Selective blockade of Ca2+ permeable AMPA receptors in CA1 area of rat hippocampus.

Using whole cell patch-clamp recording from pyramidal cells and interneurons in the CA1 area of hippocampal slices, the effect of IEM-1460, a selective channel blocker of Ca2+ permeable AMPA receptors (AMPARs), on postsynaptic currents (PSCs) was studied. Excitatory postsynaptic currents (EPSCs) were evoked by stimulation of Schaffer collaterals (SCs) in the presence of APV and bicuculline to pharmacologically isolate the EPSCs mediated by AMPAR activation. IEM-1460 (50 microM) did not affect the amplitude of EPSCs in CA1 pyramidal cells but reversibly decreased their amplitude in interneurons of pyramidal layer (15 cells), radiatum (37 cells) and border radiatum-lacunosum-moleculare (R-LM) (55 cells) layers. The ability of IEM-1460 to decrease EPSC amplitude correlated with EPSC rectification properties in CA1 interneurons, providing evidence for synaptic localization of Ca2+ permeable AMPARs at the SC synaptic input. Independent of their localization, the majority of interneurons studied exhibited only modest sensitivity to IEM-1460 (EPSC amplitude decreased by less than 30%), while in 15% of interneurons IEM-1460 induced more than 50% reduction in EPSC amplitude. To reveal possible afferent-specific localization of Ca2+ permeable AMPARs on R-LM interneurons, the effect of IEM-1460 on EPSCs evoked by stimulation of SC was compared with that of perforant path (PP). Although average sensitivities did not differ significantly, in 61% of R-LM layer interneurons, the SC-evoked EPSCs exhibited higher sensitivity to IEM-1460 than the PP-evoked EPSCs. Moreover, in 54% of R-LM layer interneurons the EPSCs evoked by SC stimulation were complex, having an initial peak followed by one or several late components. Kinetics, latency distribution and reversal potential of late components suggest di- and polysynaptic origin of the late components. Late EPSCs were strongly and reversibly inhibited by IEM-1460 indicating that Ca2+ permeable AMPARs are involved in the indirect excitation of R-LM layer interneurons. Despite the ability to decrease the excitatory synaptic input to interneurons, IEM-1460 did not affect interneuron-mediated inhibitory postsynaptic currents (IPSCs) evoked in pyramidal neurons by SC stimulation. These data suggest that interneurons with a synaptic input highly sensitive to IEM-1460 do not contribute specifically to the feed-forward inhibition of hippocampal pyramidal neurons.

Analysis of the excitatory and inhibitory components of postsynaptic currents recorded in pyramidal neurons and interneurons in the rat hippocampus.

Postsynaptic currents recorded from interneurons and pyramidal cells in hippocampal slices by local voltage clamping were found to be the sum of excitatory (EPSC) and inhibitory (IPSC) components. An approach allowing quantitative assessment of the amplitude and time course of EPSC and IPSC without pharmacological blockade of the major postsynaptic receptors involved in generating these currents was developed. The approach is based on the existence of a significant difference between reversion potentials of cationic and anionic currents and the presence of a linear zone in the voltage-current characteristics of responses to excitatory and inhibitory transmitters. Comparison of the results of this calculation-based method with those of classical pharmacological analysis of the excitatory and inhibitory components of postsynaptic currents showed them to be virtually identical, which allows synaptic currents in defined neurons to be studied without altering the state of synaptic connections throughout the brain slice. IPSC was found to make a smaller contribution to the total postsynaptic current recorded in interneurons as compared with pyramidal neurons in rat hippocampal field CA1.

[Analysis of excitatory and inhibitory components of postsynaptic currents recorded in the pyramidal neurons and interneurons of the rat hippocampus]. / Analiz vozbuzhdaiushchikh i tormoznykh komponentov postsinapticheskikh tokov, registriruemykh v pyramidnykh neironakh i interneironakh gippokampa krysy.

