Modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor desensitization by extracellular protons.

The interstitial milieu of the brain is buffered to an average pH of 7.3, but synaptic activation produces a temporal sequence of events that can affect pH in the synaptic cleft. Furthermore, pathophysiological processes such as ischemia and seizures produce global and prolonged acidification of interstitial pH. Changes in pH, in turn, can affect neuronal excitability by modulating receptors and channels. Patch-clamp recordings were made from cultured rat hippocampal neurons to determine whether physiologically relevant changes in interstitial pH (6.5-7.8) significantly affect AMPA receptor function. Acidic pH, such as that typically associated with ischemia (pH 6.5), significantly inhibited AMPA receptor-mediated responses in neurons. The effect of pH was agonist-dependent, with 2-fold greater inhibition of responses evoked by the strongly desensitizing agonists glutamate and quisqualate than the weakly desensitizing agonist kainate. Additional experiments tested the hypothesis that protons modulate AMPA receptor desensitization. In the presence of drugs that block AMPA receptor desensitization, pH 6. 5 had no effect on glutamate-evoked responses. In neuronal macropatches, protons increased equilibrium desensitization without affecting macroscopic desensitization or deactivation kinetics. The mechanisms and molecular determinants of pH-mediated effects were further investigated using human embryonic kidney 293 cells expressing recombinant AMPA receptors. Inhibition of kainate-evoked responses varied with subunit and isoform composition, ranging from 10% to >40%. Flop isoforms, which exhibit faster and more extensive desensitization, were most strongly inhibited. These findings suggest that interstitial acidification can modulate AMPA receptor-mediated synaptic transmission and that differences in receptor sensitivity to proton modulation may underlie the selective vulnerability of certain neuronal populations to ischemia.

AMPA receptor current density, not desensitization, predicts selective motoneuron vulnerability.

Spinal motoneurons are more susceptible to AMPA receptor-mediated injury than are other spinal neurons, a property that has been implicated in their selective degeneration in amyotrophic lateral sclerosis (ALS). The aim of this study was to determine whether this difference in vulnerability between motoneurons and other spinal neurons can be attributed to a difference in AMPA receptor desensitization and/or to a difference in density of functional AMPA receptors. Spinal motoneurons and dorsal horn neurons were isolated from embryonic rats and cultured on spinal astrocytes. Single-cell RT-PCR quantification of the relative abundance of the flip and flop isoforms of the AMPA receptor subunits, which are known to affect receptor desensitization, did not reveal any difference between the two cell populations. Examination of AMPA receptor desensitization by patch-clamp electrophysiological measurements on nucleated and outside-out patches and in the whole-cell mode also yielded similar results for the two cell groups. However, AMPA receptor current density was two- to threefold higher in motoneurons than in dorsal horn neurons, suggesting a higher density of functional AMPA receptors in motoneuron membranes. Pharmacological reduction of AMPA receptor current density in motoneurons to the level found in dorsal horn neurons eliminated selective motoneuron vulnerability to AMPA receptor activation. These results suggest that the greater AMPA receptor current density of spinal motoneurons may be sufficient to account for their selective vulnerability to AMPA receptor agonists in vitro.

Potentiation of late components in olfactory bulb and piriform cortex requires activation of cortical association fibers.

Previous research has demonstrated that repeated high-frequency stimulation of the granule cell layer of the olfactory bulb (OB) produces an enduring potentiation of late components (PLC) in potentials evoked in the OB and piriform cortex (PC), while leaving the monosynaptic EPSP produced by OB mitral cells in PC pyramidal cells unaltered. Two experiments were conducted using male Long-Evans rats with chronically implanted electrodes to assess the relative contribution to this potentiation of the two main fiber systems that interconnect the OB and PC: the lateral olfactory tract (LOT), which contains mitral cell axons that synapse on PC pyramidal cells, and the PC association fiber system, which consists of the axons of PC pyramidal cells that synapse on several cell populations within the PC and on granule cells in the OB. The results indicate that stimulation of PC association fibers is both necessary and sufficient to duplicate the pattern of potentiation seen following OB stimulation in previous experiments. LOT stimulation had no consistent effect, and coactivation of the LOT and PC association fibers was no more effective than activation of PC association fibers alone. Possible mechanisms underlying this effect are discussed, including (1) long-term potentiation (LTP) at synapses made by the axons of PC pyramidal cells on neurons in the OB and PC; and (2) repetitive firing in PC pyramidal cells due to regenerative excitation in a population of deep cells in the PC and endopiriform nucleus.

