptic axons or nerve terminals. 4. The joint actions of the alpha- and mu-agatoxins lead to significantly higher rates of paralysis than are obtained by either toxin class alone, and this may relate to enhancement by excitatory mu-agatoxins of use-dependent block caused by alpha-agatoxins.     

Propofol suppresses synaptic responsiveness of somatosensory relay neurons to excitatory input by potentiating GABAA receptor chloride channels

Propofol is a widely used intravenous general anesthetic. Propofol-induced unconsciousness in humans is associated with inhibition of thalamic activity evoked by somatosensory stimuli. However, the cellular mechanisms underlying the effects of propofol in thalamic circuits are largely unknown. We investigated the influence of propofol on synaptic responsiveness of thalamocortical relay neurons in the ventrobasal complex (VB) to excitatory input in mouse brain slices, using both current- and voltage-clamp recording techniques. Excitatory responses including EPSP temporal summation and action potential firing were evoked in VB neurons by electrical stimulation of corticothalamic fibers or pharmacological activation of glutamate receptors. Propofol (0.6 – 3 μM) suppressed temporal summation and spike firing in a concentration-dependent manner. The thalamocortical suppression was accompanied by a marked decrease in both EPSP amplitude and input resistance, indicating that a shunting mechanism was involved. The propofol-mediated thalamocortical suppression could be blocked by a GABA_A receptor antagonist or chloride channel blocker, suggesting that postsynaptic GABA_A receptors in VB neurons were involved in the shunting inhibition. GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) were evoked in VB neurons by electrical stimulation of the reticular thalamic nucleus. Propofol markedly increased amplitude, decay time, and charge transfer of GABA_A IPSCs. The results demonstrated that shunting inhibition of thalamic somatosensory relay neurons by propofol at clinically relevant concentrations is primarily mediated through the potentiation of the GABA_A receptor chloride channel-mediated conductance, and such inhibition may contribute to the impaired thalamic responses to sensory stimuli seen during propofol-induced anesthesia.     

Neuronal glutamate transporters control activation of postsynaptic metabotropic glutamate receptors and influence cerebellar long-term depression.

Neuronal and glial isoforms of glutamate transporters show distinct distributions on membranes surrounding excitatory synapses, but specific roles for transporter subtypes remain unidentified. At parallel fiber (PF) synapses in cerebellum, neuronal glutamate transporters and metabotropic glutamate receptors (mGluRs) have overlapping postsynaptic distributions suggesting that postsynaptic transporters selectively regulate mGluR activation. We examined interactions between transporters and mGluRs by evoking mGluR-mediated excitatory postsynaptic currents (mGluR EPSCs) in slices of rat cerebellum. Selective inhibition of postsynaptic transporters enhanced mGluR EPSCs greater than 3-fold. Moreover, impairing glutamate uptake facilitated mGluR-dependent long-term depression at PF synapses. Our results demonstrate that uniquely positioned glutamate transporters strongly influence mGluR activation at cerebellar PF synapses. Postsynaptic glutamate uptake may serve as a general mechanism for regulating mGluR-initiated synaptic depression.     

Analytical description of the activation of multi-state receptors by continuous neurotransmitter signals at brain synapses.

Chemical synaptic transmission is a fundamental component of interneuronal communications in the central nervous system (CNS). Discharge of a presynaptic vesicle containing a few thousand molecules (a quantum) of neurotransmitter into the synaptic cleft generates a transmitter concentration signal that drives postsynaptic ion-channel receptors. These receptors exhibit multiple states, with state transition kinetics dependent on neurotransmitter concentration. Here, a novel and simple analytical approach for describing gating of multi-state receptors by signals with complex continuous time courses is used to describe the generation of glutamate-mediated quantal postsynaptic responses at brain synapses. The neurotransmitter signal, experienced by multi-state N-methyl-D-aspartate (NMDA)- and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors at specific points in a synaptic cleft, is approximated by a series of step functions of different intensity and duration and used to drive a Markovian, multi-state kinetic scheme that describes receptor gating. Occupancy vectors at any point in time can be computed interatively from the occupancy vectors at the times of steps in transmitter concentration. Multi-state kinetic schemes for both the low-affinity AMPA subtype of glutamate receptor and for the high-affinity NMDA subtype are considered, and expected NMDA and AMPA components of synaptic currents are calculated. The amplitude of quantal responses mediated by postsynaptic receptor clusters having specific spatial distributions relative to foci of quantal neurotransmitter release is then calculated and related to the displacement between the center of the postsynaptic receptor cluster and the focus of synaptic vesicle discharge. Using this approach we show that the spatial relation between the focus of release and the center of the postsynaptic receptor cluster affects synaptic efficacy. We also show how variation in this relation contributes to variation in synaptic current amplitudes.     

