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Stanley A Thayer

Publications and source records attributed to Stanley A Thayer.

15 recordsLinked to original sources
PubMed2006

Delta9-tetrahydrocannabinol protects hippocampal neurons from excitotoxicity.

Excitotoxic neuronal death underlies many neurodegenerative disorders. Because cannabinoid receptor agonists act presynaptically to inhibit glutamate release, we examined the effects of Win 55212-2, a full agonist at CB(1) receptors, and Delta(9)-tetrahydrocannabinol (THC), a partial agonist, on the survival of neurons exposed to an excitotoxic pattern of synaptic activity. Reducing the extracellular Mg(2+) concentration ([Mg(2+)](o)) to 0.1 mM evoked an aberrant pattern of glutamatergic activity that produced synaptically mediated death of rat hippocampal neurons in culture. Neuronal viability was quantified with a multiwell fluorescence plate scanner equipped to detect propidium iodide fluorescence. Win 55212-2 (100 nM) and THC (100 nM) significantly reduced 0.1 mM [Mg(2+)](o)-induced cell death by 77 +/- 11% and 84 +/- 8%, respectively. Interestingly, the protection afforded by THC was not significantly different from that produced by Win 55212-2, suggesting that attenuation without a complete block of excitatory activity is sufficient for neuroprotection. The effect of prolonged drug exposure on the neuroprotection afforded by cannabinoid receptor agonists was also studied. When cultures were pretreated for 24 h with Win 55212-2 (100 nM) or THC (100 nM), inhibition of 0.1 mM [Mg(2+)](o)-induced toxicity was significantly reduced to 39 +/- 19% and 45 +/- 13%, respectively. Thus, desensitization of CB(1) receptors diminishes the neuroprotective effects of cannabinoids. This study demonstrates the importance of agonist efficacy and the duration of treatment on the neuroprotective effects of cannabinoids. It will be important to consider these effects on neuronal survival when evaluating pharmacologic treatments that modulate the endocannabinoid system.

Analysis of Variance↗
PubMed2006

Mitochondrial modulation of Ca2+ -induced Ca2+ -release in rat sensory neurons.

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.

Action Potentials↗
PubMed2006

Activation of protein kinase C in sensory neurons accelerates Ca2+ uptake into the endoplasmic reticulum.

The rate of Ca2+ clearance from the neuronal cytoplasm affects the amplitude, duration, and localization of Ca2+ signals and influences a variety of Ca2+-dependent functions. We reported previously that activation of protein kinase C (PKC) accelerates Ca2+ efflux in rat sensory neurons mediated by the plasma membrane Ca2+-ATPase isoform 4 (PMCA4). Here we show that sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-mediated Ca2+ uptake into intracellular stores is also accelerated by PKC activation. The rate of intracellular Ca2+ concentration ([Ca2+]i) clearance was studied after small (<350 nM) action potential-induced Ca2+ loads in rat dorsal root ganglion neurons. Under these conditions, mitochondrial Ca2+ uptake and Na+/Ca2+ exchange do not significantly influence [Ca2+]i recovery. Phorbol dibutyrate (PDBu) increased the rate of [Ca2+]i clearance by 71% in a manner sensitive to the selective PKC inhibitors GF109203x (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide) and calphostin. PKC-dependent acceleration was still observed (approximately 39%) when the PKC-sensitive PMCA isoform was knocked down by expression of an antisense PMCA4 cDNA (AS4). Direct measurement of Ca2+ in the endoplasmic reticulum (ER) lumen revealed that PKC increased the rate of store refilling more than twofold after depletion by treatment with cyclopiazonic acid. ER refilling was less complete in PDBu-treated cells, although, in AS4-expressing cells, PDBu accelerated the rate without reducing the ER capacity, suggesting that PMCA and SERCA compete for Ca2+. Thus, activation of PKC accelerates the clearance of Ca2+ from the cytoplasm by the concerted stimulation of Ca2+ sequestration and Ca2+ efflux.

Action Potentials↗
PubMed2006

Glutamate-induced protease-mediated loss of plasma membrane Ca2+ pump activity in rat hippocampal neurons.

