Blockade of desensitization augments quisqualate excitotoxicity in hippocampal neurons.

Glutamate neurotoxicity is thought to play a role in the pathogenesis of several neurodegenerative diseases. While prolonged activation of either NMDA or non-NMDA receptors causes neuronal damage, NMDA receptors appear to mediate most of the glutamate toxicity. The reasons why NMDA toxicity predominates are uncertain but may relate to more effective neuroprotective mechanisms acting at non-NMDA receptors. To determine whether desensitization is one such mechanism, we studied the effects of the lectin wheat germ agglutinin (WGA) on quisqualate currents and toxicity in cultured postnatal rat hippocampal neurons. After WGA treatment, quisqualate currents exhibit little desensitization and a 4- to 8-fold increase in steady-state amplitude. WGA also markedly augments the degree of acute, quisqualate-induced neuronal degeneration. These results suggest that non-NMDA desensitization serves a neuroprotective function in hippocampal neurons.

Modulation of inhibitory glycine receptors in cultured embryonic mouse hippocampal neurons by zinc, thiol containing redox agents and carnosine.

Modulation of inhibitory glycine receptors by zinc (Zn(2+)) and endogenous redox agents such as glutathione may alter inhibition in the mammalian brain. Despite the abundance of Zn(2+) in the hippocampus and its ability to modulate glycine receptors, few studies have examined Zn(2+) modulation of hippocampal glycine receptors. Whether redox agents modulate hippocampal glycine receptors also remains unknown. This study examined Zn(2+) and redox modulation of glycine receptor-mediated currents in cultured embryonic mouse hippocampal neurons using whole-cell recordings. Zn(2+) concentrations below 10 microM potentiated currents elicited by low glycine, beta-alanine, and taurine concentrations by 300-400%. Zn(2+) concentrations above 300 microM produced nearly complete inhibition. Potentiating Zn(2+) concentrations shifted the dose-response curves for the three agonists to the left and decreased the Hill coefficient for glycine and beta-alanine but not taurine. Inhibiting Zn(2+) concentrations shifted the dose-response curves for glycine and beta-alanine to the right but reduced the maximum taurine response. Histidine residues may participate in potentiation because diethyl pyrocarbonate and pH 5.4 diminished Zn(2+) enhancement of glycine currents. pH 5.4 diminished Zn(2+) block of glycine currents, but diethyl pyrocarbonate did not. These findings indicate that separate sites mediate Zn(2+) potentiation and inhibition. The redox agents glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and 5,5'-dithiobis(2-nitrobenzoic acid) did not alter glycine currents by a redox mechanism. However, glutathione and dithiothreitol interfered with the effects of Zn(2+) on glycine currents by chelating it. Carnosine had similar effects. Thus, Zn(2+) and thiol containing redox agents that chelate Zn(2+) modulate hippocampal glycine receptors with the mechanism of Zn(2+) modulation being agonist dependent.

Properties of vertebrate glutamate receptors: calcium mobilization and desensitization.

Glutamate is now recognized as a major excitatory neurotransmitter in the vertebrate CNS, participating in a number of physiological and pathological processes. The importance of glutamate in the mobilization of intracellular Ca2+ as well as the relationship between excitatory and toxic properties has made it important to understand factors that regulate the responsivity of glutamate receptors. In recent years considerable insight has been gained about regulatory sites on NMDA receptors, with the recognition that these receptors are modulated by multiple endogenous and exogenous agents. Less is known about the regulation of responses mediated by AMPA, kainate, ACPD or APB receptors. Desensitization represents a potentially powerful means by which glutamate responses may be regulated. Indeed, two agents closely linked to the physiology of NMDA receptors, glycine and Ca2+, appear to modulate different types of desensitization. In the case of glycine, alteration of a rapid form of desensitization may be important in the role of this amino acid as a necessary cofactor for NMDA receptor activation. Additionally, changes in the affinity of the receptor complex for glycine may underlie the use-dependent decline in NMDA responses under certain conditions. Likewise, Ca2+ is a crucial player in the synaptic and toxic effects mediated by NMDA receptors, and is involved in a slower form of desensitization, in effect helping to regulate its own influx into neurons. The site and mechanism of the Ca2+ regulatory effects remain uncertain with evidence supporting both intracellular and ion channel sites of action. A clear role for Ca(2+)-dependent desensitization in the function of NMDA receptors under physiological conditions has not yet been demonstrated. AMPA receptor desensitization has been an area of intense investigation in recent years. The rapidity and degree of this process, coupled with its apparent rapid recovery, has suggested that desensitization is a key mechanism for the short-term regulation of responses mediated by these receptors. Furthermore, rapid desensitization appears to be one factor determining the time course and efficacy of fast excitatory synaptic transmission mediated by AMPA receptors, highlighting the physiological relevance of the process. The molecular mechanisms underlying desensitization remain uncertain. Traditionally, desensitization, like inactivation of voltage-gated channels, has been thought to represent a conformational change in the ion channel complex (Ochoa et al., 1989). However, it is unknown to what extent desensitization, in particular rapid AMPA receptor desensitization, has mechanistic features in common with inactivation. In voltage-gated channels, conformational changes in the channel protein restrict ion flow through the channel (Stuhmer, 1991).(ABSTRACT TRUNCATED AT 400 WORDS)

Ketone bodies do not directly alter excitatory or inhibitory hippocampal synaptic transmission.