Postsynaptic currents recorded in the whole-cell configuration with patch-clamp method are actually the sum ofexcitatory (EPSC) and inhibitory (IPSC) components. An approach has been developed allowing the quantitative evaluation of the amplitude and the time course of EPSC and IPSC without treatment of the brain slice with pharmacological inhibitors. The approach is based on the substantial difference in the equilibrium potential values of incoming cationic and anionic currents as the existence of linear regions of corrent-voltage dependence of these currents. The comparison of the results obtained with the classical pharmacological method and with the suggested one demonstrated their coincidence. It allows analysing the postsynaptic currents in sigle neurons without altering the synaptic transmission in the whole brain slice. The contribution of inhibitory currents in the composite synaptic response of intemeurons turned out to be smaller in comparison with pyramidal neurons of CA1 field of the rat hippocampus.

Studies of the structure of glutamate receptor ion channels and the mechanisms of their blockade by organic cations.

The structural determinants for blockade of the AMPA and NMDA subtypes of glutamate receptors were studied by analysis of structural-functional relationships in a series of mono- and dicationic compounds. The results showed that the hydrophobic and nucleophilic components of the blocker binding sites are located close to each other in the channel of the NMDA receptor, while they are spatially distant in the channel of the AMPA receptor. Molecular mechanical methods were used to construct models of these channels satisfying these topographic criteria and providing adequate descriptions of the binding of the channel blockers. According to the models, binding of blockers to the NMDA channel occurs in the selective filter of the channel (the N/Q/R site). The nucleophilic region of the AMPA channel is formed by the oxygen atoms of glycine residues in position +2 relative to the selective filter. Identification of the major relationships between the molecular structure of the ion channels of these glutamate receptor subtypes and their blockade by organic cations allows the further synthesis of AMPA and NMDA channel blockers with specified levels of activity and selectivity to be directed.

Characterization of acid-sensitive ion channels in freshly isolated rat brain neurons.

Transient proton-activated currents induced by rapid shifts of the extracellular pH from 7.4 to < or =6.8 were recorded in different neurons freshly isolated from rat brain (hypoglossal motoneurons, cerebellar Purkinje cells, striatal giant cholinergic interneurons, hippocampal interneurons, CA1 pyramidal neurons and cortical pyramidal neurons) using whole-cell patch clamp technique. Responses of hippocampal CA1 pyramidal neurons were weak (100-300 pA) in contrast to other types of neurons (1-3 nA). Sensitivity of neurons to rapid acidification varied from pH(50) 6.4 in hypoglossal motoneurons to 4.9 in hippocampal interneurons. Proton-activated currents were blocked by amiloride (IC(50) varied from 3.6 to 9.5 microM). Reversal potential of the currents was close to E(Na), indicating that the currents are carried by sodium ions. The data obtained suggest that the proton-activated currents in the neurons studied are mediated by acid-sensitive ion channels. Strong acidification (pH<4) induced biphasic responses in all neuron types: the transient current was followed by a pronounced sustained one. Sustained current was not blocked by amiloride and exhibited low selectivity for sodium and cesium ions. Slow acidification from pH 7.4 to 6.5 did not induce detectable whole-cell currents. At pH 6.5, most of the channels are desensitized and responses to fast pH shifts from this initial level are decreased at least 10 times. This suggests that slow acidification which is well known to accompany some pathological states should rather desensitize than activate acid-sensitive ion channels and depress their function. Our results provide evidence for a widespread and neuron-specific distribution of acid-sensitive ion channels in the brain. The large amplitudes and transient character of currents mediated by these channels suggest that they could contribute to fast neuronal signaling processes.

Structural characteristics of ionotropic glutamate receptors as identified by channel blockade.

