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Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp, Philanthus triangulum. PhTX-433 non-selectively inhibits several excitatory ligand-gated ion channels, and we recently showed that its synthetic analogue, PhTX-343, exhibits strong selectivity for neuronal over muscle-type nicotinic acetylcholine receptors (nAChRs). Here we examined the action of seventeen analogues of PhTX-343 against ganglionic (?3?4) and brain (?4?2) nAChRs expressed in Xenopus oocytes by using two-electrode voltage-clamp at -100 mV. IC50 values for PhTX-343 inhibition of ?3?4 and ?4?2 receptors were 7.7 nM and 80 nM, respectively. All of the studied analogues had significantly higher potency at ?3?4 nAChRs with IC50 values as low as 0.16 nM and with up to a 91-fold selectivity for ?3?4 over ?4?2 receptors. We conclude that PhTX-343 analogues displaying both a saturated ring and an aromatic moiety in the hydrophobic headgroup of the molecule demonstrate exceptional potency and selectivity for ?3?4 nAChRs. 2

Ca2+-permeable AMPA receptors (AMPARs) are expressed throughout the adult CNS but yet their role in development is poorly understood. In the developing retina, most investigations have focused on Ca2+ influx through NMDARs in promoting synapse maturation and not on AMPARs. However, NMDARs are absent from many retinal cells suggesting that other Ca2+-permeable glutamate receptors may be important to consider. Here we show that inhibitory horizontal and AII amacrine cells lack NMDARs but express Ca2+-permeable AMPARs. Before eye-opening, AMPARs were fully blocked by philanthotoxin (PhTX), a selective antagonist of Ca2+-permeable AMPARs. After eye-opening, however, a subpopulation of Ca2+-permeable AMPARs were unexpectedly PhTX resistant. Furthermore, Joro spider toxin (JSTX) and IEM-1460 also failed to antagonize, demonstrating that this novel pharmacology is shared by several AMPAR channel blockers. Interestingly, PhTX-insensitive AMPARs failed to express in retinae from dark-reared animals demonstrating that light entering the eye triggers their expression. Eye-opening coincides with the consolidation of inhibitory cell connections suggesting that the developmental switch to a Ca2+-permeable AMPAR with novel pharmacology may be critical to synapse maturation in the mammalian retina.

The wasp venom philanthotoxin-4.3.3 (PhTX-4.3.3) is an antagonist of glutamate transmission in the insect as well as in the mammalian brain. It was recently shown that PhTX-4.3.3 inhibits the N-methyl-D-aspartate (NMDA) transmission in rat hippocampus. In this study we show that dideaza-philanthotoxin-12 (dideaza-PhTX-12), an analogue of PhTX-4.3.3, is a potent antagonist of voltage-dependent Ca2+ currents in rat hippocampal CA1 neurons. At a concentration of 10 microM it reduces the Ca2+ current to 40%. Two voltage-dependent potassium currents, the A current and the delayed rectifier, were hardly affected by dideaza-PhTX-12, indicating selectivity of the drug for Ca2+ currents. As a consequence the philanthotoxins will inhibit the calcium influx via voltage dependent as well as NMDA mediaded calcium channels and thus reduce excitability in the hippocampus.

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin, delta-philanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using voltage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A+)RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10(-11)-10(-7) M) of PhTX-343 and by 10(-5)-10(-4) M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentrations of PhTX-343 and spermine also potentiated the responses to nicotine and gamma-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate receptor.

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.

Three photosensitive, synthetic analogues of δ-philanthotoxin (PhTX-433) have been tested on the locust, excitatory nerve-muscle system. At 10 −9 M they inhibit reversibly the postjunctional currents (EPSCs) recorded from muscle fibres during motor nerve stimulation, mainly by non-competitively antagonizing postjunctional quisqualate-sensitive glutamate receptors (Quis-R), probably through open channel block. This use-dependent antagonism is characteristic of the philanthotoxins. When the preparation was irradiated with 270 nm U.V. during toxin application and nerve stimulation the EPSCs were inhibited irreversibly. Irradiation of the preparation alone or in the presence of philanthotoxins (e.g. PhTX-433) which are not photosensitive did not lead to irreversible inhibition of the EPSC. It is concluded that the three photosensitive toxins bind covalently to Quis-R in its open channel conformation during U.V. irradiation, thereby irreversibly blocking the channel gated by this receptor.

1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

The effects of intracellularly and extracellularly applied synthetic analogues of delta-philanthotoxin (PhTX-433) and the polyamine spermine on the excitatory postsynaptic current (EPSC) of glutamatergic synapses and single channel currents gated by quisqualate-sensitive glutamate receptors (QUIS-R) on locust leg muscle have been compared. When applied extracellularly all 3 compounds reversibly antagonised the EPSC and the single channel currents. Antagonism was voltage independent, but use (agonist) dependent. Antagonism also occurred when they were injected into muscle fibres, but in this case it was not use dependent. It is proposed that spermine and the two toxins bind to the closed and open channel conformations of QUIS-R at a site near the intracellular opening of the channel gated by this receptor.

M. Bruce , R. Bukownik , A. T. Eldefrawi , M. E. Eldefrawi , R. Goodnow , Jr, T. Kallimopoulos , K. Konno , K. Nakanishi , M. Niwa and P. N. R. Usherwood . Structure-activity relationships of analogues of the wasp toxin philanthotoxin: non-competitive antagonists of quisqualate receptors. Toxicon 28, 1333–1346, 1990.—Fifty-two analogues of the wasp toxin, philanthotoxin-433, have been synthesized and tested on a glutamatergic, nerve-muscle preparation from locust leg. Reduction in amplitude of the neurally-evoked muscle twitch was used to construct dose-inhibition relationships from which ic 50 s were estimated. The most active analogues were characterized by one or more of the following: increased hydrophobicity of aromatic and tyrosyl regions; an increased number of protonated groups in the polyamine region; a guanidinium instead of a spermine terminal amino moiety. The incorporation of a butyl side-group in the polyamine also enhanced potency. These results are explained on the basis of the known non-competitive antagonistic blockage by philanthotoxin-433 of the channel gated by postjunctional glutamate receptors when the channel is open.

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.

This paper provides answers to the questions which of the toxins present in the venom of the wasp Philanthus triangulum may be responsible for the previously reported blockage of transmission through the sixth abdominal ganglion of the cockroach, and whether this may occur by block of synaptic transmission or by affecting axonal exitability. In current clamp experiments the crude venom induces a slight depolarization of the membrane of the giant axon from the sixth abdominal ganglion of the cockroach and a small and irreversible decrease in the amplitude of the action potential. These marginal effects are not seen with relatively high concentrations of the philanthotoxins β-PTX and δ-PTX. It appears that neither the crude venom nor the toxins significantly affect the excitability of the cockroach giant axon. At a concentration of 20 μg ml−1 δ-PTX causes a slowly reversible block of synaptic transmission from the cercal nerve XI to a giant interneuron without any change in resting membrane potential, whereas β-PTX is inactive. Iontophoretically evoked acetylcholine potentials of the giant neuron are more sensitive to δ-PTX than excitatory postsynaptic potentials. This suggests that the toxin acts on the postsynaptic membrane.