OBJECTIVE: To determine the effect of the ketone bodies beta-hydroxybutyrate (betaHB) and acetoacetate (AA) on excitatory and inhibitory neurotransmission in the mammalian CNS. BACKGROUND: The ketogenic diet is presumed to be an effective anticonvulsant regimen for some children with medically intractable seizures. However, its mechanism of action remains a mystery. According to one hypothesis, ketone bodies have anticonvulsant properties. METHODS: The authors examined the effect of betaHB and AA on excitatory and inhibitory synaptic transmission in rat hippocampal-entorhinal cortex slices and cultured hippocampal neurons. In cultured neurons, their effect was also directly assayed on postsynaptic receptor properties. Finally, their ability to prevent spontaneous seizures was determined in a hippocampal-entorhinal cortex slice model. RESULTS: betaHB and AA did not alter synaptic transmission in these models. CONCLUSIONS: The anticonvulsant properties of the ketogenic diet do not result from a direct effect of ketone bodies on the primary voltage and ligand gated ion channels mediating excitatory or inhibitory neurotransmission in the hippocampus.

Blockade of ionotropic quisqualate receptor desensitization in rat hippocampal neurons by wheatgerm agglutinin and other lectins.

Previous experiments with wheatgerm agglutinin, an inhibitor of ionotropic quisqualate receptor desensitization, suggest that desensitization regulates quisqualate receptor-mediated synaptic transmission and excitotoxicity. Using whole-cell recordings from cultured postnatal rat hippocampal neurons, we have examined the wheatgerm agglutinin effect in further detail and compared it to other lectins. Wheatgerm agglutinin and other lectins belonging to the glucose/mannose, N-acetylglucosamine, and sialic acid classes inhibited desensitization. However, wheatgerm agglutinin was the most effective and had the most rapid onset of action. The inhibition was dose-dependent, and it was reduced and reversed by N-acetylglucosamine and N-acetylneuraminic acid. Treating neurons with neuraminidase, which cleaves sialic acid residues from the surface of cells, also diminished the effect. These results suggest that wheatgerm agglutinin reversibly inhibits ionotropic quisqualate receptor desensitization by interacting with carbohydrate residues on or near the quisqualate receptor complex. Future studies using the lectins may help to clarify the functional role of carbohydrate chains on the ionotropic quisqualate receptor.

Electrophysiological properties of identified postnatal rat hippocampal pyramidal neurons in primary culture.

Electrophysiological experiments were performed on primary cell cultures of retrogradely labelled postnatal rat hippocampal neurons. Rhodamine microspheres injected into the dorsal fornix clearly labelled the pyramidal cell layer of the hippocampus while excluding the other hippocampal layers and the dentate gyrus. In dissociated cell culture, the labelled cells were easily identified by fluorescence microscopy. Anti-neuron-specific enolase and anti-glial fibrillary acidic protein antibody staining confirmed that the labelled cells were neurons. The input resistance decreased from 2 G omega to 450 M omega, the input capacitance increased from 25 pF to 75 pF, the percentage of cells showing repetitive action potentials increased from 6% to 30%, and both peak GABA and glutamate responses increased over 100% during the 0 to 10 days time period investigated. This increase in chemosensitivity can be accounted for by an increase in cell size without an increase in the specific amino acid gated channel density. The subset of hippocampal neurons identified by the retrograde tracer technique are similar to non-labelled neurons with respect to the electrophysiological and pharmacological variables investigated. Nevertheless, it is likely that identified neurons may possess unique properties not evident in this study and refinement of the dissociated cell culture system using identified neuronal subpopulations may facilitate investigations looking at neuronal interactions such as synapse formation.

Calcium influx through N-methyl-D-aspartate channels activates a potassium current in postnatal rat hippocampal neurons.