The channels of four types of ionotropic glutamate receptor (NMDA receptors and Ca-permeable AMPA receptors of rat brain neurons, and cation-selective receptors from mollusk neurons and insect postsynaptic muscle membranes) and two subtypes of nicotinic cholinoreceptor (from frog neuromuscular junctions and cat sympathetic ganglia) were studied. The structural characteristics of channels determining their susceptibility to blockade by organic mono- and dications were identified. These studies used homologous series of adamantane and phenylcyclohexyl derivatives. These experiments showed that the receptors studied here could be divided into two groups. The first group included the AMPA receptor and the mollusk and insect receptors. These were characterized by the lack of effect on the part of monocations and a strong relationship between the activity of dications and the distance between nitrogen atoms. The second group included the NMDA receptor and both subtypes of the nicotinic cholinoreceptor (muscular and neuronal). Here, conversely, the activity of monocations and dications, regardless of their lengths, were essentially identical. A model for the binding sites of blockers in channels is proposed, which takes these observations into account.

[Structure of glutamate receptor ion channels and mechanisms of their blockade by organic cations]. / Issledovanie stroeniia ionnykh kanalov retseptorov glutamata i mekhanizmov ikh blokady organicheskimi kationami.

Structural determinants of blocking the glutamate receptors of AMPA and NMDA subtypes, were studied. Close location of hydrophobic and ammonium groups is necessary for affective blocking of the NMDA receptor channels, whereas blockers of the AMPA receptor channels have a distance of about 10 angstroms between these two groups. Models of the channels meeting these topographic data have been devised using a molecular mechanics approach. The accomplished studies revealed molecular basis of channel blockade of the NMDA and AMPA receptors. This may allow designing predictable new blocking compounds with a desired selectivity.

Pharmacological analysis of the subunit composition of the AMPA receptor in hippocampal neurons.

Experiments were performed on isolated neurons from hippocampal field CA1 and the dentate fascia to identify the subunit composition and distribution of splicing variants of AMPA receptor subunits. Currents evoked by the application of kainate were recorded using a whole-cell patch clamping method. The presence of GluR2 subunits in receptors was associated with a sharp reduction in the activity of the selective channel blocker IEM-1460. The composition of flip versions of subunits was assessed using cyclothiazide. AMPA receptors in the major cell types (pyramidal and granule cells) had low sensitivity to IEM-1460, while AMPA receptors of other cells (interneurons) had high or intermediate sensitivity. Cyclothiazide had strong potentiating effects on the main cell types in both structures as compared with interneurons. Thus, there is a correlation between the sensitivities of hippocampal neurons to IEM-1460 and cyclothiazide. The main cell types in both structures expressed large quantities of the GluR2 subunit in their AMPA receptors, with high levels of flip subunits, as compared with the other cell types, in which GluR2 subunits were virtually absent and the flop version predominated. This appears to reflect the functional features of different types of neurons.

[Structural characteristics of ionotropic glutamate receptors revealed by channel blockade]. / Strukturnye osobennosti ionotropnykh glutamatnykh retseptorov, vyiavliaemye blokadoi kanalov.

The topography of the channel binding site in glutamate receptors (AMPA and NMDA types of rat brain neurons, receptors of molluscan neurons and insect muscle), and in two subtypes of nicotinic cholinoreceptors (in frog muscle and cat sympathetic ganglion), has been investigated by comparison of the blocking effects of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The channels studied can be divided into two groups. The first one includes AMPA receptor and glutamate receptors of mollusc and insect, and is characterised by the absence of activity of monocationic drugs and the strong dependence of dicationic once on the internitrogen distance in the drug molecule. The second group includes NMDA receptor and both nicotinic cholinoreceptors. Contrary, here the blocking potency of monocations and dications are practically equal irrespective of molecule length. The data obtained suggest that hydrophobic and nucleophilic components of the binding site are located close to each other in the channels of the NMDA receptor type but are separated by approximately 10 A in the AMPA receptor channel.

Ca2+-dependent desensitization of AMPA receptors.