One component (delta-philanthotoxin (delta-PTX) of the venom from the wasp Philanthus triangulum blocks transmission postsynaptically at excitatory synapses on locust muscle. delta-PTX depresses both the iontophoretic glutamate potential and the excitatory junctional current (e.j.c.) in a glutamate receptor activation-dependent manner. The rate of recovery from the effects of the toxin is reduced following either prolonged application of L-glutamate or repetitive iontophoretic application of this amino acid or high frequency neural stimulation of the muscle in the presence of delta-PTX. The decay phase of the e.j.c. is shortened by delta-PTX. The effects of delta-PTX on the e.j.c. are not voltage dependent. The open-close kinetics of glutamate channels in extrajunctional muscle membrane are modified by delta-PTX as shown by patch clamp analysis. The mean life time of the glutamate channel is reduced, whilst the mean interval between single opening events is increased with the events often occurring in bursts. These data are consistent with glutamate channel blocking by this toxin. It is proposed that the toxin blocks open channels gated by both junctional and extrajunctional glutamate receptors on locust muscle. It is further proposed that delta-PTX enters a compartment of the muscle through the glutamate open channels and that it can also block the open channels from this site.

The beta-fraction of the venom of the solitary wasp Philanthus triangulum inhibited glutamate-induced potentials in muscle fibres of the locust in a dose-dependent manner (50% block at 2 micrograms/ml). Single channel behaviour and miniature excitatory postsynaptic currents were not affected. The blocking effect disappeared after treatment of the muscle with the lectin concanavalin A which prevents desensitization of glutamate receptors. Block of potentials induced by iontophoretic application of glutamate was strongly dependent on the frequency of stimulation: no block occurred at stimulus intervals of 30 sec or more. The nature of this inhibition is discussed and proposed to be a delay of recovery from desensitization.

Glutamate uptake in insect neuromuscular junctions is inhibited by delta-philanthotoxin. Two other components of the philanthus venom (beta- and gamma-philanthotoxin) do not affect this glutamate uptake. No synergism between the active delta-philanthotoxin and the two inactive components had to be assumed to explain the inhibition by the Philanthus venom.

Alterations in the polyamine and amino acid (tyrosine) moieties of philanthotoxin-343 (PhTX-343) were investigated for their effects on the antagonism of nicotinic acetylcholine receptors (nAChRs) isolated from the locust (Schistocerca gregaria) mushroom body. Through whole-cell patch-clamp recordings, the philanthotoxin analogues in this study were shown to cause inhibition of the inward current when co-applied with acetylcholine (ACh). PhTX-343 (IC50 = 0.80 μM at −75 mV) antagonised locust nAChRs in a use-dependent manner, suggesting that it acts as an open-channel blocker. The analogue in which both the secondary amine functionalities were replaced with methylene groups (i.e., PhTX-12) was ~6-fold more potent (IC50 (half-maximal inhibitory concentration) = 0.13 μM at −75 mV) than PhTX-343. The analogue containing cyclohexylalanine as a substitute for the tyrosine moiety of PhTX-343 (i.e., Cha-PhTX-343) was also more potent (IC50 = 0.44 μM at −75 mV). A combination of both alterations to PhTX-343 generated the most potent analogue, i.e., Cha-PhTX-12 (IC50 = 1.71 nM at −75 mV). Modulation by PhTX-343 and Cha-PhTX-343 fell into two distinct groups, indicating the presence of two pharmacologically distinct nAChR groups in the locust mushroom body. In the first group, all concentrations of PhTX-343 and Cha-PhTX-343 inhibited responses to ACh. In the second group, application of PhTX-343 or Cha-PhTX-343 at concentrations ≤100 nM caused potentiation, while concentrations ≥ 1 μM inhibited responses to ACh. Cha-PhTX-12 may have potential to be developed into insecticidal compounds with a novel mode of action.

We elucidated the mechanisms underlying the kainate receptor (KAR)-mediated facilitatory modulation of synaptic transmission in the cerebellum. In cerebellar slices, KA (3 μM) increased the amplitude of evoked excitatory postsynaptic currents (eEPSCs) at synapses between axon terminals of parallel fibers (PF) and Purkinje neurons. KA-mediated facilitation was antagonized by NBQX under condition where AMPA receptors were previously antagonized. Inhibition of protein kinase A (PKA) suppressed the effect of KA on glutamate release, which was also obviated by the prior stimulation of adenylyl cyclase (AC). KAR-mediated facilitation of synaptic transmission was prevented by blocking Ca2+ permeant KARs using philanthotoxin. Furthermore, depletion of intracellular Ca2+ stores by thapsigargin, or inhibition of Ca2+-induced Ca2+-release by ryanodine, abrogated the synaptic facilitation by KA. Thus, the KA-mediated modulation was conditional on extracellular Ca2+ entry through Ca2+-permeable KARs, as well as and mobilization of Ca2+ from intracellular stores. Finally, KAR-mediated facilitation was sensitive to calmodulin inhibitors, W-7 and calmidazolium, indicating that the increased cytosolic [Ca2+] sustaining KAR-mediated facilitation of synaptic transmission operates through a downstream Ca2+/calmodulin coupling. We conclude that, at cerebellar parallel fiber-Purkinje cell synapses, presynaptic KARs mediate glutamate release facilitation, and thereby enhance synaptic transmission through Ca2+-calmodulin dependent activation of adenylyl cyclase/cAMP/protein kinase A signaling.

Earlier findings had suggested that spontaneous and evoked glutamate release activates non-overlapping populations of NMDA receptors. Here, we evaluated whether AMPA receptor populations activated by spontaneous and evoked release show a similar segregation. To track the receptors involved in spontaneous or evoked neurotransmission, we used a polyamine agent, philanthotoxin, that selectively blocks AMPA receptors lacking GluR2 subunits in a use-dependent manner. In hippocampal neurons obtained from GluR2-deficient mice, philanthotoxin application decreased AMPA-receptor-mediated spontaneous miniature EPSCs (AMPA-mEPSCs) down to 20% of their initial level within 5 min. In contrast, the same philanthotoxin application at rest decreased the subsequent AMPA-receptor-mediated evoked EPSCs (eEPSCs) only down to 80% of their initial value. A 10-min-long perfusion of philanthotoxin further decreased AMPA-eEPSC amplitudes to 60% of their initial magnitude, which remained substantially higher than the level of AMPA-mEPSC block achieved within 5 min. Finally, stimulation after removal of philanthotoxin resulted in reversal of AMPA-eEPSC block, verifying strict use dependence of philanthotoxin. These results support the notion that spontaneous and evoked neurotransmission activate distinct sets of AMPA receptors and bolster the hypothesis that synapses harbor separate microdomains of evoked and spontaneous signaling.

