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J F MacDonald

Publications and source records attributed to J F MacDonald.

At least 55 records · Page 3Linked to original sources
PubMed1997

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons.

Neurotrophin modulation of NMDA receptors in cultured murine and isolated rat neurons. J. Neurophysiol. 78: 2363-2371, 1997. Patch-clamp and calcium imaging techniques were used to assess the acute effects of the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and nerve growth factor (NGF), on the responses of cultured and acutely isolated hippocampal and cultured striatal neurons to the glutamate receptor agonist N-methyl--aspartic acid (NMDA). The effects of BDNF on NMDA-activated currents were examined in greater detail. Currents evoked by NMDA, and the accompanying changes in intracellular calcium, were enhanced by low concentrations of the neurotrophins (1-20 ng/ml). The potentiation by the neurotrophins was rapid in onset and offset (<1 s). The neurotrophins also reduced desensitization of these currents in most cells. The enhancement of NMDA-activated currents by BDNF was observed using both perforated and whole cell patch recording techniques and could be demonstrated in outside-out patches. Furthermore, its effects were not attenuated by pretreatment with the protein kinase inhibitors genistein or 1-(5-isoquinolynesulfony)2-methylpiperazine (H7). Therefore, the actions of BDNF do not appear to be mediated by phosphorylation. Similar enhancements were observed with NT-3 and NT-4 and with NGF despite the fact that hippocampal neurons lack TrkA receptors. All together this evidence suggests that the enhancement of NMDA-evoked currents is unlikely to be mediated through the activation of growth factor receptors. Modulation of NMDA responses by BDNF was dependent on the concentration of extracellular glycine. The most pronounced potentiation by BDNF was observed at low concentrations, whereas no potentiation was observed in saturating concentrations of glycine, suggesting that BDNF may have increased the affinity of the NMDA receptor for glycine. However, the competitive glycine-site antagonist 7-chloro-kynurenic acid blocked the enhancement by BDNF without shifting the dose-inhibition relationship for this antagonist, and Mg2+ consistently depressed the potentiation of NMDA-evoked currents by BDNF, indicating that BDNF does not alter glycine affinity. BDNF also reversibly increased the probability of opening of NMDA channels recorded from outside-out patches taken from cultured hippocampal neurons. Other unrelated peptides including dynorphin and somatostatin also caused a glycine-dependent enhancement of NMDA currents and depressed the currents in saturating concentrations of glycine. In contrast, a shortened analogue dynorphin (6-17), which lacks N-terminus glycine residues, and another peptide met-enkephalin were without effects on NMDA currents recorded in low concentrations of glycine. Our results suggest that neurotrophins and other peptides can serve as glycine-like ligands for the NMDA receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Platelet-derived growth factor induces a long-term inhibition of N-methyl-D-aspartate receptor function.

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-gamma-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

Analysis of Variance↗
PubMed1996

Cloning of a G protein-activated inwardly rectifying potassium channel from human cerebellum.

Based on sequence homology with the rat atrial G protein-coupled muscarinic potassium channel (GIRK1 or KGA1/KGB1), a human cDNA encoding a G protein-activated inwardly rectifying K+ channel (HGIRK1) was isolated. The cDNA encodes a protein of 501 amino acids and shares 99% identity to rat GIRK1 in its total amino acid sequence. Southern blot analysis of genomic DNA indicates a high degree of conservation among various species. In the human population a useful NlaIII restriction fragment length polymorphism was found in the coding sequence of HGIRK1. Co-expression of HGIRK1 and the 5-HT1A receptor in Xenopus oocytes resulted in opening of the channel upon treatment with serotonin. HGIRK1 currents showed strong inward rectification and could be blocked by extracellular Ba2+. Northern blot analysis shows that HGIRK1 expression in human is most abundant in the brain, while lower levels are round in kidney and heart.

Amino Acid Sequence↗
PubMed1995

Differential effects of lectins on recombinant glutamate receptors.

