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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.

Philanthotoxin (PhTX-433), a potent noncompetitive inhibitor of the L-glutamate receptors and the nicotinic acetylcholine receptors of vertebrates and invertebrates, has a butyryl-tyrosyl-thermospermine structure. Several synthetic analogs of PhTX with hydrophobic alkyl branches in the polyamine chain exhibit 6- to 10-fold enhanced activities to various receptors. Because the branched analogs exhibit such unique activities and because of their importance in tertiary structural studies of ligand/receptor binding, methods for preparing branched PhTX analogs, including photolabile analogs, are presented

The synthetic methods for 27 novel and photolabile philanthotoxin analogs are described. Most analogs were synthesized by two general methods with modifications of these methods where necessary.

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.

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.

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.

Calcium ions play an important role in many signalling pathways involved in normal cell metabolism. Pertrebations of normal Ca++ signalling may also play a pivotal role in the initiation of cell death. In these studies I have examined the influx of 45Ca++ into the extensor tibiae muscle of the locust (Schistocerca gregaria ). 45Ca++ entry could be stimulated by the addition of glutamate receptor-agonists or by activation of voltage activated calcium channels. L-glutamate, L-quisqualate and NMDA stimulated the influx of 45Ca++ while L-aspartate had only a small effect. DL-ibotenate, kainate, AMPA and glycine had no effect on 45Ca++ uptake (all agonists were tested at concentrations up to (100μM). Glycine (1μM) enhanced the 45Ca++ entry induced by NMDA and L-glutamate. Only the glycine potentiation of L-glu-stimulated responses was abolished in the presence of Mg++ (2mM) or AP5 (10μM) whereas the NMDA-stimulated response was completely abolished by these agents. These finding suggests that in the presence of glycine, L-glutamate may activate NMDA receptors and that in the absence of glycine L-glu-stimulated 45Ca++ entry occurs via activation of the qGluR. Depolarisation of the extensor tibiae muscles (50mM KCl) stimulated 45Ca++ influx by activation of voltage-sensitive calcium channels. Philanthotoxin-343 (0.1μM) had no effect on depolarisation activated calcium entry, however, nifedipine (1μM) an L-type calcium channel antagonist inhibited this Ca++ influx. Nifedipine did not inhibit L-glu-stimulated Ca++ entry suggesting that in these muscles L-type Ca++ channels are not involved in the Ca++ influx pathway following G1uR activation. Philanthotoxin-433 (PhTX-433) and many of its synthetic analogues are potent inhibitors of locust GluR. In the future these analogues may prove as useful potential neuroprotective agents or as novel pesticides. Over 100 analogues of PhTX-433 have been synthesized with changes made in the four regions of the structure, the thermospermine moiety, the tyrosyl moiety, the butyryl moiety and the terminal amino moiety. The effects of different concentrations (10-4M to 10-14M) of synthetic analogues of PhTX-433 (PhTX-343, PhTX-343-Arg, PhTX-4) were investigated in the 45Ca++ influx assay using locust extensor tibiae muscle. PhTX-343-Arg was more potent (IC50= - 7x10-9) than PhTX- 343 (IC50= - 10-8M) or PhTX-4 in blocking 45Ca H uptake. These findings were further supported by electrophysiological studies. The interaction of these synthetic analogues of philanthotoxin with GluR of locust muscle were further investigated by examining the effect of these compounds on evoked excitatory post synaptic potentials. In recent years control of ticks have been very important issue because of the social and economical damage they cause. Neuromuscular transmission is a main target site for the chemical control of many pests. Philanthotoxin and its analogues block the glutamate receptors which are involved in arthropod neuromuscular transmission and thus may prove useful as novel pesticides. The action of synthetic analogues of philanthotoxin (C7PhTX-343, DNP12-, PhTX-343 and PhTX-343) were examined on evoked excitatory postsynaptic potential in tick coxal muscle. These compounds all antagonized the evoked EPSP. C7PhTX-343 and DNP12-PhTX-343 exhibited same potency (IC50 = 10-8M) and both were more potent than PhTX-343 (IC50 ='2X10-5M). In recent years Xenopus oocyte has taken over a new role as a test tube for the study of the biogenesis, functional architecture and modulation of plasma membrane protein. Attempts were made to express mRNA from embryonic tissue of tick and locust leg muscle in to Xenopus oocyte for pharmacological studies. Xenopus oocytes failed to translate RNA faithfully and efficiently from these sources. Rat brain RNA injected oocytes used as control, expressed routinely.