BackgroundSpontaneous "miniature" transmitter release takes place at low rates at all synapses. Long thought of as an unavoidable leak, spontaneous release has recently been suggested to be mediated by distinct pre- and postsynaptic molecular machineries and to have a specialized role in setting up and adjusting neuronal circuits. It remains unclear how spontaneous and evoked transmission are related at individual synapses, how they are distributed spatially when an axon makes multiple contacts with a target, and whether they are commonly regulated.ResultsElectrophysiological recordings in the Drosophila larval neuromuscular junction, in the presence of the use-dependent glutamate receptor (GluR) blocker philanthotoxin, indicated that spontaneous and evoked transmission employ distinct sets of GluRs. Invivo imaging of transmission using synaptically targeted GCaMP3 to detect Ca(2+) influx through the GluRs revealed little spatial overlap between synapses participating in spontaneous and evoked transmission. Spontaneous and evoked transmission were oppositely correlated with presynaptic levels of the protein Brp: synapses with high Brp favored evoked transmission, whereas synapses with low Brp were more active spontaneously. High-frequency stimulation did not increase the overlap between evoked and spontaneous transmission, and instead decreased the rate of spontaneous release from synapses that were highly active in evoked transmission.ConclusionsAlthough individual synapses can participate in both evoked and spontaneous transmission, highly active synapses show a preference for one mode of transmission. The presynaptic protein Brp promotes evoked transmission and suppresses spontaneous release. These findings suggestthe existence of presynaptic mechanisms that promote synaptic specialization to either evoked or spontaneous transmission.

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4-aminopyridine (4-AP)-evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post-synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione, and persisted after pre-treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G-protein-independent regulation further in thalamocortical slices, the KAR-mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca(2+) permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca(2+) stores by thapsigargin, or inhibition of Ca(2+) -induced Ca(2+) -release by ryanodine. Thus, the KA-mediated modulation was contingent on both Ca(2+) entry through Ca(2+) -permeable KARs and liberation of intracellular Ca(2+) stores. Finally, sensitivity to W-7 indicated that the increased cytosolic [Ca(2+) ] underpinning KAR-mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre-synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca(2+) -calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G-protein involvement.

There is accumulating evidence that sleep contributes to memory formation and learning, but the underlying cellular mechanisms are incompletely understood. To investigate the impact of sleep on excitatory synaptic transmission, we obtained whole-cell patch-clamp recordings from layer V pyramidal neurons in acute slices of somatosensory cortex of juvenile rats (postnatal days 21–25). In animals after the dark period, philanthotoxin 74 (PhTx)-sensitive calcium-permeable AMPA receptors (CP-AMPARs) accounted for ∼25% of total EPSP size, and current–voltage (I–V) relationships of the underlying EPSCs showed inward rectification. In contrast, in similar experiments after the light period, EPSPs were PhTx insensitive with linearI–Vcharacteristics, indicating that CP-AMPARs were less abundant. Combined EEG and EMG recordings confirmed that slow-wave sleep-associated delta wave power peaked at the onset of the more quiescent, lights-on phase of the cycle. Subsequently, we show that burst firing, a characteristic action potential discharge mode of layer V pyramidal neurons during slow-wave sleep has a dual impact on synaptic AMPA receptor composition: repetitive burst firing without synaptic stimulation eliminated CP-AMPARs by activating serine/threonine phosphatases. Additionally, repetitive burst-firing paired with EPSPs led to input-specific long-term depression (LTD), affecting Ca2+impermeable AMPARs via protein kinase C signaling. In agreement with two parallel mechanisms, simple bursts were ineffective after the light period but paired bursts induced robust LTD. In contrast, incremental LTD was generated by both conditioning protocols after the dark cycle. Together, our results demonstrate qualitative changes at neocortical glutamatergic synapses that can be induced by discharge patterns characteristic of non-rapid eye movement sleep.

Although the N-methyl-D-aspartate (NMDA) subtype of L-glutamate receptor is well characterized, the significance of non-NMDA glutamate-sensitive binding sites is not well documented. In this study, a new tricyclic quinoxalinedione (NBQX) and an arthropod toxin (philanthotoxin) were shown to block responses of spinal neurones in vivo to kainate, quisqualate, and AMPA in parallel but had little effect on responses to NMDA. Philanthotoxin appeared to be a use-dependent antagonist consistent with a channel-blocking mode of action. On cortical wedges in vitro, however, NBQX proved to be a more potent antagonist of AMPA and quisqualate than of kainate (pA2 values of 7.1, 7.0, and 5.6, respectively) with no effect at 10 μM on responses to NMDA. These studies provide evidence that on cortical neurones, but not on spinal neurones, AMPA and kainate depolarize by pharmacologically different mechanisms.Key words: glutamate receptors, quinoxalinediones, philanthotoxin, AMPA, kainate.

The α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) are glutamate-gated cation channels that mediate fast excitatory synaptic transmission in the central nervous system. AMPARs are tetramers formed by homo- or heteromeric assembly of GluA1-4 subunits to produce multiple subtypes with varying biophysical properties. Polyamine toxins such as joro spider toxins, philanthotoxins (PhTXs), and argiotoxins are use-dependent ion channel blockers of AMPARs widely employed as highly potent antagonists of GluA2-lacking receptor subtypes. In addition to this use, recent findings have indicated that a philanthotoxin analog, PhTX-74, can distinguish among GluA2-containing AMPAR subtypes in the presence of the prototypical transmembrane AMPAR regulatory protein γ-2 (or stargazin). Thus, PhTX-74 may be of potential use in studies of the neurobiological role of GluA2-containing subtypes. We have evaluated the pharmacological profile of PhTX-74 and related polyamine toxins at homo- and heteromeric AMPARs in the presence and absence of γ-2. Determination of IC(50) values for inhibition of glutamate-evoked currents from Xenopus oocytes expressing recombinant homo- or heteromeric combinations of GluA1, GluA2, and GluA3 in the presence of γ-2 shows that PhTX-74 inhibits homomeric GluA1 and GluA3 receptors nonselectively, with IC(50) values in the nanomolar range (252-356 nM), and heteromeric GluA1/A2 and GluA2/A3 receptors nonselectively, with IC(50) values in the micromolar range (22 μM). Thus, in contrast to earlier findings, we find that PhTX-74 cannot pharmacologically discriminate between GluA2-containing AMPAR subtypes.