The effects of the lectins concanavalin A, succinyl concanavalin A, wheat-germ agglutinin and soybean agglutinin were studied at recombinant ionotropic glutamate receptors expressed in Xenopus oocytes. Homomeric and heteromeric receptors from each of the three major classes of ionotropic glutamate receptors (N-methyl-D-asparate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainate) were studied. The lectins potentiated homomeric configurations of kainate, AMPA and NMDA receptors to a greater degree than the corresponding heteromeric configurations although the rank order of the lectin potentiating effects was the same for both homomeric and heteromeric receptors within a given glutamate receptor class. The most profound effects of the lectins were observed with the kainate receptors; the rank order of potentiating effects of the lectins at the homo- and heteromeric kainate receptors (Glu6 and Glu6/KA-2) was concanavalin A > succinyl concanavalin A > wheat-germ agglutinin > soybean agglutinin. At the recombinant Glu3 and Glu2/3 AMPA receptor complexes, wheat-germ agglutinin and concanavalin A produced the largest enhancements of the glutamare-activated currents followed by succinyl concanavalin A; soybean agglutinin had no significant potentiating effect. Agonistevoked currents recorded from oocytes expressing the homo- and heteromeric NMDA receptors were only slightly enhanced by concanavalin A and succinyl concanavalin A but not by wheat-germ agglutinin or soybean agglutinin. These results demonstrate that kainate. AMPA and NMDA receptors display dramatic differences in their responses to lectins, and suggest that the receptor-bound oligosaccharide side chains may play different roles in the functional responses mediated by the three major classes of ionotropic glutamate receptors.

Animals↗
PubMed1995

Modulation by magnesium of the affinity of NMDA receptors for glycine in murine hippocampal neurones.

1. The effects of the divalent cation Mg2+ on NMDA currents recorded from cultured fetal mouse and acutely isolated neonatal rat hippocampal neurones were studied using the whole-cell patch-clamp technique. 2. Current-voltage relations were measured in the presence or absence of applied Mg2+ and added glycine. NMDA-evoked currents were studied in the absence or in a low concentration (0.2 mM) of applied Ca2+ in order to minimize Ca(2+)-dependent inactivation of the responses. Mg2+ unexpectedly enhanced NMDA-activated currents at positive membrane potentials. At negative membrane potentials Mg2+ caused a previously characterized voltage-dependent block of inward NMDA-activated currents. 3. The potentiation by Mg2+ of outward currents activated by NMDA was concentration dependent (EC50, approximately 3 mM; Hill coefficient, approximately 2). Mg2+ also reduced the desensitization of the NMDA receptor. The maximal enhancement of steady-state NMDA-activated currents was 2.7-fold and at 6 mM the time constant of desensitization was doubled. 4. Comparisons of concentration-response curves for glycine and 7-chloro-kynurenic acid demonstrated that Mg2+ significantly increased the affinity of the NMDA receptor for glycine. The EC50 for glycine was 380 nM in the absence of Mg2+ and 163 nM in 3 mM Mg2+. Mg2+ had little effect on the forward rate of the glycine response but halved the off-rate (2.34 to 1.15 s-1) and thus similarly reduced the apparent dissociation constant. 5. There was a good correlation between the concentration of extracellular Ca2+ and a reduction in the time constant of the glycine-sensitive component of NMDA receptor desensitization. Ca2+ could enhance these NMDA-activated currents briefly following exposure to high concentrations of Ca2+. These results are consistent with a Ca(2+)-dependent enhancement of the affinity of the NMDA receptor for glycine. 6. Mg2+ can enhance NMDA-mediated currents and reduce desensitization of this receptor by allosterically interacting with the glycine binding site. This interaction may be a key physiological mechanism through which modulation of the NMDA receptor is achieved.

Animals↗
PubMed1995

NMDA receptor-dependent excitotoxicity: the role of intracellular Ca2+ release.

Massive activation of glutamate receptors can result in excessive rises in cytoplasmic Ca2+ that are thought to underlie the fundamental processes ultimately leading to neuronal death. Preventing such cellular Ca2+ rises in the brain may reduce considerably the neuronal damage produced by stroke, head trauma, or epilepsy. Activation of NMDA receptors is instrumental in this type of neurotoxicity. Recent findings, discussed here by Istvan Mody and John MacDonald, indicate that a large proportion of the neurotoxic Ca2+ that enters nerve cells following NMDA receptor activation originates from an intracellular Ca2+ pool. The release of Ca2+ from this pool is sensitive to the skeletal muscle relaxant dantrolene, and this may constitute a novel and alternative therapeutic approach against NMDA receptor-mediated excitotoxicity.

Animals↗
PubMed1995

Blockade by sigma site ligands of N-methyl-D-aspartate-evoked responses in rat and mouse cultured hippocampal pyramidal neurones.