The effect of changes in the external concentrations (0.4-10 mM) of Ca2+ ions on AMPA receptors (AMPARs) of different subunit composition was studied on freshly isolated rat brain neurones. Ca2+ produces rapid and reversible voltage-independent inhibition of AMPARs. Ca2+-permeable and Ca2+-impermeable AMPARs are equally sensitive to external Ca2+ suggesting that the effect is not addressed to the ion channel. The inhibition of responses evoked by AMPA is significantly larger than those evoked by kainate or glutamate. Cyclothiazide and aniracetam, which are known to prevent AMPAR desensitization, both greatly diminish inhibition of AMPARs by Ca2+. Cyclothiazide is more potent than aniracetam in both preventing of AMPAR desensitization and protecting against the Ca2+ inhibitory effect on hippocampal pyramidal cells. On giant cholinergic interneurones of striatum, aniracetam but not cyclothiazide significantly prevents inhibition by Ca2+. This agrees with available data on relative abundance of flip and flop splice variants in these cell types. The results suggest that Ca2+ may allosterically increase AMPA receptor desensitization independently on subunit composition and splice variants.

Voltage-dependent block of native AMPA receptor channels by dicationic compounds.

1. The kinetics of open channel block of GluR2-containing and GluR2-lacking AMPA receptors (AMPAR) by dicationic compounds (IEM-1460, IEM-1754, and IEM-1925) have been studied in rat hippocampal neurones using whole-cell patch clamp recording and concentration-jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation. 2. The dicationic compounds were approximately 100 - 200 times more potent as blockers of GluR2-lacking AMPAR than as blockers of GluR2-containing AMPAR. The subunit specificity of channel block is determined by the blocking rate constant of a dicationic compound, whereas differences in unblocking rate constants account for differences in potency. 3. Hyperpolarization may decrease the block produced by IEM-1460 and IEM-1754 block due to the voltage-dependence of the unblocking rate constants for these compounds. This suggests that dicationic compounds permeate the AMPAR channel at negative membrane potentials. The effect was particularly apparent for GluR2-lacking AMPAR. These findings indicate that the presence of GluR2-subunit(s) in AMPAR hinders the binding of the cationic compounds and their permeation through the channel. 4. The most potent compound tested was IEM-1925. The presence of a phenylcyclohexyl moiety instead of an adamantane moiety, as in IEM-1460 and IEM1754, is probably responsible for the higher potency of IEM-1925. Dicationic compounds are important not only as pharmacological tools, but also as templates for the synthesis of new selective AMPAR blockers which may be potential therapeutic agents.

The open channel blocking drug, IEM-1460, reveals functionally distinct alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors in rat brain neurons.

The properties of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors were examined in various cell types isolated from young rat hippocampus, striatum and cerebellum using patch-clamp and fast application techniques. A dicationic adamantane derivative, IEM-1460, reversibly inhibited kainate-induced currents. In the presence of 100 microM IEM-1460, kainate currents in striatal giant cholinergic interneurons and hippocampal non-pyramidal neurons were inhibited by 95% and 81%, respectively, at Vh = - 70 mV. Striatal GABAergic principal cells, hippocampal pyramidal neurons and cerebellar Purkinje cells had low sensitivity to IEM-1460 (inhibition by 4-15%). Analysis of averaged data from the cell types studied revealed a highly significant positive correlation (r= 0.93, P < 0.01) between percentage inhibition by 100 microM IEM-1460 and relative calcium permeability of AMPA receptors, P(Ca)/P(Na). Also, within each brain structure, the sensitivity of IEM-1460 block was lower the stronger the outward rectification of kainate currents. Some hippocampal neurons exhibited intermediate sensitivity to IEM-1460. Kainate currents were suppressed by 40% in the presence of 100 microM IEM-1460. Meanwhile, AMPA receptors in this cell type had low calcium permeability (P(Ca)/P(Na) = 0.13) and demonstrated outwardly rectifying kainate currents. The interrelation of different properties of AMPA receptors considering their assembly is discussed. The data obtained suggest that IEM-1460 may be a convenient and promising marker of native AMPA receptor assembly: it selectively inhibits Ca(2+)-permeable, GluR2-lacking AMPA receptors.

Characterization of AMPA receptor populations in rat brain cells by the use of subunit-specific open channel blocking drug, IEM-1460.