Neurite outgrowth is a fundamental step in establishing proper neuronal connections in the developing central nervous system. Dynamic control of outgrowth has been attributed to changes in growth cone Ca2+ levels in response to extracellular cues. Here we have investigated a possible role for Ca2+ permeable kainate (KA) receptors in regulating neurite outgrowth of nociceptive-like dorsal root ganglion (DRG) neurons. To identify KA receptor subunits likely to be involved, we used quantitative RT-PCR on acutely dissociated DRG and dorsal horn neurons. DRG neurons expressed more GluK1, particularly the GluK1b spice variant, than dorsal horn neurons. Conversely, dorsal horn neurons expressed more GluK2, particularly GluK2a, than DRG neurons. Further, an RNA editing assay indicated that the majority of GluK1 and GluK2 mRNA transcripts in DRG were unedited. Imaging Ca2+ transients following application of a KA receptor agonist to DRG and dorsal horn co-cultures revealed increases in intracellular Ca2+ in the growth cones of DRG neurons. In the majority of cases, this increase in Ca2+ was partly or completely blocked by Joro spider toxin (JSTX), an antagonist for Ca2+-permeable AMPA and KA receptors. Treatment of DRG/dorsal horn co-cultures with KA for 18 hours suppressed neurite outgrowth while application of the rapidly desensitizing KA receptor agonist SYM 2081, the competitive AMPA/KA receptor antagonist, CNQX, and JSTX or philanthotoxin enhanced neurite outgrowth and prevented KA effects on neurite outgrowth. Thus, Ca2+ entry through KA receptors at the growth cone of DRG neurons may be an important regulator of neurite outgrowth.

1. The blocking action of δ-philanthotoxin (PhTX-4.3.3), a polyamine amide wasp toxin, and 33 structural analogues was studied at the nicotinic acetylcholine receptor of isolated neuronal somata from locust thoracic ganglia and at the cockroach cercal nerve-giant interneuron nicotinic cholinergic synapse. 2. A comparison of the structure-activity relationships reported here for the locust somal and cockroach synaptic nicotinic receptors and for the glutamatergic neuromuscular synapses of housefly larvae reported previously suggests a generally similar pharmacology for the channel-blocking action at ligand-activated ion channels, but with differences that might be due to variation in accessibility between synaptic receptors and those on the neuronal somata or to genuine divergence in ligand-activated ion channel pharmacology.

Voltage-dependent, non-competitive inhibition by philanthotoxin-343 (PhTX-343) analogues, with reduced charge or length, of nicotinic acetylcholine receptors (nAChR) of TE671 cells and ionotropic glutamate receptors (N-methyl-D-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR)) expressed in Xenopus oocytes from rat brain RNA was investigated. At nAChR, analogues with single amine-to-methylene or amine-to-ether substitutions had similar potencies to PhTX-343 (IC(50)=16.6 microM at -100 mV) whereas PhTX-(12), in which both secondary amino groups of PhTX-343 were replaced by methylenes, was more potent than PhTX-343 (IC(50)=0.93 microM at -100 mV). Truncated analogues of PhTX-343 were less potent. Inhibition by all analogues was voltage-dependent. PhTX-343 (IC(50)=2.01 microM at -80 mV) was the most potent inhibitor of NMDAR. At AMPAR, most analogues were equipotent with PhTX-343 (IC(50)=0.46 microM at -80 mV), apart from PhTX-83, which was more potent (IC(50)=0.032 microM at -80 mV), and PhTX-(12) and 4,9-dioxa-PhTX-(12), which were less potent (IC(50)s>300 microM at -80 mV). These studies show that PhTX-(12) is a selective nAChR inhibitor and PhTX-83 is a selective AMPAR antagonist.

Presynaptic kainate receptors (KARs) exert a modulatory action on transmitter release. This effect can be switched from facilitation to inhibition by an increased concentration of KAR agonists. We here report that activation of presynaptic GluK1-containing KARs facilitates GABA release on oxytocin and vasopressin neurons in the supraoptic nucleus of the hypothalamus. Increase in ambient levels of glutamate associated with the physiological reduction of astrocytic coverage of oxytocin neurons in lactating rats switches this KAR-mediated facilitation to inhibition of GABAergic transmission. This effect was reproduced in both oxytocin and vasopressin neurons of virgin rats when glutamate transporters were blocked pharmacologically, thereby establishing that enhanced levels of extracellular glutamate induce the switch in KAR-mediated action. The facilitation of GABA release was inhibited with philanthotoxin, a Ca2+-permeable KAR antagonist, suggesting that this effect was associated with an ionotropic mode of action. Conversely, KAR-mediated inhibition was compromised in the presence of U73122, a phospholipase C inhibitor, in agreement with the involvement of a metabotropic pathway. We thus reveal that physiological astrocytic plasticity modifies the mode of action of presynaptic KARs, thereby inversing their coupling with GABA release.

Spermine, a polyamine based on a 12-carbon motif, is an effective inhibitor of E. coli OmpF porin. Here we study the inhibition of porin by two polyamine toxins commonly used as modulators of polyamine-sensitive channels: Philanthotoxin-433 (PhTX) from wasp venom and Joro spider toxin (JSTX). Both are highly asymmetric molecules, with at one end a 12-carbon chain polyamine targeting the molecule to the porin constriction zone, and at the other end large aromatic groups conferring to this extremity a size in the order of the OmpF constriction zone. Here we report that PhTX, but not Joro toxin, induces a high degree of flickering in the OmpF-mediated current. The effect is concentration and voltage-dependent, and greatly diminished in a mutant lacking D113 on the constriction loop, a residue previously shown to be required for spermine sensitivity. Possible reasons for the distinct sensitivity of OmpF to PhTX and Joro toxin are discussed.

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.