1. The effects of a range of structurally-dissimilar compounds which possess affinity for sigma binding sites were examined on the responses of cultured hippocampal pyramidal neurones to the excitatory amino acid analogues N-methyl-D-aspartate (NMDA), kainate and (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). 2. In mouse hippocampal neurones under whole-cell voltage-clamp, the compounds tested reversibly attenuated NMDA-, but not kainate- or AMPA-, evoked currents with a rank order potency (IC50 values in microM): ifenprodil (0.8) > (+)-N-allylnormetazocine (1.1) > dextromethorphan (1.8) = haloperidol (1.9) > (+)-pentazocine (7.2) > 1S,2R-(-)-cis-N-methyl-N-[2-(3, 4-dichlorophenyl) ethyl]-2-(1-pyrrolidinyl)cyclohexylamine (17) = rimcazole (18) > 1,3-di(2-tolyl)guanidine (37) > opipramol (96) > caramiphen (110) = carbetapentane (112) > > (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (485). 3. The attenuation of NMDA-evoked responses was not mediated through interactions with the agonist, glycine (except haloperidol) or polyamine (except ifenprodil) binding sites on the NMDA receptor-channel complex but, in the light of the voltage- and, in some cases, use-dependent nature of their antagonism, an interaction with the ion channel appears to be a likely mechanism of action for many of the compounds. 4. Micromolar concentrations of selected sigma site ligands also reduced NMDA-evoked rises in intracellular free calcium concentration in Fura-2-loaded cultured hippocampal neurones of the rat with the same rank order potency as observed in the electrophysiological studies. 5. The data indicate that, at micromolar concentrations, the sigma site ligands tested act as NMDA receptor antagonists, an action which does not appear to be mediated by high-affinity sigma binding site(s). The functional effects of micromolar concentrations of sigma site ligands cannot, therefore, be attributed exclusively to interactions with high-affinity sigma binding sites.

Animals↗
PubMed1995

Inhibition by propofol (2,6 di-isopropylphenol) of the N-methyl-D-aspartate subtype of glutamate receptor in cultured hippocampal neurones.

1. The effects of propofol (2,6 di-isopropylphenol) on responses to the selective glutamate receptor agonists, N-methyl-D-aspartate (NMDA) and kainate, were investigated in cultured hippocampal neurones of the mouse. Whole cell and single channel currents were recorded by patch-clamp techniques. Drugs were applied with a multi-barrel perfusion system. 2. Propofol produced a reversible, dose-dependent inhibition of whole cell currents activated by NMDA. The concentration of propofol which induced 50% of the maximal inhibition (IC50) was approximately 160 microM. The maximal inhibition was incomplete leaving a residual current of about 33% of the control response. This inhibitory action of propofol was neither voltage- nor use-dependent. 3. Analysis of the dose-response relation for whole cell NMDA-activated currents indicated that propofol caused no significant change in the apparent affinity of the receptor for NMDA. 4. Outside-out patch recordings of single channel currents evoked by NMDA (10 microM) revealed that propofol (100 microM) reversibly decreased the probability of channel opening but did not influence the average duration of channel opening or single channel conductance. 5. Whole-cell currents evoked by kainate (50 microM) were insensitive to propofol (1 microM-1 mM). 6. These results indicate that propofol inhibits the NMDA subtype of glutamate receptor, possibly through an allosteric modulation of channel gating rather than by blocking the open channel. Depression of NMDA-mediated excitatory neurotransmission may contribute to the anaesthetic, amnesic and anti-convulsant properties of propofol.

Animals↗
PubMed1994

Alteration of neuronal calcium homeostasis and excitotoxic vulnerability by chronic depolarization.

Free intracellular Ca2+ concentration ([Ca2+]i, Ca2+ currents, and excitatory amino acid (EAA) currents were studied in spinal neurons cultured in low (4.5 mM) and high (25 mM) extracellular potassium. When challenged with lethal concentrations of N-methyl-D-aspartate (NMDA) or kainate, neurons cultured in 25 mM K+ exhibited markedly attenuated Ca2+ currents and [Ca2+]i responses, and survived the EAA challenge more readily than controls. Surprisingly, NMDA and Kainate currents remained comparable between neurons grown in high- and low K+. The disparity between the observed [Ca2+]i increases and EAA currents suggests that chronic depolarization induces a fundamental alteration in intracellular Ca2+ handling. This phenomenon may provide clues for the development of neuroprotective strategies against excitotoxin excess.

Animals↗
PubMed1994

Expression of functional metabotropic and ionotropic glutamate receptors in baculovirus-infected insect cells.

An ionotropic glutamate receptor of the kainate subtype (GluR6) and a G-protein coupled metabotropic glutamate receptor (mGluR1 alpha) were expressed and studied in two insect cell lines: sf9 cells from Spodoptera frugiperda and MG1 cells from Trichoplusia ni. Application of kainate to GluR6-infected MG1 cells produced kainate-activated currents. Glutamate activation of mGluR1 alpha in MG1- and sf9-infected cells caused rapid, transient increases in intracellular calcium levels. This effect was more pronounced in MG1 cells compared to sf9 cells. These results indicate that functional glutamate receptors can be expressed in the baculovirus system, and that MG1 cells may have several advantages over the widely used sf9 cells for studying the functional properties of receptors and channels.

