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John F MacDonald

Publications and source records attributed to John F MacDonald.

18 recordsLinked to original sources
PubMed2006

Recent advances in basic neurosciences and brain disease: from synapses to behavior.

Understanding basic neuronal mechanisms hold the hope for future treatment of brain disease. The 1st international conference on synapse, memory, drug addiction and pain was held in beautiful downtown Toronto, Canada on August 21-23, 2006. Unlike other traditional conferences, this new meeting focused on three major aims: (1) to promote new and cutting edge research in neuroscience; (2) to encourage international information exchange and scientific collaborations; and (3) to provide a platform for active scientists to discuss new findings. Up to 64 investigators presented their recent discoveries, from basic synaptic mechanisms to genes related to human brain disease. This meeting was in part sponsored by Molecular Pain, together with University of Toronto (Faculty of Medicine, Department of Physiology as well as Center for the Study of Pain). Our goal for this meeting is to promote future active scientific collaborations and improve human health through fundamental basic neuroscience researches. The second international meeting on Neurons and Brain Disease will be held in Toronto (August 29-31, 2007).

Behavior↗
PubMed2006

D2-class dopamine receptor inhibition of NMDA currents in prefrontal cortical neurons is platelet-derived growth factor receptor-dependent.

NMDA receptor function is modulated by both G-protein-coupled receptors and receptor tyrosine kinases. In acutely isolated rat hippocampal neurons, direct activation of the platelet-derived growth factor (PDGF) receptor or transactivation of the PDGF receptor by D4 dopamine receptors inhibits NMDA-evoked currents in a phospholipase C (PLC)-dependent manner. We have investigated further the ability of D2-class dopamine receptors to modulate NMDA-evoked currents in isolated rat prefrontal cortex (PFC). We have demonstrated that, similar to isolated hippocampal neurons, the application of PDGF-BB or quinpirole to isolated PFC neurons induces a slow-onset and long-lasting inhibition of NMDA-evoked currents. However, in contrast to hippocampal neurons, the inhibition of NMDA-evoked currents by quinpirole in PFC neurons is dependent upon D2/3, rather than D4, dopamine receptors. In PFC slices, application of both PDGF-BB and quinpirole induced a phosphorylation of the PDGF receptor at the PLCgamma binding and activation site, Tyr1021. The PDGF receptor kinase inhibitor, tyrphostin A9, and the D2/3 dopamine receptor antagonist, raclopride, inhibited quinpirole-induced Tyr1021 phosphorylation. These finding suggest that quinpirole treatment inhibits NMDAR signaling via PDGF receptor transactivation in both the hippocampus and the PFC, and that the effects of quinpirole in these regions are mediated by D4 and D2/3 dopamine receptors, respectively.

Animals↗
PubMed2006

Protein kinase C enhances glycine-insensitive desensitization of NMDA receptors independently of previously identified protein kinase C sites.

Protein kinase C (PKC) phosphorylates the NR1 and NR2A subunits of NMDARs at consensus sites located within their intracellular C-terminal tails. However, the functional consequences of these biochemical events are not well understood. In HEK293 cells expressing NR1/NR2A, activation of endogenous PKC by 4beta-phorbol 12-myristate 13-acetate (PMA) increased NMDAR desensitization as evidenced by a reduced steady-state current without any change in peak. The effects of PMA on NMDAR-mediated responses were prevented by specific PKC inhibitors and were not mimicked by an inactive enantiomer of PMA. The effects of PMA were preserved despite mutagenesis of the major PKC sites on the NR1 subunit (S889A, S890A, S896A and S897A) or removal of the entire NR1 C-terminal tail (NR1(stop838)). When co-expressing NR1(stop838)/NR2A the effects of PMA could only be observed with agonist concentrations sufficient to induce glycine-insensitive desensitization. Moreover, the effects of PMA were observed in receptors composed of NR1/NR2A and NR1/NR2B, but not NR1/NR2C, a subunit combination in which desensitization is absent. The NR2 subunit dependence suggested that the actions of PMA might require specific PKC sites previously identified within NR2A. However, a C-terminal truncated form of NR2A (NR2A(stop905)) remained responsive to PMA. We conclude that activation of PKC increases NMDAR glycine-insensitive desensitization independently of previously identified sites located within the NR1 C-terminus and distal segment of the NR2A C-terminus.

Binding Sites↗
PubMed2006

Paradox of Ca2+ signaling, cell death and stroke.

