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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 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.

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.

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.

Philanthotoxin-433 (PhTX-433) is a polyamine wasp toxin that antagonizes certain ionotropic receptors noncompetitively. Four analogues of PhTX-433, C-methylated in the polyamine chain, were synthesized from (RS)-1,3-butanediol, two diamine building blocks, and an activated/protected tyrosine derivative. Use of a phosphane reagent more bulky than trimethylphosphane gave a high intramolecular selectivity between primary and secondary hydroxy groups in the Fukuyama−Mitsunobu reaction. Thus, trimethylphosphane proved to be the only phosphane reagent that enabled alkylation of 2-nitrobenzenesulfonamides with a wide range of secondary alcohols, whereas tributylphosphane was selective for primary alcohol groups. This selectivity was utilized to obtain orthogonally protected, asymmetric, branched tetraamines, employed for solution-phase synthesis of philanthotoxin analogues. The branched philanthotoxin analogues thus obtained were tested in an electrophysiological assay using rat brain ionotropic glutamate receptors expressed in Xenopus laevis oocytes. Their potencies proved to be similar to the corresponding nonbranched analogues. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003)

Several polyamine derivatives were synthesized in order to produce novel antagonists of muscular nicotinic acetylcholine receptors. Their affinities were compared with those of philanthotoxin PhTX-343.

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.

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.

In this brief review on Koji Nakanishi's remarkable career in natural products chemistry, we have highlighted a number of his accomplishments that illustrate the broad diversity of his interests. These include the isolation, structure determination, and biological mechanism of action of many natural products including the triterpenoid pristimerin; the diterpenoid ginkgolides; insect and crustacean molting hormones; phytoalexins; the toxic red tide principle brevetoxin; the vanadium tunicate pigments; philanthotoxin from killer wasps; antisickling agents; mitomycin DNA adducts; insect antifeedants; a mitotic hormone, the small molecule fish attractants from the sea anemone; new isolation and purification technologies; molecular chemistry of vision; age-related macular degeneration; and the development of the exciton circular dichroism (CD) chirality method for microscale determination of absolute configuration of natural products and chirality of other chiral molecules and supramolecular assembly.

A study was undertaken to characterize the plasticity of AMPA receptor (AMPAR)-mediated neurotransmission in the hippocampus during status epilepticus (SE).SE was induced by pilocarpine, and animals were studied 10 minutes (refractory SE) or 60 minutes (late SE) after the onset of the first grade 5 seizures. AMPAR-mediated currents were recorded from CA1 pyramidal neurons and dentate granule cells (DGCs) by voltage clamp technique. The surface expression of GluA2 subunit on hippocampal membranes was determined using a biotinylation assay. GluA2 internalization and changes in intracellular calcium ([Ca](i)) levels were studied in hippocampal cultures using immunocytochemical and live-imaging techniques. AMPAR antagonist treatment of SE was evaluated by video and electroencephalography.AMPAR-mediated currents recorded from CA1 neurons from refractory and late SE animals were inwardly rectifying, and philanthotoxin-sensitive; similar changes were observed in recordings obtained from DGCs from refractory SE animals. GluA2 subunit surface expression was reduced in the hippocampus during refractory and late SE. In cultured hippocampal pyramidal neurons, recurrent bursting diminished surface expression of the GluA2 subunit and enhanced its internalization rate. Recurrent bursting-induced increase in [Ca](i) levels was reduced by selective inhibition of GluA2-lacking AMPARs. GYKI-52466 terminated diazepam-refractory SE.During SE, there is rapid, ongoing plasticity of AMPARs with the expression of GluA2-lacking AMPARs. These receptors provide another source of Ca(2+) entry into the principal neurons. Benzodiazepam-refractory SE can be terminated by AMPAR antagonism. The data identify AMPARs as a potential therapeutic target for the treatment of SE.

After fear conditioning, plastic changes of excitatory synaptic transmission occur in the amygdala. Fear-related memory also involves the GABAergic system, although no influence on inhibitory synaptic transmission is known. In the present study we assessed the influence of Pavlovian fear conditioning on the plasticity of GABAergic synaptic interactions in the lateral amygdala (LA) in brain slices prepared from fear-conditioned, pseudo-trained and naive adult mice. Theta-burst tetanization of thalamic afferent inputs to the LA evoked an input-specific potentiation of inhibitory postsynaptic responses in projection neurons; the cortical input was unaffected. Philanthotoxin (10 microM), an antagonist of Ca2+-permeable AMPA receptors, disabled this plastic phenomenon. Surgical isolation of the LA, extracellular application of a GABA(B) receptor antagonist (CGP 55845A, 10 microM) or an NMDA receptor antagonist (APV, 50 microM), or intracellular application of BAPTA (10 mM), did not influence the plasticity. The plasticity also showed as a potentiation of monosynaptic excitatory responses in putative GABAergic interneurons. Pavlovian fear conditioning, but not pseudo-conditioning, resulted in a significant reduction in this potentiation that was evident 24 h after training. Two weeks after training, the potentiation returned to control levels. In conclusion, a reduction in potentiation of inhibitory synaptic interactions occurs in the LA and may contribute to a shift in synaptic balance towards excitatory signal flow during the processes of fear-memory acquisition or consolidation.

Philanthotoxin?433 (PhTX?433) is a known potent inhibitor of ionotropic glutamate receptors, and analogues have been synthesised to identify more potent and selective antagonists. Herein we report the synthesis of four PhTXs with a cyclopropane moiety introduced into their polyamine chain, and their inhibition of an ??amino?3?hydroxy?5?methyl?4?isoxazolepropionic acid (AMPA) receptor subtype by using two?electrode voltage?clamp assays on Xenopus oocytes expressing the GluA1flop subunit. All analogues were found to be more potent than PhTX?343, with trans?cyclopropyl?PhTX?343 being the most potent (?28?fold) and cis?cyclopropyl?PhTX?343 least potent (?4?fold). Both cis? and trans?cyclopropyl?PhTX?444 had intermediate potency (both ?12?fold). Molecular modelling indicates that a cyclopropane moiety confers a favourable steric constraint to the polyamine part, but this is compromised by a cis conformation due to enhanced intramolecular folding. Elongated PhTX?444 analogues alleviate this to some extent, but optimal positioning of the amines is not permitted.