Ca2+ dysregulation is a hallmark of excitotoxicity, a process that underlies multiple neurodegenerative disorders. The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Here, we show that the rate of PMCA-mediated Ca2+ efflux from rat hippocampal neurons decreased following treatment with an excitotoxic concentration of glutamate. PMCA-mediated Ca2+ extrusion following a brief train of action potentials exhibited an exponential decay with a mean time constant (tau) of 8.8 +/- 0.2 s. Four hours following the start of a 30 min treatment with 200 microm glutamate, a second population of cells emerged with slowed recovery kinetics (tau = 16.5 +/- 0.3 s). Confocal imaging of cells expressing an enhanced green fluorescent protein (EGFP)-PMCA4b fusion protein revealed that glutamate treatment internalized EGFP and that cells with reduced plasma membrane fluorescence had impaired Ca2+ clearance. Treatment with inhibitors of the Ca2+-activated protease calpain protected PMCA function and prevented EGFP-PMCA internalization. PMCA internalization was triggered by activation of NMDA receptors and was less pronounced for a non-toxic concentration of glutamate relative to one that produces excitotoxicity. PMCA isoform 2 also internalized following exposure to glutamate, although the Na+/K+ ATPase did not. These data suggest that glutamate exposure initiated protease-mediated internalization of PMCAs with a corresponding loss of function that may contribute to the Ca2+ dysregulation that accompanies excitotoxicity.

Adenosine Triphosphatases↗
PubMed2005

Delta(9)-Tetrahydrocannabinol-induced desensitization of cannabinoid-mediated inhibition of synaptic transmission between hippocampal neurons in culture.

Prolonged exposure to cannabinoids results in desensitization of cannabinoid receptors. Here, we compared the desensitization produced by the partial agonist, Delta(9)-tetrahydrocannabinol (THC) to that produced by the full agonist Win55,212-2 on cannabinoid-mediated inhibition of glutamatergic synaptic transmission. Synaptic activity between rat hippocampal neurons was determined from network-driven increases in the intracellular Ca(2+) concentration ([Ca(2+)](i) spikes). To assess the effects of prolonged treatment, cultures were incubated with cannabinoids, washed in 0.5% fatty-acid-free bovine serum albumin to ensure the removal of the lipophilic drug and then tested for inhibition of [Ca(2+)](i) spiking by Win55,212-2. In control experiments, 0.1 microM Win55,212-2 inhibited [Ca(2+)](i) spiking by 93 +/- 5%. Win55,212-2 produced significantly less inhibition of [Ca(2+)](i) spiking following 18-24h treatment with 1 microM THC (48 +/- 5%) or treatment with 1 microM Win55,212-2 (29 +/- 6%). Thus, THC produced significantly less functional desensitization than Win55,212-2. The desensitization produced by THC was maximal at 0.3 microM, remained stable between 1 and 7 days of preincubation and shifted the EC(50) of acute inhibition by Win55,212-2 from 27 to 251 nM. Differences in the long-term effects of cannabinoid receptor agonists on synaptic transmission may prove important for evaluating their therapeutic and abuse potential.

Animals↗
PubMed2004

Anandamide transport inhibitor AM404 and structurally related compounds inhibit synaptic transmission between rat hippocampal neurons in culture independent of cannabinoid CB1 receptors.

N-(hydroxyphenyl)-arachidonamide (AM404) is an inhibitor of endocannabinoid transport. We examined the effects of AM404 on glutamatergic synaptic transmission using network-driven increases in intracellular Ca2+ concentration ([Ca2+] spikes) as an assay. At a concentration of 1 microM AM404 inhibited [Ca2+]i spiking by 73+/-8%. The cannabinoid CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A), the vanilloid VR1 receptor antagonist capsazepine (CPZ), and treatment with pertussis toxin failed to block AM404-mediated inhibition. AM404 (3 microM) inhibited action-potential-evoked Ca2+ influx by 58+/-3% but failed to affect calcium influx evoked by depolarization with 30 mM K+, suggesting that the inhibition of electrically evoked [Ca2+]i increases and that [Ca2+]i spiking was due to inhibition of Na+ channels. Palmitoylethanolamide (PMEA), capsaicin (CAP) and (5Z,8Z,11Z,14Z)-N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide (VDM11), compounds structurally similar to AM404, inhibited [Ca2+]i spiking by 34+/-10%, 42+/-18% and 67+/-12%, respectively. Thus, AM404 and related compounds inhibit depolarization-induced Ca2+ influx independent of cannabinoid receptors, suggesting caution when using these agents as pharmacological probes to study synaptic transmission.

Animals↗
PubMed2004

Delta 9-tetrahydrocannabinol antagonizes endocannabinoid modulation of synaptic transmission between hippocampal neurons in culture.