Since the early 1960s it has been known that restoring extracellular Ca2+ following a period of low Ca2+ concentrations paradoxically causes an increase in intracellular Ca2+ levels that can lead to cell death. The mystery of this 'Ca2+ paradox' is made more intriguing by observations that lowering concentrations of extracellular Ca2+ and/or Mg2+ paradoxically enhances the entry of Ca2+ into hippocampal neurons. Until recently, the entry of Ca2+ through NMDA receptors was accepted as the major pathway leading to the excitotoxic, delayed cell death associated with the ischemic periods of stroke. Here, we discuss how several transient receptor potential (TRP) channels are likely to contribute to both the Ca2+ paradox and the delayed death of neurons following an ischemic stroke.

Animals↗
PubMed2006

Gabapentin increases a tonic inhibitory conductance in hippocampal pyramidal neurons.

BACKGROUND: The mechanisms underlying the therapeutic actions of gabapentin remain poorly understood. The chemical structure and behavioral properties of gabapentin strongly suggest actions on inhibitory neurotransmission mediated by gamma-aminobutyric acid (GABA); however, gabapentin does not directly modulate GABAA or GABAB receptors. Two distinct forms of GABAergic inhibition occur in the brain: postsynaptic conductance and a persistent tonic inhibitory conductance primarily generated by extrasynaptic GABAA receptors. The aim of this study was to determine whether gabapentin increased the tonic conductance in hippocampal neurons in vitro. As a positive control, the effects of vigabatrin, which irreversibly inhibits GABA transaminase, were also examined. METHODS: GABAA receptors in hippocampal neurons from embryonic mice were studied using whole cell patch clamp recordings. Miniature inhibitory postsynaptic currents and the tonic current were recorded from cultured neurons that were treated for 36-48 h with gabapentin, vigabatrin, or gabapentin and vigabatrin. To determine whether gabapentin increased the expression of GABAA receptors, Western blots were stained with antibodies selective for alpha1, alpha2, and alpha5 subunits. RESULTS: GABAA receptors were insensitive to the acute application of gabapentin, whereas chronic treatment increased the amplitude of the tonic current threefold (EC50 = 209 microm) but did not influence miniature inhibitory postsynaptic currents. Vigabatrin increased the tonic conductance, and the maximally effective concentration did not occlude the actions of gabapentin, which suggests that these compounds act by different mechanisms. Neither gabapentin nor vigabatrin increased the expression of GABAA receptors in the neurons. CONCLUSIONS: Gabapentin increases a tonic inhibitory conductance in mammalian neurons. High-affinity GABAA receptors that generate the tonic conductance may detect small increases in the ambient concentration of neurotransmitter caused by gabapentin.

4-Aminobutyrate Transaminase↗
PubMed2004

Selective enhancement of tonic GABAergic inhibition in murine hippocampal neurons by low concentrations of the volatile anesthetic isoflurane.

Volatile (inhaled) anesthetics cause amnesia at concentrations well below those that cause loss of consciousness and immobility; however, the underlying neuronal mechanisms are unknown. Although many anesthetics increase inhibitory GABAergic synaptic transmission, this effect occurs only at high concentrations (>100 microm). Molecular targets for low concentrations of inhaled anesthetics have not been identified. Here, we report that a tonic inhibitory conductance in hippocampal pyramidal neurons generated by alpha5 subunit-containing GABA(A) receptors is highly sensitive to low concentrations of the volatile anesthetic isoflurane (ISO) (25 and 83.3 microm). The alpha5 subunit is necessary for enhancement of the tonic current by these low concentrations of isoflurane because potentiation is absent in neurons from alpha5-/- mice. Furthermore, ISO (25 microm) potentiated recombinant human alpha5beta3gamma2L GABA(A) receptors, whereas this effect was not seen with alpha1beta3gamma2L GABA(A) receptors. These studies suggest that an increased tonic inhibition in the hippocampus may contribute to amnestic properties of volatile anesthetics.

Anesthetics, Inhalation↗
PubMed2004

Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels.

Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.

Acid Sensing Ion Channels↗
PubMed2004

Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by alpha5 subunit-containing gamma-aminobutyric acid type A receptors.

The principal inhibitory neurotransmitter in the mammalian brain, gamma-aminobutyric acid (GABA), is thought to regulate memory processes by activating transient inhibitory postsynaptic currents. Here we describe a nonsynaptic, tonic form of inhibition in mouse CA1 pyramidal neurons that is generated by a distinct subpopulation of GABA type A receptors (GABA(A)Rs). This tonic inhibitory conductance is predominantly mediated by alpha5 subunit-containing GABA(A)Rs (alpha5GABA(A)Rs) that have different pharmacological and kinetic properties compared to postsynaptic receptors. GABA(A)Rs that mediate the tonic conductance are well suited to detect low, persistent, ambient concentrations of GABA in the extracellular space because they are highly sensitive to GABA and desensitize slowly. Moreover, the tonic current is highly sensitive to enhancement by amnestic drugs. Given the restricted expression of alpha5GABA(A)Rs to the hippocampus and the association between reduced alpha5GABA(A)R function and improved memory performance in behavioral studies, our results suggest that tonic inhibition mediated by alpha5GABA(A)Rs in hippocampal pyramidal neurons plays a key role in cognitive processes.