Philanthotoxin-433 (PhTX-433) is an active component of the Egyptian solitary digger wasp, Philanthus triangulum, venom which non-selectively inhibits several excitatory ion channels. With the aim of improving potency and selectivity, several synthetic analogues were developed based on the single or multiple modifications in the hydrophilic polyamine tail and hydrophobic aromatic head group of the PhTX. \ud In the first part of this study, we investigated the pharmacological actions of two synthetic analogues, Philanthotoxin-343 (PhTX-343) and Philanthotoxin-12 (PhTX-12), on mammalian hetero- and homooligomeric nicotinic acetylcholine receptor (nAChR) subunit combinations expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX-analogue were studied electrophysiologically using two-electrode voltage-clamp at three different membrane holding potentials (VH = -60 mV, -80 mV and -100 mV). Concentration-inhibition curves were constructed and IC50 values estimated for each holding potential. The IC50 (95% CI, n=oocytes) values for PhTX-343 inhibition of α3β4, α3β2, α4β2, α4β4, α7, α3β4(F255V), α4β2(V253F) and α1β1δγ peak currents at -100 mV were 0.07 µМ (0.05-0.10 µМ, n=9), 3.20 µМ (1.69-6.26 µМ, n=8), 0.14 µМ (0.09-0.21 µМ, n=7), 0.28 µМ (0.17-0.46 µМ, n=6), 8.7 µМ (6.9-11.0 µМ, n =9), 0.01 µМ (0.01-0.02 µМ, n=8), 0.10 µМ (0.06-0.16 µМ, n=8) and 3.2 µМ (2.5-4.1 µМ, n=9) respectively; for PhTX-12 they were 2.03 µМ (1.22-3.26 µМ, n=8), 74.0 µМ (21-259 µМ, n=10), 1.6 µМ (0.7-3.6 µМ, n=11), 1.82 µМ (0.81-4.11 µМ, n=9), 12.1 µМ (9.0-16.0 µМ, n =10), 1.0 µМ (0.5-2.0 µМ, n=5), 3.4 µМ (1.3-8.7 µМ, n=9) and 5.0 µМ (2.8-8.8 µМ, n=8) respectively; i.e. in contrast to M-nAChR, PhTX-343 was more potent than PhTX-12 in all cases. All IC50s were lower when inhibition was measured after 1 minute of application indicating use dependence. For inhibition of heteromeric nAChRs, the peak potency of PhTX-343 was strongly augmented by holding the cell at more negative VH while this was not the case for PhTX-12 where only weak voltage-dependence was observed. The inhibition of homomeric α7 receptors by PhTX-343 was voltage-independent, whereas the block by PhTX-12 was voltage-dependent. In addition, these two synthetic analogues were different in their recovery rates (PhTX-12 was generally faster than PhTX-343) but they were similar in their mechanism of action, non-competitive inhibition.\ud In the second part of the project, I explored the structure-activity relations of twenty one synthetic analogues, with a view to improving their activity and selectivity on rat neuronal α4β2 and α3β4 nAChRs at VH = -100 mV. We showed that the presence of positive charge in the polyamine tail of PhTX compounds is essential for nAChR subtype selectivity and its removal made the molecule lose its selectivity. Also, it appears that adding a bulky group to the terminal ammonium drastically reduced activity whereas a similar addition to the head region it increased their potency. In addition, we identified the key regions and substitutions responsible for increasing PhTX activity, cyclohexylalanine in place of tyrosine, and selectivity, phenyl group. Analogues having cyclohexylalanine and a phenolic group in the head region showed IC50 values in the low nano-molar and pico-molar (160-400 pM) range. These data suggest that PhTXs could serve as lead compounds for highly potent and selective inhibitors of N-nAChRs.

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, “spontaneous” transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.

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.

Recent studies in Jurkat T cells and in rat basophilic leukemia cells revealed an Mg(2+)-inhibited cation (MIC) channel that has electrophysiological properties similar to TRPM7 Eyring rate model expressed exogenously in mammalian cells. Here we compare the characteristics of several polyvalent cations and Mg(2+) to block monovalent MIC current from the outside. Putrescine, spermidine, spermine, PhTX-343 (a derivative of the naturally occurring polyamine toxin philanthotoxin), and Mg(2+) each blocked in a dose- and voltage-dependent manner, indicating a blocking site within the electric field of the ion channel. Spermine and the relatively bulky PhTX-343 exhibited voltage dependence steeper than that expected for the number of charges on the molecule. Polyamines and Mg(2+) are permeant blockers, as judged by relief of block at strongly negative membrane potentials. Intracellular dialysis with spermine (300 microM) had no effect, indicating an asymmetrical pore. At the single-channel level, spermine and Mg(2+) induced flickery block of 40-pS single channels. I/V characteristics and polyamine block are similar in expressed TRPM7 and in native MIC currents, consistent with the conclusion that native MIC channels are composed of TRPM7 subunits. An Eyring rate model is developed to account for I/V characteristics and block of MIC channels by polyvalent cations from the outside.

Background Ca2+-permeable non-N-methyl-D-aspartate receptors are found in the spinal dorsal horn and represent a presumptive target for glutamatergic transmission in nociceptive processing. This study characterized the analgesic profile associated with the blockade of these spinal receptors by intrathecally delivered agents known to act at these receptors, the spider venom Joro toxin (JST) and philanthotoxin. Methods Philanthotoxin (0.5, 2.5, or 5 microg) or JST (5 microg) was given spinally before thermal injury to the paw. JST (5 microg) was also given 10 min before subcutaneous formalin injection, after intraplantar administration of carrageenan, and to rats that were allodynic due to tight ligation of spinal nerves. Lower doses of JST (0.25 and 1.0 microg) were given before formalin injection and testing of thermal latencies. Thermal latencies were measured using a Hargreaves box, mechanical thresholds using von Frey hairs, and formalin response by means of counting flinches. Results Both agents blocked thermal injury-induced mechanical allodynia. JST (5 microg) given 1 h after carrageenan blocked induction of thermal hyperalgesia and mechanical allodynia. JST (5 microg) had no effect in the formalin test, on allodynia after spinal nerve ligation, or when given 3 h after carrageenan. The lowest dose (0.25 microg JST) at pretreatment intervals of 60-120 min resulted in modest hypoalgesia during phase 1 formalin and thermal testing. Conclusions The behavioral effect of intrathecal Ca2+-permeable non-N-methyl-D-aspartate antagonists indicates an important role for this spinal receptor in regulating hyperalgesic states induced by tissue injury and inflammation and reveals an action that is distinct from those observed with other glutamate receptor antagonists.

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.

The synthesis of the non-competitive glutamate receptor antagonist, argiotoxin-636 is described. Furthermore, synthetic routes are presented for the preparation of structural analogs including argiotoxin-philanthotoxin hybrids. Biological activities on glutamate receptors are discussed.

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.