1. Block by philanthotoxin-343 (PhTX-343), a neurotoxin from wasps, of ionic currents mediated through neuronal nicotinic acetylcholine (ACh) receptor/channels was characterized in rat phaeochromocytoma PC12 cells, by use of whole cell voltage-clamp techniques. 2. In the cells held at -60 mV, PhTX-343 at 0.1 and 1 microM inhibited an inward current activated by 100 microM ACh. The current inhibition was relieved by depolarizing steps, and augmented at negative potentials, suggesting that PhTX-343 blocks the channel in a voltage-dependent manner. Joro spider toxin-3 (JSTX-3) also exerted voltage-dependent inhibition of ACh-activated currents in a similar concentration range, but argiotoxin636 did not affect the currents. 3. Analysis of the current decay during hyperpolarizing steps indicated that the current inhibition by 100 nM PhTX-343 develops in an order of several hundres of milliseconds. On the other hand, the recovery from the current inhibition during depolarizing steps developed in an order of about 100 ms. 4. The results suggest that PhTX-343 blocks neuronal nicotinic receptor channels in PC12 cells at concentrations lower than those required for channel block in non-mammalian cells, and the block exhibits clear voltage-dependence. Estimated from the voltage-dependence, the binding site of PhTX-343 may be located near the outer mouth of the channel.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA-R)-mediated neurotoxicity was studied in relation to subunit expression and the presence of Ca(2+)-permeable receptor channels. AMPA-mediated toxicity had two components: 1) a direct AMPA-R-mediated component, which was not due to Ca(2+) influx through voltage-gated Ca(2+) channels, reversal of the Na(+)/Ca(2+) exchanger or release of calcium from dantrolene-sensitive intracellular Ca(2+) stores, and 2) a minor, indirect component involving activation of NMDA receptor channels, because of glutamate release and removal of the Mg(2+) block of the NMDA receptor on AMPA-R stimulation. The involvement of Ca(2+) influx through AMPA-R was also examined. The number of neurons possessing Ca(2+)-permeable AMPA-R increased during culture development, concurrently with an increasing susceptibility for AMPA-induced toxicity during development. GluR2(R) levels also increased during development, and channel blockers of Ca(2+)-permeable AMPA-R lacking the GluR2(R) subunit (spermine and philanthotoxin) failed to prevent neurotoxicity or increases in [Ca(2+)](i). Thus, the direct AMPA-R-mediated toxicity may be explained by initiation of cell death by Ca(2+) fluxing through AMPA-R containing GluR2(R). The components of direct AMPA-R-mediated toxicity are proposed to be 1) toxicity mediated by GluR2(R)-lacking AMPA-R and 2) toxicity mediated by low-Ca(2+)-permeability AMPA-R containing GluR2(R).

AMPA receptors mediate rapid glutamatergic synaptic transmission. In the mammalian cochlear nuclei, neurons receive excitatory input from either auditory nerve fibers, parallel fibers, or both fiber systems. The functional correlates of differences in the source of input were examined by recording AMPA receptor-mediated, miniature EPSCs (mEPSCs) in whole-cell voltage-clamp mode from identified neurons. Bushy, octopus, and T-stellate cells of the ventral cochlear nucleus (VCN) and tuberculoventral cells of the dorsal cochlear nucleus (DCN) receive most of their excitatory input from the auditory nerve; fusiform cells receive excitatory inputs from both the auditory nerve and parallel fibers; cartwheel cells receive excitatory input from parallel fibers alone. mEPSCs from bushy, octopus, T-stellate, and tuberculoventral cells had significantly faster decay time constants (0.35–0.40 msec) than did those from fusiform and cartwheel cells (1.32–1.79 msec). Some fusiform cells had two populations of mEPSCs with distinct time courses. mEPSCs in cells with auditory nerve input alone were inhibited by philanthotoxin, a blocker of calcium-permeable AMPA receptors, whereas mEPSCs in cells with parallel fiber input were not. Thus AMPA receptors postsynaptic to the auditory nerve differ from those postsynaptic to parallel fibers both in channel-gating kinetics and in their permeability to calcium. These results confirm the conclusion that synaptic AMPA receptors are specialized according to the source of input (Hunter et al., 1993; Rubio and Wenthold, 1997;Wang et al., 1998).

At most excitatory, glutamatergic synapses in the brain, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs) make distinct contributions to the postsynaptic response: AMPARs mediate transient conductances that are well suited to fast information transfer; NMDARs associate glutamate binding with postsynaptic depolarization to mediate a slower conductance that, unlike that of most AMPARs, includes Ca2+ influx that can trigger long-lasting changes in synaptic strength. This delegation of postsynaptic roles has been blurred recently by evidence implicating Ca2+-permeable AMPARs (CP-AMPARs) in different forms of synaptic transmission and plasticity. AMPARs are heterotetramers comprising various combinations of subunits GluR1–4, each of which is coded to express an uncharged glutamine in the pore region that permits permeation of divalent cations. In GluR2, RNA editing recodes this glutamine to a positively charged arginine, rendering GluR2-containing AMPARs Ca2+ impermeable (Dingledine et al. 1992). Many neurons, however, express AMPARs that lack GluR2 and are, consequently, Ca2+ permeable. Particularly in interneurons, CP-AMPARs often replace NMDARs as the primary source of postsynaptic Ca2+ influx. For example, Ca2+ influx through CP-AMPARs induces long-term potentiation (LTP) of excitatory synapses onto interneurons in the amygdala (Mahanty & Sah, 1998) and triggers GABA release from A17 amacrine cells in the retina (Chavez et al. 2006). CP-AMPAR channels are distinguished electrophysiolgically via voltage-dependent block by (and eventual permeation of) endogenous intracellular polyamines that confers biphasic rectification on an otherwise linear I–V relationship (Bahring et al. 1997). CP-AMPARs also are blocked by exogenous extracellular polyamines such as philanthotoxin (Washburn et al. 1997). Ca2+ permeability and polyamine sensitivity typically go hand in hand, but a paper in this issue of The Journal of Physiology reports CP-AMPARs in rat retinal interneurons that are insensitive to philanthotoxin (Osswald et al. 2007). The expression of these receptors coincides with eye opening and is delayed by rearing animals in darkness, suggesting that they may participate in a critical phase of retinal development. Functional CP-AMPARs are detected using cobalt staining and electrophysiology. Cobalt permeates CP-AMPARs and so fills cells expressing activated receptors. Following exposure to cobalt and glutamate, retinas were fixed according to a protocol that rendered cobalt-positive horizontal and amacrine cells a deep orange. Particularly strong staining was observed in AII amacrine cells, glycinergic interneurons known to express CP-AMPARs. Cobalt staining was not induced by membrane depolarization in the absence of glutamate and was unaffected by NMDAR and metabotropic glutamate receptor (mGluR) antagonists but was abolished by AMPAR antagonists, including philanthotoxin. During the third postnatal week, around the time of eye opening (∼P14), cobalt staining persisted, but philanthotoxin sensitivity disappeared in horizontal cells and was reduced in AII cells, particularly in processes ramifying in the outer portion of the inner plexiform layer (IPL), where synapses transmitting ‘OFF’ visual signals are made. AII cells, but not horizontal cells, recovered philanthotoxin sensitivity 2 weeks later. Decreased philanthotoxin sensitivity did not occur in dark-reared animals, suggesting that light-induced activity may trigger the change. Interestingly, voltage clamp recordings of spontaneous synaptic currents in AII cells also reflected the transient disappearance of philanthotoxin sensitivity. This, together with the morphological data described above, would suggest that most spontaneous activity in AIIs arises at synapses in the ‘OFF’ region of the IPL. This is a bit surprising, as most synapses onto AIIs are made by rod bipolar cells (RBCs) in the ON region, and spontaneous events in AIIs are indistinguishable from evoked quantal events from RBCs (Singer et al. 2004). The molecular mechanism underlying this peculiar developmental switch remains unknown, but the authors propose the possibility of a novel AMPAR subtype (or novel RNA editing of known subunits), a reasonable suggestion given that other glutamate receptors (e.g. mGluR6) and transporters (e.g. EAAT5) appear unique to the retina. The functional impact of these philanthotoxin-insensitive CP-AMPARs also remains to be determined. Relief of polyamine sensitivity could change postsynaptic responsivity to patterns of synaptic input (Rozov & Burnashev, 1999) during a time when excitatory synaptic connections with bipolar cells are first being established (Horsburgh & Sefton, 1987). Alternatively, the authors suggest that these receptors may act as temporary molecular cues to direct formation of synaptic connections within specific sublaminae of the IPL, a critical step in the proper wiring of ON and OFF visual pathways.