Calcium-activated potassium conductances play important roles in modulating neuronal excitability. Indeed, the effects of some neurotransmitters such as acetylcholine and norepinephrine are, in part, due to actions on these conductances. We have found that the N-methyl-D-aspartate (NMDA) class of excitatory amino acid receptors also is coupled to a calcium activated potassium current. In voltage-clamped postnatal rat hippocampal neurons, NMDA responses consist of an initial inward cationic current followed by a slowly developing outward current carried by potassium ions. The slow outward current always follows the inward current, is associated with an increase in membrane conductance and is dependent on the influx of calcium ions. Similar responses are produced by other agonists active at NMDA receptors, including aspartate, glutamate and ibotenate, but are not activated by kainate, quisqualate or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA). Inhibition of the NMDA gated inward current by a competitive antagonist, 2-amino-5-phosphonovalerate (APV), eliminates the outward current. From these results we conclude that calcium influx through NMDA channels activates a potassium current. The extended time course of this outward current suggests that NMDA receptors may modulate neuronal excitability long after the opening of the NMDA channel.

Characterization of quisqualate receptor desensitization in cultured postnatal rat hippocampal neurons.

The quisqualate class of glutamate receptors is thought to play an important role in excitatory synaptic transmission, synaptic plasticity, and neuronal death. Since desensitization is a prominent feature of the responses mediated by this class of receptors, we have characterized the rapidly desensitizing quisqualate response in cultured postnatal rat hippocampal neurons using the whole-cell patch-clamp technique. Quisqualate and its structural analogs elicit a peak current that rapidly decays to a steady-state level. In contrast, currents induced by kainate, NMDA, and their structural analogs exhibit either no decay or a much slower decay. The biophysical and pharmacological properties of the peak and steady-state quisqualate currents indicate that both are mediated by an ionotropic quisqualate receptor. Quisqualate currents desensitized monoexponentially by approximately 70% with a time constant near 80 msec. Both the rate and percentage of desensitization showed slight voltage dependence and were concentration dependent, reaching maximal values at saturation. Additionally, the overlap of the dose-response curves for activation of the steady-state current and desensitization of the peak current by a conditioning dose suggests that the two processes are related. Furthermore, desensitizing quisqualate currents were observed when Ca2+, Mg2+, Na+, K+, and Cl- were removed from the extracellular solution or their concentrations greatly reduced. These results suggest that the decline in the response is not caused by a simple open channel block mechanism. Despite the lack of desensitization by kainate, our observations are consistent with the hypothesis that quisqualate and kainate act at a single receptor-channel complex. Kainate and quisqualate appeared to interact competitively when applied simultaneously and noncompetitively when quisqualate was applied first. In addition, saturating doses of quisqualate and kainate gave steady-state currents of equal amplitude in neurons treated with the lectin WGA, an inhibitor of quisqualate receptor desensitization.

Blockade of ionotropic quisqualate receptor desensitization by wheat germ agglutinin in cultured postnatal rat hippocampal neurons.

1. The effects of the lectin wheat germ agglutinin (WGA) on quisqualate-gated currents were examined in postnatal rat hippocampal neurons using recordings from whole cells and outside-out membrane patches. 2. Rapid applications of quisqualate to whole cells and outside-out patches evoked a current that desensitized to a steady-state level. WGA blocked desensitization by increasing the steady-state current amplitude without altering the current-voltage (I-V) relationship or pharmacology of the current. 3. In outside-out patches quisqualate (2.5-1,000 microM) elicited bursts of channel openings having conductances of 2.7, 6.3, and 13 pS. The mean burst length for all conductances was 8.6 +/- 0.6 ms (mean +/- SE) and exhibited little voltage (-110 to +80 mV) or concentration (2.5-1,000 microM) dependence. Treating patches with 580 nM WGA produced no change in conductance, but the mean burst length for 100 microM quisqualate increased from 9.0 +/- 1.1 to 16 +/- 3.2 ms. 4. Fluctuation analysis of whole cell currents evoked by 1 microM quisqualate at -80 mV revealed an increase in the time constant from 8.7 +/- 0.5 to 13 +/- 1.0 ms after treatment with 580 nM WGA. This treatment produced no change in the estimated single-channel conductance. 5. These findings suggest that an increase in channel burst length, rather than an increase in single-channel conductance, contributes to the WGA-induced augmentation of the steady-state quisqualate current.

Effects of bromowillardiine and willardiine on non-N-methyl-D-aspartate receptors in postnatal rat hippocampal neurons.

The physiology and pharmacology of willardiine and bromowillardiine, structural analogues of quisqualate, were studied in cultured postnatal rat hippocampal neurons using whole-cell voltage-clamp techniques. These agonists appear to act at a shared non-N-methyl-D-aspartate (non-NMDA) receptor-channel complex and gate nonselective cationic currents. Willardiine currents desensitize rapidly and to a much greater degree than bromowillardiine currents. In addition, the brominated compound produces steady state currents that are 5 times larger than those produced by willardiine at saturation. Bromowillardiine is also a more efficacious excitotoxin, producing about 3-fold greater acute neuronal damage than willardiine at saturating concentrations. These results suggest that agonist structure affects the ability of non-NMDA agonists to induce desensitization and add support to the hypothesis that receptor desensitization serves to limit acute excitotoxicity in cultured neurons.