Animals↗
PubMed2003

A key role for TRPM7 channels in anoxic neuronal death.

Excitotoxicity in brain ischemia triggers neuronal death and neurological disability, and yet these are not prevented by antiexcitotoxic therapy (AET) in humans. Here, we show that in neurons subjected to prolonged oxygen glucose deprivation (OGD), AET unmasks a dominant death mechanism perpetuated by a Ca2+-permeable nonselective cation conductance (IOGD). IOGD was activated by reactive oxygen/nitrogen species (ROS), and permitted neuronal Ca2+ overload and further ROS production despite AET. IOGD currents corresponded to those evoked in HEK-293 cells expressing the nonselective cation conductance TRPM7. In cortical neurons, blocking IOGD or suppressing TRPM7 expression blocked TRPM7 currents, anoxic 45Ca2+ uptake, ROS production, and anoxic death. TRPM7 suppression eliminated the need for AET to rescue anoxic neurons and permitted the survival of neurons previously destined to die from prolonged anoxia. Thus, excitotoxicity is a subset of a greater overall anoxic cell death mechanism, in which TRPM7 channels play a key role.

Animals↗
PubMed2003

Acidosis decreases low Ca(2+)-induced neuronal excitation by inhibiting the activity of calcium-sensing cation channels in cultured mouse hippocampal neurons.

The effects of extracellular pH (pHo) on calcium-sensing non-selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole-cell voltage-clamp and current-clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride-sensitive transient proton-gated currents which decayed to baseline in several seconds. With proton-gated channels inactivated by pre-perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose-inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose-inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current-clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o-induced csNSC channel-mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o-induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.

Acidosis↗
PubMed2003

Activation of PI3-kinase is required for AMPA receptor insertion during LTP of mEPSCs in cultured hippocampal neurons.

Hippocampal CA1 homosynaptic long-term potentiation (LTP) is expressed specifically at activated synapses. Increased insertion of postsynaptic alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) appears to be crucial for CA1 LTP. However, the mechanism underlying AMPAR insertion during LTP remains largely unknown. We now report that phosphatidylinositol 3-kinase (PI3K) is complexed with AMPARs at synapses and activated by selective stimulation of synaptic N-methyl-D-aspartate (NMDA) receptors. Activation of the AMPAR-associated PI3K is required for the increased cell surface expression of AMPARs and LTP. Thus, our results strongly suggest that the AMPAR-PI3K complex may constitute a critical molecular signal responsible for AMPAR insertion at activated CA1 synapses during LTP, and consequently, this lipid kinase may serve to determine the polarity of NMDA receptor-dependent synaptic plasticity.

Androstadienes↗
PubMed2003

Co-stimulation of mGluR5 and N-methyl-D-aspartate receptors is required for potentiation of excitatory synaptic transmission in hippocampal neurons.

In the central nervous system, excitatory synaptic transmission is mediated by the neurotransmitter glutamate and its receptors. Interestingly, stimulation of group I metabotropic glutamate receptors (mGluRs) can either enhance or depress synaptic transmission at CA1 hippocampal synapses. Here we report that co-activation of mGluR5, a member of the group I mGluR family, and N-methyl-d-aspartate receptors (NMDARs) potentiates NMDAR currents and induces a long lasting enhancement of excitatory synaptic transmission in primary cultured hippocampal neurons. Unexpectedly, activation of mGluR5 alone fails to enhance evoked NMDAR currents and synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) AMPAR currents. The observed potentiation requires an mGluR5-induced, inositol 1,4,5-trisphosphate receptor-mediated mobilization of intracellular Ca2+, which acts in concert with a protein kinase C, calcium-activated tyrosine kinase cascade to induce a long lasting enhancement of NMDAR and AMPAR currents.

Alkaloids↗
PubMed2003

Enhanced vulnerability to NMDA toxicity in sublethal traumatic neuronal injury in vitro.