Cannabinoids inhibit excitatory synaptic transmission between hippocampal neurons in culture. Delta9-tetrahydrocannabinol (THC), the principal psychoactive component in marijuana, acts as a partial agonist at these synapses. Thus, THC inhibited but did not block synaptic transmission when applied alone and, when applied in combination with WIN552212-2, it partially reversed the effects of this full agonist. Here, we address the question of how THC might interact with endocannabinoid signaling. Reducing the extracellular Mg2+ concentration to 0.1 mM elicited a repetitive pattern of glutamatergic synaptic activity that produced intracellular Ca2+ concentration spikes that were measured by indo-1-based microfluorimetry. The endocannabinoid, 2-arachidonyl glycerol (2-AG) produced a concentration-dependent and complete inhibition of spike frequency with an EC50 of 63 +/- 13 nM. 2-AG (1 microM) inhibition of spiking was blocked by SR141716A (1 microM). THC (100 nM) antagonized the actions of 2-AG producing a parallel shift in the concentration-response relationship for 2-AG (EC50 of 1430 +/- 254 nM). The attenuation of 2-AG (1 microM) inhibition of synaptic activity by THC was concentration-dependent with an IC50 of 42 +/- 9 nM. These results demonstrate that THC can antagonize endocannabinoid signaling. Thus, the effects of THC on synaptic transmission are predicted to depend on the level of endocannabinoid tone.

Animals↗
PubMed2003

NMDA-evoked consumption and recovery of mitochondrially targeted aequorin suggests increased Ca2+ uptake by a subset of mitochondria in hippocampal neurons.

Activation of NMDA receptors produces large increases in cytosolic Ca(2+) that are taken up into mitochondria. We used recombinant aequorin targeted to mitochondria to report changes in matrix Ca(2+) in rat hippocampal neurons in culture. Upon binding Ca(2+), aequorin emits a photon in a one-shot reaction that consumes the indicator. Here we show that stimulation with NMDA produced a mitochondrial Ca(2+) response that rapidly inactivated. However, following a 30-min recovery period the response was restored, suggesting the presence of a pool of indicator that was not exposed to high Ca(2+) during the initial stimulus. We speculate that aequorin distant from the Ca(2+) source was protected from microdomains of high Ca(2+) near the plasmalemma and that this aequorin moved, either by movement of individual mitochondria or via the mitochondrial tubular network, to replenish consumed indicator during the recovery time. A large Ca(2+) increase in a subset of mitochondria could produce local changes in energy metabolism, regional Ca(2+) buffering, and foci that initiate neurotoxic processes.

Aequorin↗
PubMed2003

Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in culture.

The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 micro m, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+-induced toxicity and participate in local ATP production.

Adenosine Triphosphate↗
PubMed2002

Modulating Ca2+ clearance from neurons.

Neurons are exquisitely sensitive to the duration, amplitude and localization of transient increases in intracellular Ca2+ concentration ([Ca2+]i). Modulation of Ca2+ uptake into the mitochondrion and endoplasmic reticulum, and efflux via the plasma membrane Ca2+ pump and Na+/Ca2+ exchange profoundly affect the shape of [Ca2+]i signals. Ca2+ clearance mechanisms are modulated by other signaling pathways, are sensitive to metabolic state and have a memory of the recent history of cell activation. We present here examples of pharmacologic and endogenous regulation of Ca2+ sequestration and efflux in neurons. Ca2+ clearance mechanisms differentially shape [Ca2+]i signals based on their affinity, capacity and location; their modulation alters specific neuronal functions. The increasingly apparent diversity of the molecular entities that make up the [Ca2+]i regulatory system reveals new sites for modulation. Specialized Ca2+ clearance mechanisms participate in unique cellular functions and thus, are important targets for pharmacological and physiological regulation of the neuron.

Animals↗
PubMed2002

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.

Action Potentials↗
PubMed2002

Transient rise in intracellular calcium produces a long-lasting increase in plasma membrane calcium pump activity in rat sensory neurons.

The plasma membrane Ca2+ ATPase (PMCA) plays a major role in clearing Ca2+ from the neuronal cytoplasm. Calmodulin stimulates PMCA activity and for some isoforms this activation persists following clearance of Ca2+ owing to the slow dissociation of calmodulin. We tested the hypothesis that PMCA-mediated Ca2+ efflux from rat dorsal root ganglion (DRG) neurons in culture would remain stimulated following increases in intracellular Ca2+ concentration ([Ca2+]i). PMCA-mediated Ca2+ extrusion was recorded following brief trains of action potentials using indo-1-based photometry in the presence of cyclopiazonic acid. A priming stimulus that increased [Ca2+]i to 506 +/- 28 nm (>15 min) increased the rate constant for [Ca2+]i recovery by 47 +/- 3%. Ca2+ clearance from subsequent test stimuli remained accelerated for up to an hour despite removal of the priming stimulus and a return to basal [Ca2+]i. The acceleration depended on the magnitude and duration of the priming [Ca2+]i increase, but was independent of the source of Ca2+. Increases in [Ca2+]i evoked by prolonged depolarization, sustained trains of action potentials or activation of vanilloid receptors all accelerated Ca2+ efflux. We conclude that PMCA-mediated Ca2+ efflux in DRG neurons is a dynamic process in which intense stimuli prime the pump for the next Ca2+ challenge.