Determinants of directionality in lambda site-specific recombination.

The DNA structural features governing directionality in lambda site-specific recombination are shown to reside in regions of the phage attachment site more than 70 bp to the left and more than 40 bp to the right of the cross-over region. Disposition of these sequences on the same attachment site in integration, and on different attachment sites in excision, determines the opposite effects of Xis protein upon the two reactions (stimulation of excision and inhibition of integration). The binding of Xis to two adjacent directly repeated sequences in the left phage arm is shown to occur in a highly cooperative manner, to alter the conformation of the DNA, and to produce a 32-fold stimulation of Int binding to an adjacent locus.

Wheat germ agglutinin enhances EPSCs in cultured postnatal rat hippocampal neurons by blocking ionotropic quisqualate receptor desensitization.

1. The effect of the lectin wheat germ agglutinin (WGA), an inhibitor of ionotropic quisqualate receptor desensitization, on both evoked and spontaneous fast excitatory postsynaptic events was examined in cultured postnatal rat hippocampal neurons with the use of whole cell recordings. 2. WGA, at 580 nM, potentiated evoked fast excitatory postsynaptic currents (EPSCs) by increasing the amplitudes by 100 +/- 27% (mean +/- SE) and the time constant of decay from 5.8 +/- 0.6 to 7.9 +/- 0.5 ms. The increases in these parameters were not accompanied by changes in the current-voltage (I-V) relationship or pharmacological profile of the fast EPSCs. 3. WGA did not alter the amplitude or time course of decay of inhibitory postsynaptic currents (IPSCs), and it did not alter neuronal input resistance or action potentials. 4. WGA increased the amplitude of spontaneous fast miniature EPSCs (MEPSCs), defined as spontaneous EPSCs recorded in the presence of tetrodotoxin, by 53 +/- 11% and increased the time required to decay to 50% of the peak amplitude by 48 +/- 23%. These changes were not associated with a change in the rate of MEPSC occurrence. 5. These results suggest that WGA augments hippocampal excitatory postsynaptic events via a postsynaptic mechanism. The results further imply that ionotropic quisqualate receptor desensitization can modulate the amplitude and time course of decay of fast excitatory synaptic events. Thus desensitization may be one factor that regulates fast excitatory synaptic transmission.

Rapid desensitization of glutamate receptors in vertebrate central neurons.

We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.

Rapid desensitization of glutamate receptors in vertebrate central neurons.

We have examined glutamate receptor desensitization in voltage-clamped embryonic chicken spinal cord neurons and postnatal rat hippocampal neurons maintained in culture. Rapid currents that rose in 0.8-3.6 msec were evoked when glutamate was ionophoresed with 0.5- to 1.0-msec pulses. With prolonged pulses or brief, repetitive pulses, glutamate-evoked currents decayed rapidly in a manner that was independent of holding potential. A similar desensitization occurred following close-range pressure ejection of glutamate. The rapid, desensitizing glutamate current exhibited a linear current-voltage relation and it was not blocked by 2-amino-5-phosphonovalerate, suggesting that it was mediated by N-methyl-D-aspartate-insensitive (G2) receptors. Desensitization of G2 receptors may be agonist-dependent: currents evoked by kainate, a selective G2 agonist, did not decay, whereas prior application of glutamate did reduce the size of kainate responses. The appearance of the rapid current depended critically on the position of the ionophoretic pipette. Such glutamate-receptor "hot spots" often corresponded to points of contact with neighboring neurites, which raises the possibility that they are located at synapses.

Nicotinic acetylcholine currents in cultured postnatal rat hippocampal neurons.

Nicotinic acetylcholine (ACh) currents were studied in cultured postnatal rat hippocampal neurons, using whole-cell voltage-clamp techniques. In most cells, ACh produces one of two types of response. One class of ACh currents exhibits rapid and profound desensitization and is sensitive to inhibition by alpha-bungarotoxin (alpha BTXN). The second class activates slowly and exhibits no desensitization during prolonged agonist applications. This slow current is insensitive to alpha BTXN. Both the fast and slow responses exhibit inwardly rectifying current-voltage relationships and pass little current at positive membrane potentials. Both currents can be recorded in the presence of 1 microM atropine but are blocked by 0.1-1.0 mM d-tubocurarine and 0.1-1.0 mM mecamylamine. These observations suggest heterogeneity of nicotinic ACh receptors in rat hippocampal neurons and provide support for functional alpha BTXN-sensitive nicotinic receptors in this region.