Dicationic adamantane derivative, IEM-1460, which selectively blocks GluR2-lacking, Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors, was used to characterize the distribution of AMPA receptors among populations of rat brain cells. IEM-1460 inhibited kainate-induced inward currents (at -80 mV) in a dose-dependent manner. IEM-1460 concentrations producing 50% inhibition of kainate-induced current amplitude (IC50) varied greatly depending on the cell type studied. Striatal giant cholinergic interneurons and putative Bergmann glial cells isolated from the cerebellum were found to be highly sensitive to IEM-1460 block (IC50=2.6 microM), indicating the expression of GluR2-lacking AMPA receptor subtype. Among hippocampal and cortical non-pyramidal neurons, there were cell-to-cell differences in the pattern of AMPA receptor subtype expression. Some cells which are known to express AMPA receptors lacking GluR2 subunit exhibited high sensitivity of IEM-1460 block (IC50 about 1 microM) but in the others, the part of AMPA receptor population seemed to be represented by GluR2-having receptor subtype. The latter subtype was mainly expressed by pyramidal neurons isolated from hippocampus (IC50=1102 microM) and sensorimotor cortex (IC50=357 microM) which showed low affinity for IEM-1460 block. In conclusion, IEM-1460 can be utilized as an indicator of the distribution of AMPA receptor subtypes among populations of rat brain cells, and pharmacological detection of the absence of GluR2 subunit in AMPA receptor assembly can provide useful information for the interpretation of physiological events.

[Pharmacologic analysis of the subunit composition of AMPA-receptors in the hippocampal neurons]. / Farmakologicheskii analiz sub''edinichnogo sostava AMPA-retseptorov v neironakh gippokampa.

Subunit composition and abundance of flip version of different AMPA receptor subunits were studied in neurons acutely isolated from hippocampal area CA1 and dentate gyrus. Whole cell recordings were made to record kainate unduced currents. Presence of GluR2 in the receptor complex led to significant decrease of selective channel blocker IEM-1460 potency. Flip versions of AMPA receptor subunits were discriminated on the basis of their sensitivity to cyclothiazide. Principal cell AMPA receptors in both areas were characterized by low sensitivity to IEM-1460 while AMPA receptors of nonprinciple cells exhibited high or intermediate sensitivity to IEM-1460. We observed significantly larger potentiating effect of cyclothiazide on principal cells. Our data indicate that there is a correlation between low sensitivity to IEM-1460 and high sensitivity to cyclothiazide among AMPA receptors of different cells. Principal cells in both regions possess more GluR2 subunits in their AMPA receptor complexes and more abundant flip versions of their subunits in comparison with nonprincipal cells. This correlation is obviously related to functional pecularities of different neurons.

Selective block of AMPA/kainate receptors of hippocampal interneurons as a new approach to the investigation of inhibitory system.

Effects of open channel blockers of AMPA/kainate receptors have been examined using whole cell recordings and kainate application in the neurons freshly isolated by vibrodissociation from the rat hippocampal slice preparation. Although the hippocampal neurons differed little in the voltage-current relations and sensitivity to kainate, a prominent difference was found in their susceptibility to the blocking action of adamantane derivatives studied. The pyramidal neurons had low sensitivity to the open channel blockers but the neurons which might be assigned most probably to the group of inhibitory interneurons proved to be highly sensitive. A group of neurons of intermediate sensitivity have also been found. The ability of the same blocking drugs to depress the excitatory inputs in the inhibitory interneurons has been demonstrated in the experiments on the hippocampal slice preparation. Enhancement of the field spike and excitatory postsynaptic potential amplitude was observed in the presence of adamantane derivatives. An additional treatment of the preparation with a GABA receptor antagonist, bicuculline, did not potentiate this effect. In conclusion, the observed difference in the pharmacological properties of inhibitory interneurons may be effectively used for detailed analysis of the brain synaptic transmission.

Block of open channels of recombinant AMPA receptors and native AMPA/kainate receptors by adamantane derivatives.