The effects of extracellularly-applied synthetic philanthotoxin-343 (PhTX-343) on transmission and long-term potentiation (LTP) at Schaffer-collateral/commissural-CA1 synapses were investigated. PhTX-343 was ineffective in antagonizing CA1 field-EPSPs mediated by AMPA/kainate receptors. However, when a micromolar concentration of the toxin was present during tetanization, the induction of LTP was suppressed. In contrast, when PhTX-343 was applied either immediately after or long after tetanization no effect on LTP could be found. It appears that the synaptic, non-NMDA receptors of the CA1-region are insensitive to PhTX-343. Suppression of LTP induction could result from antagonism of postsynaptic NMDA receptors, but the results do not rule out other possibilities such as presynaptic block.

Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of Ph TX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did Ph TXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in Ph TX structure may possibly produce derivatives that can be potential insecticides. © 1993 Wiley-Liss, Inc.

We report the isolation and functional characterization of cDNAs encoding a Drosophila kainate-selective glutamate receptor. The deduced mature 964-residue protein (DGluR-I) is 108,482 Da and exhibits significant homology to mammalian glutamate receptor subunits. Injection of DGluR-I cRNA into Xenopus oocytes generated kainate-operated ion channels which were blocked by the selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione and philanthotoxin. DGluR-I transcripts are differentially expressed during Drosophila development and, in late embryogenesis, accumulate in the central nervous system.

1. The blocking action of delta-philanthotoxin (PhTX-4.3.3), a wasp toxin composed of a polyamine chain, an aromatic amino acid (tyrosyl) nucleus and an aliphatic (butanoic) acid was studied, together with 33 structural analogues, at the glutamatergic neuromuscular synapse in larvae of the house fly, Musca domestica. 2. Shortening of the polyamine or the aliphatic acid chain, or removing the aromatic nucleus caused a decrease in potency. 3. An increase in potency was obtained by elongating the molecule in any of several directions: elongation of the polyamine chain with amine containing groups, by iodination of the aromatic moiety and by aliphatic or aromatic elongation of the butanoic acid.

Piek and co-workers reported that the solitary digger wasp Philanthus triangulum F., a wasp found in the Sahara Desert that preys on honeybees, makes a venom which blocks glutamate receptors (GluR) on locust muscles.1 A toxin, δ-philanthotoxin (δ-PhTX), which is an antagonist of GluR, was isolated from the venom sacs of the female wasp and assigned structure 1 (Fig. 1) from spectroscopic data and the synthesis of its DL-form.2 Using a honeybee worker paralysis assay, we independently isolated the toxin PhTX-433 (numerals denote the number of methylenes in the polyamine moiety) and established its structure as 1, with the S configuration, by synthesis and circular dichroic spectroscopy. Natural PhTX-433 and two other isomers, -343 and -334, were also synthesized.3 PhTX-433 and its analogs are efficient noncompetitive reversible inhibitors of the quisqualate-sensitive glutamate receptor (QUISR) in locust skeletal muscle.3 A number of similar but structurally more complex toxins have been isolated from various spider venoms4-8 (Fig. 1). The isolation and characterization of polyamine toxins from spiders and wasps has stimulated considerable interest in view of the important involvement of GluR in mammalian central nervous systems and in insect neurotransmission.9,10

1. Effects of structural philanthotoxin (PTX) analogues on the high affinity uptake of [ 3 H]- l -glutamate by slices of the rat hippocampus were studied using light microscopy surface autoradiography. 2. Considering the various effects of the PTX-analogues it can be concluded that the presence of an aromatic nucleus in the molecule is an important moiety, while the number and distribution of positive charges in the polyamine chain seems to be of a still not understood importancy.