Animals↗
PubMed1994

Regulation of NMDA receptors in cultured hippocampal neurons by protein phosphatases 1 and 2A.

Phosphorylation of glutamate receptors is probably an important mechanism for modulating excitatory transmission. However, there is little direct evidence to indicate which protein phosphatases can dephosphorylate glutamate or other ligand-gated channels, although it is known that protein phosphatases 1 and 2A play a major part in modulating voltage and second-messenger-gated channels. Here we report that in cultured hippocampal neurons, the N-methyl-D-aspartate (NMDA) receptor can be regulated by endogenous and exogenous serine/threonine protein phosphatases. Phosphatase inhibitors enhanced NMDA currents recorded using the perforated patch technique or in cell-attached patches, whereas protein phosphatases 1 or 2A decreased the open probability of these channels in inside-out patches.

Cells, Cultured↗
PubMed1994

Sensitivity of AMPA receptors to pentobarbital.

The inhibitory effects of pentobarbital on various AMPA receptors expressed (GluR1, GluR3, GluR1/3, GluR1/2, and GluR2/3) in Xenopus oocytes were examined. Combinations of AMPA receptor subunits that included GluR2 demonstrated a much higher sensitivity to blockade by this barbiturate and the apparent co-operativity of the interaction of pentobarbital with the receptor was reduced. This evidence demonstrates that the GluR2 subunit alters the structure of AMPA receptors in such a way as to facilitate any interaction with this barbiturate.

Animals↗
PubMed1994

A transmembrane model for an ionotropic glutamate receptor predicted on the basis of the location of asparagine-linked oligosaccharides.

Several different models have been proposed for the transmembrane structure of receptors for the neurotransmitter L-glutamate. In this study, the sites of N-linked oligosaccharides on GluR6, a member of the kainate class of ionotropic glutamate receptors, were examined. Site-directed mutagenesis was utilized to alter the consensus sequence at three potential sites for N-linked glycosylation in the carboxyl-terminal half of the molecule. The presence of a carbohydrate substitution was monitored by shifts in the relative molecular weight of the mutant receptors on immunoblots. Molecular weight shifts were observed for the mutants N515Q and N720Q and for two companion mutants, T517A and T722A, which also eliminate the consensus sequence for N-linked glycosylation. No shift in molecular weight was observed in the the mutant N574Q. These results indicate that asparagines 515 and 720 are glycosylated and thus are likely located extracellularly. In immunocytochemical analyses of GluR6 expressed in baculovirus-infected cells, permeabilization with detergents was required for immunostaining with a carboxyl-terminal antibody, indicating that the carboxyl terminus is located intracellularly. Electrophysiological recordings of the mutant receptors expressed in human embryonic kidney cells demonstrated that the amplitudes of the kainate-activated currents mediated by the N574Q, N720Q, and the T722A mutants were not significantly different from currents mediated by wild type GluR6 receptors, while the currents mediated by the N515Q and T517A mutants were significantly depressed. Based on these findings, we propose a model for the transmembrane topology of GluR6.

Amino Acid Sequence↗
PubMed1994

Haloperidol blocks voltage-activated Ca2+ channels in hippocampal neurones.

The Ca2+ channel antagonist action of the antipsychotic haloperidol was investigated using two functional assays of Ca2+ channel activity. Haloperidol dose dependently attenuated the rise in intracellular free Ca2+ ([Ca2+]i) evoked by 50 mM extracellular K+ in Fura-2 loaded cultured rat hippocampal neurones with an IC50 (+/- S.E.M.) of 7.8 +/- 0.5 microM and similarly reduced whole-cell Ba2+ currents (IBa) in voltage-clamped mouse hippocampal neurones with an IC50 value of 15.6 +/- 1.1 microM. Block of whole-cell IBa by haloperidol was rapid, fully reversible, and was greater at more depolarized membrane potentials. Our data indicate that haloperidol non-selectively blocks neuronal voltage-gated Ca2+ channels at micromolar concentrations.

Animals↗
PubMed1994

Modulation of AMPA/kainate receptors in cultured murine hippocampal neurones by protein kinase C.