1. The effects of two adamantane derivatives, 1-trimethylammonio-5-(1-adamantane-methyl-ammoniopentane dibromide) (IEM-1460) and 1-ammonio-5-(1-adamantane-methylammoniopentane dibromide) (IEM-1754) on kainate-induced currents were studied in Xenopus oocytes expressing recombinant ionotropic glutamate receptors and in freshly isolated neurones from rat hippocampal slices. 2. The adamantane derivatives caused use- and voltage-dependent block of open channels of recombinant AMPA receptors. This antagonism was dependent on receptor subunit composition; channels gated by recombinant, homomeric GluR1 and GluR3 receptors exhibited a higher sensitivity to block than those gated by receptors containing edited GluR2 subunits. In the former cases, IEM-1460 had an IC50 of 1.6 microM at a holding potential (Vh) of -80 mV and IEM-1754 was 3.8 times less potent than IEM-1460. In contrast, 100 microM IEM-1460 inhibited responses to 100 microM kainate of receptors containing edited GluR2 subunits by only 7.8 +/- 2.4% (n = 5 oocytes at a Vh of -80 mV. 3. Native AMPA/kainate receptors in isolated hippocampal cells were inhibited by adamantane derivatives in a use- and voltage-dependent manner. This antagonism was dependent on cell type: pyramidal neurones were less sensitive to IEM-1460 (IC50 = 1617 microM at Vh = -80 mV) than interneurones (IC50 = 1.6 microM at Vh = -80 mV). IEM-1460 and IEM-1754 were equipotent when applied to pyramidal neurones, but IEM-1754 was less potent (approximately 3 times) than IEM-1460 when applied to interneurones. 4. It is concluded that the presence of the edited GluR2 subunit in recombinant AMPA receptors and native AMPA/kainate receptors inhibits channel block by organic cations and that adamantane derivatives are potentially valuable tools for identifying classes of AMPA/kainate receptors and their roles in synaptic transmission.

[Temperature-evoked long-term potentiation in hippocampal slices]. / Vyzvannaia temperaturoi dlitel'naia potentsiia v srezakh gippokampa.

The effect of temperature changes on the activity of CA1 pyramidal cells has been investigated on completely submerged rat hippocampal slices. Fast and slow increase in the temperature from 24-28 to 37-38 degrees C decreased the amplitude of population spikes (PS), but increased the number of active pyramidal cells. The decrease in PS is more evident from 32 to 37 degrees C. After cooling to 24-28 degrees C, a long (more than 30 min) and considerable (up to 12%) increase of PS was observed. Increasing the temperature from 24-28 to 37-38 degrees C does not affect double facilitation, nor the maintenance of a long post-tetanic potentiation. It is suggested that the mechanism of temperature-induced long-term potentiation is similar to that of a long post-tetanic potentiation.

[Changes in the ultrastructural indices of the axospinal synapses and in the reactions of long-term posttetanic potentiation in surviving slices of mouse sensorimotor cortex]. / Izmeneniia ul'trastrukturnykh pokazatelei aksoshipikovykh sinapsov i reaktsii dlitel'noi posttetanicheskoi potentsitsii na perezhivaiushchikh srezakh sensomotornoi kory myshei.

Electron microscopic and morphometrical methods were used to study axospinal synapses as well as reactions of continuous posttetanic potentiation of neurons in surviving slices of sensomotor cerebral cortex in white mice. Under the same experimental conditions electron microscopic and electrophysiological studies have established that the result of high frequency electrical stimulation consisted in the appearance of continuous posttetanic potentiation, and quantitative and morphometrical changes in the ultrastructure of axospinal synapses: decreased active zone, increased summary greatest width of the spine apparatus cisterns. A supposition is made that high frequency electrical stimulation results in changed content of Ca2+ in spine cytoplasm, which may be considered as a trigger factor influencing the changes of cable properties and synaptic efficiency of axospinal synapses at the initial period of the continuous posttetanic potentiation.