AMPA receptor heterogeneity in rat hippocampal neurons revealed by differential sensitivity to cyclothiazide.

1. The kinetics of onset of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor desensitization by glutamate, and the extent of attenuation of AMPA receptor desensitization by cyclothiazide, showed pronounced cell-to-cell variation in cultures of rat hippocampal neurons. Cultures prepared from area CA1 stratum radiatum tended to show weaker modulation by cyclothiazide than cultures prepared from the whole hippocampus. 2. Kinetic analysis of concentration jump responses to glutamate revealed multiple populations of receptors with fast (approximately 400 ms), intermediate (approximately 2-4 s), and slow (> 20 s) time constants for recovery from modulation by cyclothiazide. The amplitudes of these components varied widely between cells, suggesting the existence of at least three populations of AMPA receptor subtypes, the relative density of which varied from cell to cell. 3. The complex patterns of sensitivity to cyclothiazide seen in hippocampal neurons could be reconstituted by assembly of recombinant AMPA receptor subunits generated from cDNAs encoding the flip (i) and flop (o) splice variants of the GluR-A and GluR-B subunits. Recovery from modulation by cyclothiazide was slower for GluR-AiBi and GluR-AoBi than for GluR-AiBo and GluR-AoBo. 4. Coexpression of the flip and flop splice variants of GluR-A, in the absence of GluR-B, revealed that heteromeric AMPA receptors with intermediate sensitivity to cyclothiazide, similar to responses observed for the combinations GluR-AoBi or GluR-AiBo, could be generated independently of the presence of the GluR-B subunit. However, recovery from modulation by cyclothiazide was twofold slower for GluR-AiBi than for homomeric GluR-Ai, indicating that the GluR-A and GluR-B subunits are not functionally equivalent in controlling sensitivity to cyclothiazide. 5. These results demonstrate that AMPA receptors expressed in hippocampal neurons are assembled in a variety of subunit and splice variant combinations that might serve as a mechanism to fine-tune the kinetics of synaptic transmission.

Cyclothiazide differentially modulates desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor splice variants.

Agonist responses for flip splice variants of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR-A, -C, and -D are more strongly potentiated by cyclothiazide than are those for the flop forms. Cyclothiazide shows both greater efficacy and higher apparent affinity for potentiation of GluR-Aflip versus GluR-Aflop. Consistent with higher affinity for the flip splice variant, recovery from potentiation by cyclothiazide proceeds 30 times more slowly for GluR-Aflip than for GluR-Aflop. In the presence of 300 microM cyclothiazide a 6-fold leftward shift in the kainate dose-response curve for GluR-Aflip but not GluR-Aflop additionally contributes to a difference in potentiation for these splice variants. Although control responses to glutamate show strong desensitization for both splice variants of GluR-A, in the presence of 100 microM cyclothiazide desensitization is strongly attenuated for GluR-Aflip, whereas for GluR-Aflop desensitization remains pronounced but with a rate of onset slowed 50-fold, compared with control. In heteromeric AMPA receptors formed from GluR-A and GluR-B, the flip splice variants are dominant for controlling both recovery from potentiation of responses to kainate and block of desensitization of responses to glutamate. Our results suggest that the flip/flop module could directly contribute to the binding site for cyclothiazide, raising the possibility that this site is located in an extracellular receptor domain.

Excitatory amino acid receptors in glial progenitor cells: molecular and functional properties.

We have analyzed the molecular and biophysical properties of glutamate-gated channels in cells of the oligodendrocyte lineage, using both the CG-4 primary cell line (Louis et al: J. Neurosci. Res. 31:193-204, 1992a) and oligodendrocyte progenitors purified from the rat cerebral cortex. CG-4 progenitor cells, as well as primary progenitors, were stained with a specific anti-GABA antibody. In whole-cell patch-clamp recordings, rapid perfusion of the agonists L-glutamate, kainate, and AMPA produced rapidly desensitizing currents in CG-4 cells. NMDA was ineffective. Both rapidly desensitizing and steady-state components of responses to kainate were inhibited by the kainate/AMPA receptor antagonist CNQX. Northern blot analysis of total mRNA isolated from CG-4 cells revealed co-expression of both AMPA- and kainate-preferring glutamate receptor subunits. The activation of glutamate receptors in CG-4 cells caused a rapid and transient elevation of mRNAs for the immediate early gene NGFI-A.