1. The patch clamp technique, together with intracellular perfusion of the catalytic fragment of protein kinase C (PKCM), was employed to investigate the role of this enzyme in the intracellular regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors in cultured hippocampal neurones. 2. The responses evoked by near-maximal concentrations of kainate (250 microM) and AMPA (100 microM) were potentiated by the introduction of PKCM, whilst co-application of the inhibitory peptide fragment PKCI(19-36) prevented this action. 3. Modulation of kainate responses by PKCM was dependent upon the concentration of agonist applied. Currents evoked by kainate were potentiated at concentrations above those which caused 50% of the maximal response (EC50) and depressed at lower concentrations. Furthermore, okadaic acid, a specific inhibitor of phosphatases 1 and 2A, had a similar effect upon concentration-response relationships when currents activated by kainate were recorded using the perforated patch technique. 4. In addition, the mean amplitude and/or time constant of decay of miniature excitatory synaptic currents (mediated by AMPA/kainate receptors) was increased by the intracellular injection of PKCM. 5. These observations suggest that the function of postsynaptic excitatory amino acid receptors can be modulated by the activity of PKC as well as by endogenous phosphatases. This regulation may contribute to some forms of synaptic plasticity within the central nervous system.

Animals↗
PubMed1994

Blockade by ifenprodil of high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones: comparison with N-methyl-D-aspartate receptor antagonist actions.

1. The block by ifenprodil of voltage-activated Ca2+ channels was investigated in intracellular free calcium concentration ([Ca2+]i) evoked by 50 mM K+ (high-[K+]o) in Fura-2-loaded rat hippocampal pyramidal neurones in culture and on currents carried by Ba2+ ions (IBa) through Ca2+ channels in mouse cultured hippocampal neurones under whole-cell voltage-clamp. The effects of ifenprodil on voltage-activated Ca2+ channels were compared with its antagonist actions on N-methyl-D-aspartate- (NMDA) evoked responses in the same neuronal preparations. 2. Rises in [Ca2+]i evoked by transient exposure to high-[K+]o in our preparation of rat cultured hippocampal pyramidal neurones are mediated predominantly by Ca2+ flux through nifedipine-sensitive Ca2+ channels, with smaller contributions from nifedipine-resistant, omega-conotoxin GVIA-sensitive Ca2+ channels and Ca2+ channels sensitive to crude funnel-web spider venom (Church et al., 1994). Ifenprodil (0.1-200 microM) reversibly attenuated high-[K+]o-evoked rises in [Ca2+]i with an IC50 value of 17 +/- 3 microM, compared with an IC50 value of 0.7 +/- 0.1 microM for the reduction of rises in [Ca2+]i evoked by 20 microM NMDA. Tested in the presence of nifedipine 10 microM, ifenprodil (1-50 microM) produced a concentration-dependent reduction of the dihydropyridine-resistant high-[K+]o-evoked rise in [Ca2+]i with an IC50 value of 13 +/- 4 microM. The results suggest that ifenprodil blocks Ca2+ flux through multiple subtypes of high voltage-activated Ca2+ channels. 3. Application of the polyamine, spermine (0.25-5 mM), produced a concentration-dependent reduction of rises in [Ca2+]i evoked by high-[K+]o. The antagonist effects of ifenprodil 20 micro M on high-[K+]0-evoked rises in [Ca2+]. were attenuated by spermine 0.25 mM but not by putrescine 1 or 5 mM. In contrast,spermine 0.1 mM increased rises in [Ca2+]i evoked by NMDA and enhanced the ifenprodil (5 micro M) block of NMDA-evoked rises in [Ca2+]i.4. Similar results were obtained in mouse cultured hippocampal pyramidal neurones under whole-cell voltage-clamp. Ifenprodil attenuated both the peak and delayed whole-cell IB. with an IC% value of 18 +/- 2 micro M, whilst it attenuated steady-state NMDA-evoked currents with an IC50 of 0.8 +/- 0.2 micro M. Block of IBa by ifenprodil 10 JaM was rapid in onset, fully reversible and occurred without change in thecurrent-voltage characteristics of Ba. The ifenprodil block of IBa was enhanced on membrane depolarization and was weakly dependent on the frequency of current activation. Spermine 0.1 mM potentiated control NMDA-evoked currents but attenuated IB,. In agreement with the microspectrofluorimetric studies, co-application of spermine produced a small enhancement of the inhibitory effect of ifenprodil 10 micro M on NMDA-evoked responses whereas the reduction of I4 by ifenprodil 10 micro M in the presence of spermine was less than expected if the inhibitory effects of ifenprodil and spermine on IBa were simply additive.5. The results indicate that ifenprodil blocks high voltage-activated Ca2+ channels in rat and mouse cultured hippocampal pyramidal neurones. Although the Ca2+ channel blocking actions of ifenprodil are observed at higher concentrations than those associated with NMDA antagonist activity, Ca2+ channel blockade may contribute, at least in part, to the established neuroprotective and anticonvulsant properties of the compound.

Animals↗