Mechanisms and Expression of Receptors.- Glutamate Receptors in Cultures of Mouse Hippocampus Studied with Fast Applications of Agonists, Modulators and Drugs.- Measuring and Controlling the Extracellular Glycine Concentration at the NMDA Receptor Level.- The Glycine Coagonist Site of the NMDA Receptor.- The PCP Site of the NMDA Receptor Complex.- Antagonists of NMDA-Activated Current in Cortical Neurons: Competition with Glycine and Blockade of Open Channels.- Types of EAA Receptors.- Experiments with Kainate and Quisqualate Agonists and Antagonists in Relation to the Sub-Classification of 'Non-NMDA' Receptors.- Homocysteic Acid as Transmitter Candidate in the Mammalian Brain and Excitatory Amino Acids in Epilepsy.- Specific Quisqualate Receptor Ligand Blocks Both Kainate and Quisqualate Responses.- Molecular Characterization, Ultrastructural Localization and Gene Cloning of the Chick Cerebellar Kainate Receptor.- Intracellular Messengers Associated with Excitatory Amino Acid (EAA) Receptors.- The Glycine Site on the NMDA Receptor: Pharmacology and Involvement in NMDA Receptor-Mediated Neurodegeneration.- Evidence for Glutamate Receptor Subtypes from in Vivo Electrophysiology: Studies with HA-966, Quinoxalinediones and Philanthotoxin.- Characterization of Membranal and Purified NMDA Receptors.- Ca++ and Excitatory Amino Acids.- Mechanisms Underlying Excitatory Amino Acid-Evoked Calcium Entry in Cultured Neurons from the Embryonic Rat Spinal Cord.- Topographical Heterogeneity of Glutamate Agonist-Induced Calcium Increase in Hippocampus.- Protection by Natural and Semisynthetic Gangliosides from Ca2+-Dependent Neurotoxicity Caused by Excitatory Amino Acid (EAA) Neurotransmitters.- EAA and in Vivo Development.- Periodic Inward Currents Triggered by NMDA in Immature CA3 Hippocampal Neurones.- GABA Mediated Synaptic Events in Neonatal Rat CA3 Pyramidal Neurons in Vitro: Modulation by NMDA and Non-NMDA Receptors.- Neural Networks and Synaptic Transmission in Immature Hippocampus.- A Role of NMDA Receptors and Ca2+ Influx in Synaptic Plasticity in the Developing Visual Cortex.- Spontaneous and Evoked NMDA-Receptor Mediated Potentials in the Entorhinal Cortex of the Neonate Rat in Vitro.- Learning by Seeing: N-Methyl-D-Aspartate Receptors and Recognition Memory.- The Role of the NMDA Receptor in the Development of the Frog Visual System.- EAA and in Vitro Development.- Excitatory Amino Acids, Growth Factors, and Calcium: A Teeter-Totter Model for Neural Plasticity and Degeneration.- Trophic Effects of Excitatory Amino Acids in the Developing Nervous System.- Mechanisms of Excitatory Amino Acid-Induced Stimulation of GABAergic Synaptic Activity in Cultures from the Rat Superior Colliculus.- The Role of Taurine and Glutamate during Early Postnatal Cerebellar Development of Normal and Weaver Mutant Mice.- Regulation of Neurite Outgrowth from Cerebellar Granule Cells in Culture: NMDA Receptors and Protein Kinase C.- Regulation of GABAA Currents by Excitatory Amino Acids.- EAA and LTP.- Long-Term Potentiation in the Dentate Gyrus in Vivo is Associated with a Sustained Increase in Extracellular Glutamate.- Long Term Potentiation is not Associated with a Sustained Enhanced Release of Glutamate in the Rat Hippocampus in Vivo and in Vitro.- Postsynaptic Mechanisms Involved in Long-Term Potentiation.- Identifying and Localizing Protein Kinases Necessary for LTP.- Delayed Onset of Potentiation in Neocortical EPSPs during Long-Term Potentiation (LTP) - A Postsynaptic Mechanism or Heterogeneous Synaptic Inputs ?.- Local Circuit Connections Mediated by NMDA and Non-NMDA Receptors in Slices of Neocortex.- Modulation of the Responsiveness of Cerebellar Purkinje Cells to Excitatory Amino Acids.- Proteins, Second Messengers and LTP.- The Role of Ependymin in Neuronal Plasticity and LTP.- The Role of Protein Kinase C Substrate B-50 (GAP-43) in Neurotransmitter Release and Long-Term Potentiation.- Different Mechanisms and Multiple Stages of LTP.- Extracellular Proteases and S100 Protein in Long-Term Potentiation in the Dentate Gyrus of the Anaesthetized Rat.- Modulation of the Induction of Long-Term Potentiation in the Hippocampus.- Roles of Metabotropic and Ionotropic Glutamate Receptors in the Long-Term Potentiation of Hippocampal Mossy Fiber Synapses.- Effects of a Neurotrophic Factor (FGF) on Development, Regeneration and Synaptic Plasticity of Central Neurons.- Trans-Synaptophobia.- EAA and Reactive Plasticity.- Kindling, Prenatal Exposure to Ethanol and Postnatal Development Selectively Alter Reponses of Hippocampal Pyramidal Cells to NMDA.- Sprouting of Mossy Fibers in the Hippocampus of Epileptic Human and Rat.- Molecular Markers of Reactive Plasticity.- Transplantation of Developing Hippocampal Neurons to Ischemic and Excitotoxic Lesions of the Adult Rat Hippocampus.- Changes in Synaptic Transmission in the Kindled Hippocampus.- NMDA Receptor Plasticity in the Kindling Model.- NMDA-Receptors are Involved in Synaptic Plasticity Following Partial Denervation of CA1 Hippocampal Cells.- EAA and Anoxia.- Anoxia and NMDA Receptors.- Modulation of ATP Sensitive K+ Channels: A Novel Strategy to Reduce the Deleterious Effects of Anoxia.- Effects of Transient Forebrain Ischemia in Area CA1 of the Gerbil Hippocampus: An in Vitro Study.- Acute Brain Injury, NMDA Receptors, and Hydrogen Ions: Observations in Cortical Cell Cultures.- Mechanisms of Excitatory Amino Acid Neurotoxicity in Rat Brain Slices.

The effects of philanthotoxin-343 (PhTX-343; tyrosyl-butanoyl-spermine) and photolabile analogues of this synthetic toxin on locust (Schistocerca gregaria) skeletal muscle have been investigated using whole muscle preparations (twitch contractions), single muscle fibres (excitatory postsynaptic currents (EPSCs)) and muscle membrane patches containing single quisqualate-sensitive glutamate receptors (qGluR). Analogues containing an azido group attached to either the butanoyl side-chain of PhTX-343 or as a substitute for the hydroxyl moiety of the tyrosyl residue were about 6 fold more potent antagonists than PhTX-343; those with an azido group located at the distal end of the toxin molecule were generally 2-3 fold less potent than PhTX-343. When these compounds were tested in subdued light, they were reversible antagonists of the muscle twitch, EPSC and qGluR. When a muscle was irradiated with U.V. during application of photolabile toxin combined with either neural stimulation of the muscle or L-glutamate application, antagonism of the twitch, EPSC and qGluR was complete and irreversible.

A low molecular weight toxin isolated from the venom of the digger wasp Philanthus triangulum, first noted by T. Piek, is a potent antagonist of transmission at quisqualate-sensitive glutamate synapses of locust leg muscle. This philanthotoxin 433 (PTX-433) has been purified, chemically characterized, and subsequently synthesized along with two closely related analogues. It has a butyryl/tyrosyl/spermine sequence and a molecular weight of 435. Its two analogues, PTX-343 and PTX-334 (the numerals denoting the number of methylenes between the amino groups of the spermine moiety), are also active on the glutamate synapse of the locust leg muscle; PTX-334 was more potent and PTX-343 was less potent than the natural toxin. Such chemicals are useful for studying, labeling, and purifying glutamate receptors and may become models for an additional class of therapeutic drugs and possibly insecticides.

The GluA2 subunit in heteromeric AMPA receptor channels restricts Ca2+ permeability and block by polyamines, rendering linear the current-voltage relationship of these glutamate-gated cation channels. Although GluA2-lacking synaptic AMPA receptors occur in GABA-ergic inhibitory neurons, hippocampal CA1 pyramidal cell synapses are widely held to feature only GluA2 containing AMPA receptors. A controversy has arisen from reports of GluA2-lacking AMPA receptors at hippocampal CA3-to-CA1 cell synapses and a study contesting these findings. Here we sought independent evidence for the presence of GluA2-lacking AMPA receptors in CA1 pyramidal cell synapses by probing the sensitivity of their gated cation channels in wild-type mice and gene-targeted mouse mutants to philanthotoxin, a specific blocker of GluA2-lacking AMPA receptors. The mutants either lacked GluA2 for maximal philanthotoxin sensitivity, or, for minimal sensitivity, expressed GluA1 solely in a Q/R site-edited version or not at all. Our comparative electrophysiological analyses provide incontrovertible evidence for the presence in wild-type CA1 pyramidal cell synapses of GluA2-less AMPA receptor channels.