Glial cells of the oligodendrocyte lineage express both kainate- and AMPA-preferring subtypes of glutamate receptor.

mRNAs for AMPA- and kainate-preferring glutamate receptor subunits are expressed abundantly in the CNS, yet functional studies of neurons and glia from brain suggest selective expression of AMPA receptors. We now show that glial cells of the O-2A lineage express rapidly desensitizing responses to kainate, mRNAs for GluR6, GluR7, KA-1, and KA-2, rapidly desensitizing responses to AMPA, and mRNAs for GluR-B, -C, and -D. Analysis of glutamate receptor currents in single cells reveals two receptor populations with high and low affinity for kainate and different sensitivity for potentiation by concanavalin A and for block of desensitization by cyclothiazide. Our experiments describe the characterization of native kainate-preferring receptors in glia and reveal coexpression in single cells of functional AMPA- and kainate-preferring receptors.

Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A.

Potentiation by cyclothiazide of recombinant glutamate receptor responses in Xenopus oocytes showed absolute selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors. Rapid desensitization in HEK 293 cells transfected with AMPA receptors was blocked by cyclothiazide, but only weakly attenuated by concanavalin A. Desensitization at kainate receptors was blocked by concanavalin A but unaffected by cyclothiazide. Selective effects of these modulators following coexpression of subunits from different families suggest independent assembly of functional AMPA and kainate receptors. Northern blot analysis of mRNA for dorsal root ganglia revealed a predominant expression of GluR5, indicating that modulation of desensitization by concanavalin A but not cyclothiazide in sensory neurons accurately predicts subunit expression for native glutamate receptors.

Hippocampal neurons exhibit cyclothiazide-sensitive rapidly desensitizing responses to kainate.

In whole-cell recordings from mammalian CNS neurons, AMPA-preferring glutamate receptors exhibit strong desensitization in response to AMPA, glutamate, and quisqualate, but not to kainate or domoate. Such desensitization is reduced by lectins, by the nootropic drug aniracetam, and by diazoxide. None of these compounds strongly modulate responses to kainate and domoate, consistent with the apparent lack of desensitization to these agonists. We now report experiments on hippocampal neurons in which responses to kainate were strongly potentiated by cyclothiazide, a benzothiadiazine diuretic and antihypertensive drug structurally related to diazoxide. Cyclothiazide increased the maximum response to a saturating concentration of kainate by approximately 300% and produced a shift to the left in the kainate dose-response curve. Because cyclothiazide was considerably more effective than aniracetam in reducing desensitization evoked by glutamate, we tested the possibility that potentiation of responses to kainate was due to block of a previously undetected component of desensitization in the response to kainate itself. In outside-out patches responses to rapid perfusion of 3 mM kainate showed 34% desensitization, the onset of which developed with a time constant of 2.2 msec. Desensitization of responses to kainate was abolished by 100 microM cyclothiazide, as was the much stronger desensitization evoked by glutamate and AMPA. Cyclothiazide also slowed the rate of deactivation of responses to kainate recorded after return to agonist-free solution. Current-voltage plots for control responses to kainate exhibited outward rectification that was associated with a reduction in the amount of desensitization on depolarization. Both effects were absent in the presence of cyclothiazide, suggesting that rectification of responses to kainate was due to the voltage dependence of desensitization. The complete block of desensitization produced by cyclothiazide provides a powerful new tool for analysis of allosteric regulatory mechanisms at AMPA-preferring glutamate receptors.

Functional correlates of selective long-term potentiation in the olfactory cortex and olfactory bulb.

High-frequency stimulation of the granule cell layer of the olfactory bulb (OB) has previously been shown to result in a selective long-term potentiation (LTP) of late components of potentials evoked in the OB and piriform cortex (PC). The functional impact of this potentiation was explored in male Long-Evans rats with chronically implanted electrodes by comparing the effects of paired-pulse stimulation of the OB in potentiated and control animals. Effects were examined on two components of the potential evoked in the PC: A1, which represents the population EPSP produced by OB mitral cells in PC pyramidal cells via the lateral olfactory tract (LOT), and B1, which represents the subsequent population EPSP produced by PC pyramidal cells in other pyramidal cells. Two separate functional correlates of selective LTP were found. First, there was enhanced paired-pulse depression of B1, indicating increased inhibition of PC pyramidal cells. Second, there was a shift from paired-pulse facilitation to depression of A1, which was accompanied by a decrease in amplitude of the LOT volley, indicating that fewer mitral cells were activated by the stimulation. This shift was most prominent in animals with stimulating electrodes closest to the mitral cell layer, suggesting that it is dependent upon direct stimulation of mitral cell somata. These observations, together with other results reported in the manuscript, support the conclusion that there is an enhanced inhibition of mitral cells following selective LTP. Thus a primary consequence of selective LTP appears to be enhanced inhibition of principal neurons in both the PC and OB. These findings are consistent with our previous proposal that selective LTP represents potentiation at excitatory synapses made by PC pyramidal cells on inhibitory interneurons in the PC and OB.