Structural requirements for maximal inhibitory allosteric effect of estrogens and estrogen analogues on glutamate dehydrogenase

The inhibition of glutamate dehydrogenase by estrogens, estrogen analogues or polyphenylethylene derivatives (about one hundred molecules, most of them having estrogenic or antiestrogenic activities) was measured. The efficiency of these compounds in inducing allosteric inhibition of the enzyme was compared and correlated to their chemical structure: an aromatic ring A, a free phenolic group in the region of carbon 3 of the steroid nucleus and a lipophilic substitution in the region of C-12, C-13 or C-17 were found to be the main structural features required for maximal efficiency on glutamate dehydrogenase. A tentative model for the relative orientation of the main inhibitor families is proposed. It accounts for most of the kinetic results and can be used as a tool for the selection of affinity labels directed towards the estrogen binding site of glutamate dehydrogenase.     

Severe deficits in 5-HT2A -mediated neurotransmission in BDNF conditional mutant mice

BDNF is thought to provide critical trophic support for serotonin neurons. In order to determine postnatal effects of BDNF on the serotonin system, we examined a line of conditional mutant mice that have normal brain content of BDNF during prenatal development but later depletion of this neurotrophin in the postnatal period. These mice show a behavioral phenotype that suggests serotonin dysregulation. However, as shown here, the presynaptic serotonin system in the adult conditional mutant mice appeared surprisingly normal from histological, biochemical, and electrophysiological perspectives. By contrast, a dramatic and unexpected postsynaptic 5-HT2A deficit in the mutant mice was found. Electrophysiologically, serotonin neurons appeared near normal except, most notably, for an almost complete absence of expected 5-HT2A -mediated glutamate and GABA postsynaptic potentials normally displayed by these neurons. Further analysis showed that BDNF mutants had much reduced 5-HT2A receptor protein in dorsal raphe nucleus and a similar deficit in prefrontal cortex, a region that normally shows a high level of 5-HT2A receptor expression. Recordings in prefrontal slice showed a marked deficit in 5-HT2A -mediated excitatory postsynaptic currents, similar to that seen in the dorsal raphe. These findings suggest that postnatal levels of BDNF play a relatively limited role in maintaining presynaptic aspects of the serotonin system and a much greater role in maintaining postsynaptic 5-HT2A and possibly other receptors than previously suspected.     

Electrophysiology of the Fetal Spinal Cord : II. Interaction among peripheral inputs and recurrent inhibition

Interactions of peripheral inputs to the motoneuron of the kitten fetus as young as 3 weeks prenatal were studied by reflex discharge from the ventral root as well as by recording from single motoneurons. Facilitation was found between two synergists in fetuses 1 to 2 weeks before birth. Intracellular recording showed that the facilitation could be explained by summation of excitatory postsynaptic potentials. Inhibition was found between antagonists in the fetuses 2 to 3 weeks before birth and was accompanied by inhibitory postsynaptic potentials. Recurrent inhibition was very powerful in the fetal spinal cord as shown by large motoneuron hyperpolarization by antidromic stimulation. Cells presumed to be "Renshaw cells" and which responded to both ortho- and antidromic stimulation with repetitive firing were shown in the 2 weeks prenatal fetus. These results lead to the conclusion that there is considerable effective synaptic connection of afferent collaterals already established by the later stage of intrauterine life and that this may be achieved independently of external stimuli.