Traumatic brain injury causes neuronal disruption and triggers secondary events leading to additional neuronal death. To study injuries triggered by secondary events, we exposed cultured cortical neurons to sublethal mechanical stretch, thus eliminating confounding death from primary trauma. Sublethally stretched neurons maintained cell membrane integrity, viability, and electrophysiological function. However, stretching induced in the cells a heightened vulnerability to subsequent challenges with L-glutamate or NMDA. This heightened vulnerability was specifically mediated by NMDA receptors (NMDARs), as stretched neurons did not become more vulnerable to either kainate toxicity or to that induced by the Ca(2+) ionophore A23187. Stretch-enhanced vulnerability to NMDA occurred independently of endogenous glutamate release, but required Ca(2+) and Na(+) influx through NMDARs. Stretch did not affect the electrophysiological properties of NMDARs nor excitatory synaptic activity, indicating that specificity of enhanced vulnerability to NMDA involves postsynaptic mechanisms downstream from NMDARs. To test whether this specificity requires physical interactions between NMDARs and cytoskeletal elements, we perturbed actin filaments and microtubules, both of which are linked to NMDARs. This had no effect on the stretch-induced vulnerability to NMDA, suggesting that sublethal stretch does not affect cell survival through the cytoskeleton. Our data illustrate that sublethal in vitro stretch injury triggers distinct signaling pathways that lead to secondary injury, rather than causing a generalized increase in vulnerability to secondary insults.

Animals↗
PubMed2003

Tonically activated GABAA receptors in hippocampal neurons are high-affinity, low-conductance sensors for extracellular GABA.

In the hippocampus, two distinct forms of GABAergic inhibition have been identified, phasic inhibitory postsynaptic currents that are the consequence of the vesicular release of GABA and a tonic conductance that is activated by low ambient concentrations of extracellular GABA. It is not known what accounts for the distinct properties of receptors that mediate the phasic and tonic inhibitory conductances. Moreover, the physiological role of the tonic inhibitory conductance remains uncertain because pharmacological tools that clearly distinguish tonic and phasic receptors are lacking. Here, we demonstrate that GABAA receptors that generate a tonic conductance in cultured hippocampal neurons from embryonic mice have different pharmacological properties than those in cerebellar granule neurons or pyramidal neurons in the dentate gyrus. The tonic conductance in cultured hippocampal neurons is enhanced by the benzodiazepine, midazolam, and is insensitive to the inhibitory effects of the competitive antagonist, gabazine (< or =10 microM). We also identify penicillin as an uncompetitive antagonist that selectively inhibits the synaptic but not tonic conductance. GABA was applied to hippocampal neurons to investigate the properties of synaptic and extrasynaptic receptors. GABA-evoked current was composed of two components: a rapidly desensitizing current that was blocked by penicillin and a nondesensitizing current that was insensitive to penicillin blockade. The potency of GABA was greater for the penicillin-insensitive nondesensitizing current. Single-channel studies show that the gabazine-insensitive GABAA receptors have a lower unitary conductance (12 pS) than that estimated for synaptic receptors. Thus, specialized GABAA receptors with an apparent higher affinity for GABA that do not readily desensitize mediate the persistent tonic conductance in hippocampal neurons. The receptors underlying tonic and phasic inhibitory conductances in hippocampal neurons are pharmacologically and biophysically distinct, suggesting that they serve different physiological roles.

Animals↗
PubMed2002

A D2 class dopamine receptor transactivates a receptor tyrosine kinase to inhibit NMDA receptor transmission.

Receptor tyrosine kinases (RTKs) are membrane spanning proteins with intrinsic kinase activity. Although these receptors are known to be involved in proliferation and differentiation of cells, their roles in regulating central synaptic transmission are largely unknown. In CA1 pyramidal neurons, activation of D2 class dopamine receptors depressed excitatory transmission mediated by the NMDA subtype of glutamate receptor. This depression resulted from the quinpirole-induced release of intracellular Ca(2+) and enhanced Ca(2+)-dependent inactivation of NMDA receptors. The dopamine receptor-mediated depression was dependent on the "transactivation" of PDGFRbeta. Therefore, RTK transactivation provides a novel mechanism of communication between dopaminergic and glutamatergic systems and might help to explain how reciprocal changes in these systems could be linked to the deficits in cognition, memory, and attention observed in schizophrenia and attention deficit hyperactivity disorder.

Animals↗
PubMed2002

Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice.

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, we demonstrate a significant role for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, we show that the knockout mice exhibited significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also showed altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function.

Actin Cytoskeleton↗
PubMed2002

Tyrosine kinases enhance the function of glycine receptors in rat hippocampal neurons and human alpha(1)beta glycine receptors.

Glycine receptors (GlyRs) are transmitter-gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR beta subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole-cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the beta subunit was examined using site-directed mutagenesis and expression of recombinant (alpha(1)beta(Y413F)) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine-evoked currents (I(Gly)) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced I(Gly) in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2-fold (control EC(50) = 143 microM; cSrc EC(50) = 74 microM). Exogenous cSrc, applied intracellularly, upregulated heteromeric alpha(1)beta receptors but not homomeric alpha(1) receptors. Substitution mutation of the tyrosine to phenylalanine at position beta-413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down-regulated wild-type alpha(1)beta but not alpha(1)beta(Y413F) receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine-413 residue of the beta subunit.

Animals↗