Activation and desensitization of AMPA/kainate receptors by novel derivatives of willardiine.

Willardiine [(S)-1-(2-amino-2-carboxyethyl)pyrimidine-2,4-dione] is a naturally occurring heterocyclic excitatory amino acid present in the seeds of Acacia and Mimosa. A series of 5-substituted willardiines were synthesized in single enantiomeric forms and tested for activity at AMPA/kainate receptors, using whole-cell recording from mouse embryonic hippocampal neurons. The (S)- but not (R)-isomers of willardiine and 5-bromowillardiine were potent agonists, producing rapidly but incompletely desensitizing responses. At equilibrium, (S)-5-fluorowillardiine (EC50, 1.5 microM) was seven times more potent than (R,S)-AMPA (EC50, 11 microM) and 30 times more potent than willardiine (EC50, 45 microM); the potency sequence was fluoro greater than nitro greater than chloro approximately bromo greater than iodo greater than willardiine. Willardiines produce strikingly different degrees of desensitization: at saturating doses the equilibrium response to the weakly desensitizing agonist (S)-5-iodowillardiine was similar in amplitude to the response to kainate and 10 times larger than the response to the strongly desensitizing agonist (S)-willardiine. The desensitization sequence was fluoro greater than willardiine greater than nitro approximately chloro greater than bromo greater than iodo greater than kainate. Cross-desensitization experiments confirm that willardiines bind to the same receptors activated by kainate and AMPA, and show that both the rapidly desensitizing and equilibrium responses to willardiines are mediated by the same receptor: (S)-5-iodowillardiine blocked activation of the rapidly desensitizing response evoked by (S)-willardiine and (S)-5-fluorowillardiine, while the latter agonists blocked the equilibrium response to (S)-5-iodowillardiine. A slowly decaying inward tail current was recorded after a brief application of (S)-5-fluorowillardiine but not (S)-willardiine, consistent with a model in which willardiines bind with different affinity to desensitized receptors, such that following removal of agonist, receptors trapped in the desensitized state can return to the open state before dissociation of agonist terminates receptor activation. Willardiines are the first compounds characterized in which simple changes in molecular structure are associated with marked differences in the ability of agonists to produce desensitization of AMPA/kainate receptors.

Modulation of excitatory synaptic transmission by drugs that reduce desensitization at AMPA/kainate receptors.

Desensitization at AMPA/kainate receptors has been proposed to contribute to the decay of excitatory synaptic currents. We examined the action of aniracetam, wheat germ agglutinin (WGA), and concanavalin A (Con A), drugs that act via separate mechanisms to reduce desensitization evoked by L-glutamate in rat hippocampal neurons. The decay of excitatory synaptic currents, and sucrose-evoked miniature excitatory postsynaptic currents (EPSCs) was slowed 2- to 3-fold by aniracetam. In contrast, WGA increased the EPSC decay time constant only 1.3-fold and Con A had no effect. Aniracetam increased the magnitude of stimulus-evoked EPSCs 1.9-fold; variance analysis suggests a postsynaptic mechanism of action. WGA and Con A reduced EPSC amplitude via a presynaptic mechanism. Aniracetam increased the burst length of L-glutamate-activated single-channel responses. Simulations suggest that aniracetam either slows entry into a desensitized state or decreases the closing rate constant for ion channel gating.

Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons.

AMPA but not kainate produces a rapidly desensitizing response in mouse hippocampal neurons. The characteristic action of these agonists appears to arise from activation of a single receptor with active and desensitized states, for which AMPA and kainate have different relative affinity. The equilibrium potency of a series of five agonists that produce rapidly desensitizing responses at non-NMDA receptors (EC50 1 microM to 4 mM) was similar to their equilibrium potency for block of kainate responses. Increasing the concentration of kainate overcame such block, but in the presence of AMPA the rate of activation of responses to kainate was slowed. Conversely, in the presence of kainate the amplitude of rapidly desensitizing responses evoked by AMPA was reduced, and the rate of onset of desensitization was slowed.

Characterization and anatomical distribution of selective long-term potentiation in the olfactory forebrain.