Mechanically isolated neuronal somata from the thoracic ganglia of the locust Locusta migratoria remain electrophysiologically viable under current-or voltage-clamp in vitro for many hours. Nicotine and muscarine evoke different responses when pressure-microapplied to these somata. The response to acetylcholine is mainly nicotinic but contains a small muscarinic component. The nicotinic (AChl) response is a rapid depolarisation accompanied by a decrease in membrane resistance. In voltage-clamped somata, the current mediating the AChl response is inward over the membrane potential range −30 to − 110 mV, decreasing with depolarisation and with a projected reversal potential of about +20 mV. The muscarinic (ACh2) response is a slow depolarisation accompanied by a decrease in membrane resistance. In voltage-clamped somata, the current mediating the ACh2 response is inward, decreasing to zero at potentials of −80 to −90 mV. The AChl response is evoked by nicotine, anabasine, tetramethylammonium, DMPP and relatively high concentrations of the nitromethylene heterocycle insecticide, PMNI. Suberyldicholine or decamethonium evoke the response only when acetylcholine is present in the bathing saline. Nicotinic antagonists of the AChl response, in descending order of potency, are PMN1 > α-bungarotoxin⩾lobeline⩾mecamylamine>trimethaphan camsylate>chlorisondamine⩾d-tubo-curarine⩾hexamethomium⩾gallamine triethiodide⩾tetracthylammonium. This response is also potently blocked by strychnine and more weakly blocked by δ-philanthotoxin, bicuculline and picrotoxin. The ACh2 response is evoked by muscarine, oxotremorine, arecoline, pilocarpine and, very weakly, by the Mj-selective agonist McN-A-343. Muscarinic antagonists of the ACh2 response, in descending order of potency, are QNB> scopolamine>atropine>4-DAMP (M3) ⩾benactyzine⩾HHSiD (M1/M3) ⩾ pirenzepine (M1). QNX (M1), AF-DX116 (M2), gallamine triethiodide (M2) and methoctramine (M2) are almost or completely inactive. With the exception of pirenzepine and QNX, all of the muscarinic antagonists used in this study also block the nicotinic AChl response with EC50 values in the range 5 to 50μmol l−1, similar to those for δ-philanthotoxin, bicuculline and picrotoxin. Pirenzepine is inactive (10μmol l−1), but QNX is potently active, with an EC50 value of approximately 20 nmol l−1, similar to that of α-bungarotoxin. The extrasynaptic nicotinic and muscarinic receptors of Locusta migratoria neurones are pharmacologically distinct from the corresponding mammalian receptors studied so far.

Prolonged blockade of AMPA-type glutamate receptors in hippocampal neuron cultures leads to homeostatic enhancements of pre- and postsynaptic function that appear correlated at individual synapses, suggesting some form of transsynaptic coordination. The respective modifications are important for overall synaptic strength but their interrelationship, dynamics, and molecular underpinnings are unclear. Here we demonstrate that adaptation begins postsynaptically but is ultimately communicated to presynaptic terminals and expressed as an accelerated turnover of synaptic vesicles. Critical postsynaptic modifications occur over hours, but enable retrograde communication within minutes once AMPA receptor (AMPAR) blockade is removed, causing elevation of both spontaneous and evoked vesicle fusion. The retrograde signaling does not require spiking activity and can be interrupted by NBQX, philanthotoxin, postsynaptic BAPTA, or external sequestration of BDNF, consistent with the acute release of retrograde messenger, triggered by postsynaptic Ca 2+ elevation via Ca 2+ -permeable AMPARs.

GABAergic inhibitory transmission is very abundant within the insect brain. We, therefore, studied the functional properties of the ionotropic GABA receptor of honeybee mushroom body Kenyon cells in vitro. GABA applications elicit rapidly activating and desensitizing currents, which are concentration-dependent between 10 and 500 microM. The mean peak amplitude induced by 500 microM GABA at a holding potential of -110 mV is -1.55 +/- 0.23 nA (SEM, n = 29). The GABA-induced current is mediated by Cl(-) ions because (1) the reversal potential of the GABA-induced current of -40.6 mV is very close to the calculated Nernst potential of chloride (-44.8 mV). (2) With equimolar chloride concentrations the reversal potential shifted to about 0 mV. GABA or muscimol are equally efficient channel agonists, whereas CACA is a partial agonist. Picrotoxin or philanthotoxin (100 microM) completely and reversibly block the GABA-induced current, bicuculline (100 microM) has no effect. Elevating the intracellular Ca(2+) concentration increases the GABA current amplitude. This modulatory effect is blocked by the kinase blocker K 252a, but not by blockers of CaMkinaseII (KN-93), PKC (bisindolylmaleimide) or PKA (KT 5720). We conclude that Kenyon cells express functional GABA receptors whose properties support an inhibitory role of GABAergic transmission.

1. Like the natural toxin, synthetic delta-philanthotoxin, now called PTX-4.3.3 acts as a reversible postsynaptic open ion-channel blocker of the glutamatergic neuromuscular system of the locust. 2. It also inhibits the high-affinity re-uptake of glutamate in the nerve endings and glial cells. 3. To study the structure-activity relationship, three parts of the PTX-4.3.3 molecule were changed. 4. One of these PTX-analogues, trinuoromethyl-PTX-4.3.3, proved to be a more potent postsynaptic blocker. 5. Moreover, compared with PTX-4.3.3 a delayed recovery period is seen with trifluoromethyl-PTX-4.3.3. 6. A number of PTX-analogues were equipotent to PTX-4.3.3 regarding the inhibition of iontophoretically evoked, postsynaptic glutamate potentials. 7. However, complete inactivation was achieved by reducing the length of the polyamine chain, moreover dideaza-PTX-12 was nearly completely inactive and a reduced activity was seen with dephenol-PTX-4.3.3. 8. A decrease of the decay time constant of glutamate potentials, normally seen by open ion-channel blockers in Con A pretreated preparations, was unaffected during application of the latter two analogues. 9. Possibly these two toxins act as weak receptor antagonists. 10. The presynaptic inhibition of the glutamate re-uptake, seemed to be a very specific property of PTX-4.3.3. Only one of the tested analogues (dehydroxy-PTX-4.3.3) exhibited this capacity.

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.