Action Potentials↗
PubMed2002

Desensitization of cannabinoid-mediated presynaptic inhibition of neurotransmission between rat hippocampal neurons in culture.

Prolonged exposure to cannabinoids results in tolerance in vivo and desensitization of cannabinoid receptors in vitro. We show here that cannabinoid-induced presynaptic inhibition of glutamatergic neurotransmission desensitized after prolonged exposure to the cannabinoid receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (Win55,212-2). Synaptic activity between hippocampal neurons in culture was determined from network-driven increases in intracellular Ca(2+) concentration ([Ca(2+)](i) spikes) and excitatory postsynaptic currents. Win55,212-2-induced (100 nM) inhibition partially desensitized after 2 h and completely desensitized after 18- to 24-h exposure. The desensitization could be overcome by higher concentrations of agonist as indicated by a parallel rightward shift of the concentration response curve from an EC(50) of 2.7 +/- 0.3 nM to 320 +/- 147 nM for inhibition of [Ca(2+)](i) spiking and from 43 +/- 17 nM to 4505 +/- 403 nM for inhibition of synaptic currents, suggesting that this phenomenon may underlie tolerance. Presynaptic expression of dominant negative G-protein-coupled-receptor kinase (GRK2-Lys220Arg) or beta-arrestin (319-418) reduced the desensitization produced by 18- to 24-h pretreatment with 100 nM, Win55,212-2 suggesting that desensitization followed the prototypical pathway for G-protein-coupled receptors. Prolonged treatment with Win55,212-2 produced a modest increase in the EC(50) for adenosine inhibition of synaptic transmission and pretreatment with cyclopentyladenosine produced a slight increase in the EC(50) for Win55,212-2, suggesting a reciprocal ability to produce heterologous desensitization. The long-term changes in synaptic function that accompany chronic cannabinoid exposure will be an important factor in evaluating the therapeutic potential of these drugs and will provide insight into the role of the endocannabinoid system.

Animals↗
PubMed2002

NMDA-induced calcium loads recycle across the mitochondrial inner membrane of hippocampal neurons in culture.

Mitochondria sequester N-methyl-D-aspartate (NMDA)-induced Ca(2+) loads and regulate the shape of intracellular Ca(2+) concentration ([Ca(2+)](i)) responses in neurons. When isolated mitochondria are exposed to high [Ca(2+)](,) Ca(2+) enters the matrix via the uniporter and returns to the cytosol by Na(+)/Ca(2+) exchange. Released Ca(2+) may re-enter the mitochondrion recycling across the inner membrane dissipating respiratory energy. Ca(2+) recycling, the continuous uptake and release of Ca(2+) by mitochondria, has not been described in intact neurons. Here we used single-cell microfluorimetry to measure [Ca(2+)](i) and mitochondrially targeted aequorin to measure matrix Ca(2+) concentration ([Ca(2+)](mt)) to determine whether Ca(2+) recycles across the mitochondrial inner membrane in intact neurons following treatment with NMDA. We used ruthenium red and CGP 37157 to block uptake via the uniporter and release via Na(+)/Ca(2+) exchange, respectively. As predicted by the Ca(2+) recycling hypothesis, blocking the uniporter immediately following challenge with 200 microM NMDA produced a rapid and transient increase in cytosolic Ca(2+) without a corresponding increase in matrix Ca(2+). Blocking mitochondrial Ca(2+) release produced the opposite effect, depressing cytosolic Ca(2+) levels and prolonging the time for matrix Ca(2+) levels to recover. The Ca(2+) recycling hypothesis uniquely predicts these reciprocal changes in the Ca(2+) levels between the two compartments. Ca(2+) recycling was not detected following treatment with 20 microM NMDA. Thus Ca(2+) recycling across the inner membrane was more pronounced following treatment with a high relative to a low concentration of NMDA, consistent with a role in Ca(2+)-dependent neurotoxicity.

Animals↗