High-frequency stimulation of the granule cell layer of the olfactory bulb (OB) has previously been shown to result in a form of long-term potentiation in the piriform cortex (PC) that is selective to late components of the potential evoked in the PC58. This phenomenon was explored in male Long-Evans rats with chronically implanted electrodes by recording potentials evoked in the OB and in various sites in the ipsilateral and contralateral PC before and after repeated high-frequency stimulation of the OB. Recordings at all sites exhibited a gradually developing potentiation that was selective to late components of the evoked potential. In the OB and ipsilateral PC this potentiation had an overt long-term component that lasted for days, and all sites exhibited a latent potentiation that enabled the reestablishment of substantial levels of potentiation by mild patterns of stimulation that had no effect in control animals. No potentiation of the population EPSP representing input from the lateral olfactory tract to the PC was seen. Available evidence concerning the neuronal elements activated by the stimulation and the neuronal events likely to underlie the potentiated components of the evoked potentials suggests that this potentiation may represent an enhancement of inhibitory interactions within the PC and between the PC and OB.

Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors.

Dose-response curves for activation of excitatory amino acid receptors on mouse embryonic hippocampal neurons in culture were recorded for 15 excitatory amino acids, including the L-isomers of glutamate, aspartate, and a family of endogenous sulfur amino acids. In the presence of 3 microM glycine, with no extracellular Mg, micromolar concentrations of 11 of these amino acids produced selective activation of N-methyl-D-aspartate (NMDA) receptors. L-Glutamate was the most potent NMDA agonist (EC50 2.3 microM) and quinolinic acid the least potent (EC50 2.3 mM). Dose-response curves were well fit by the logistic equation, or by a model with 2 independent agonist binding sites. The mean limiting slope of log-log plots of NMDA receptor current versus agonist concentration (1.93) suggests that a 2-site model is appropriate. There was excellent correlation between agonist EC50S determined in voltage clamp experiments and KdS determined for NMDA receptor binding (Olverman et al., 1988). With no added glycine, and 1 mM extracellular Mg, responses to NMDA were completely blocked; responses to kainate and quisqualate were unchanged. Under these conditions, glutamate and the sulfur amino acids activated a rapidly desensitizing response, similar to that evoked by micromolar concentrations of quisqualate and AMPA, but mM concentrations of L-aspartate, homoquinolinic acid, and quinolinic acid failed to elicit a non-NMDA receptor-mediated response. Except for L-glutamate (EC50 480 microM), the low potency of the sulfur amino acids prevented the study of complete dose-response curves for the rapidly desensitizing response at quisqualate receptors. Small-amplitude nondesensitizing quisqualate receptor responses were activated by much lower concentrations of all quisqualate receptor agonists. Full dose-response curves for the nondesensitizing response were obtained for 9 amino acids; L-glutamate was the most potent endogenous agonist (EC50 19 microM). Domoate (EC50 13 microM) and kainate (EC50 143 microM) activated large-amplitude, nondesensitizing responses.

Selective long-term potentiation in the pyriform cortex.

Electrical stimulation of the olfactory bulb (OB) produces an evoked potential in the pyriform cortex (PC) characterized by an initial surface-negative wave (period 1) representing activation of PC pyramidal cells via the lateral olfactory tract, followed by a surface-positive wave (period 2) which is temporally associated with recurrent and feed-forward inhibition. The experiment reported here examined the changes that occur in the PC evoked potential following a pattern of stimulation that has been found to produce short- and long-term potentiation (LTP) in other areas of the forebrain. Male Long-Evans rats with electrodes in the OB and PC were divided into two groups. LTP animals received high-frequency stimulation of the OB (30 trains of 10 pulses each at a frequency of 100 Hz). Control animals received the same number of pulses at a lower frequency (1 Hz). This procedure was repeated 6 times at 2-day intervals. Neither high- nor low-frequency stimulation altered period 1 of the PC evoked potential, indicating that synaptic input arriving via the lateral olfactory tract was unaffected. However, LTP animals exhibited a marked increase in the amplitude and duration of period 2 which appeared to reflect two separate processes: a short-term change that peaked within 30 min of the trains; and a long-term change that accumulated across the 6 treatments. LTP of period 2 persisted in latent form for at least 32 days after the last treatment. Control animals exhibited only small changes that were attributed to the paired-pulse stimulation used for testing rather than the 1 Hz Control trains. These results suggest that repeated high-frequency stimulation of the OB causes a persistent alteration in the way information is processed within the PC. The form of LTP demonstrated here is markedly different from that found in the hippocampal formation, where potentiation of the monosynaptic excitatory postsynaptic potential is a prominent effect. The functional significance of this change cannot be determined with certainty from the present experiment, but available evidence suggests that it represents an enhancement of inhibitory processes within the PC.