James R. Brorson

The effect of capsaicin on voltage‐gated calcium currents and calcium signals in cultured dorsal root ganglion cells

1 The effects of capsaicin on voltage‐gated Ca2+ currents (ICa), and intracellular Ca2+ concentrations ([Ca2+]i) in cultured dorsal root ganglion (DRG) neurones of the rat were examined in vitro by use of combined patch clamp‐microfluorometric recordings. 2 Under voltage‐clamp conditions, capsaicin (0.1–10 μm) caused a concentration‐dependent decrease in the magnitude of the ICa, an elevation in the holding current (Ih) and a concomitant rise in the [Ca2+]i in most cells examined. Repeated application of capsaicin produced marked desensitization. 3 Some decrease in the ICa produced by capsaicin was also observed when the rise in [Ca2+]i was buffered with EGTA or BAPTA and when Ba2+ was used as the charge carrier; under these conditions the desensitization previously observed was smaller. 4 The decrement in voltage‐gated current was smaller in Ba2+ containing solutions than in Ca2+ containing solutions suggesting that the capsaicin‐induced influx of Ca2+ partially mediated the observed decrease in the voltage‐gated current. In cells which showed a marked response to capsaicin an outward (positive) current was sometimes observed upon depolarization from −80 to 0mV. This effect was consistent with an outward movement of cations through the capsaicin conductance pathway which may also account, in part, for the apparent reduction in ICa by capsaicin. 5 The effects of capsaicin under voltage‐clamp conditions were prevented by ruthenium red (1 μm). 6 Under current clamp conditions, capsaicin depolarized and caused a rise in [Ca2+]i in the majority of DRG cells examined. Both of these effects could be prevented by ruthenium red (500 nm). 7 It is concluded that capsaicin reduces the ICa of rat DRG neurones primarily by indirect mechanisms.

Delayed Antagonism of Calpain Reduces Excitotoxicity in Cultured Neurons

BACKGROUND AND PURPOSE Glutamate receptor antagonists can produce protection against the neurotoxicity of excessive glutamate stimulation. However, antagonism of the postreceptor processes that produce cell damage may provide a longer window of opportunity for protecting neurons after the initiation of excitotoxic injury. Among various processes that have been thought to mediate the toxic effects of glutamate are activation of the Ca(2+)-dependent proteases calpain I and II and the activation of nitric oxide synthase. We tested the potential for neuroprotection by delayed application of calpain antagonists after excitotoxic treatment. METHODS Primary cultures of cerebellar and hippocampal neurons were exposed to the glutamate receptor agonists kainate and N-methyl-D-aspartate (NMDA) for 20-minute periods, and survival was examined by fluorescent assay after 24 hours. Enzyme antagonists were applied at various time points during this interval. RESULTS The neurotoxic effects of NMDA in cultured hippocampal neurons and of kainate in cultured cerebellar neurons have been previously shown to be Ca2+ dependent. Here we show that in both of these examples of glutamate receptor-mediated toxicity, activation of a calpainlike proteolytic activity occurred, which was blocked by the calpain inhibitor MDL-28170. This inhibitor also limited the toxicity, even when applied at times up to 1 hour after the onset of the toxic exposure. Another protease inhibitor, E-64, also blocked the proteolysis and toxicity produced by kainate in cerebellar neurons. Blocking nitric oxide synthase activity after 1 hour with the antagonist NG-nitro-L-arginine was also protective of cerebellar and hippocampal neurons, as was the combination of MDL-28170 and NG-nitro-L-arginine. CONCLUSIONS The activation of calpain is among several enzymatic processes that contribute to the toxicity of glutamate receptor stimulation, and blocking these postreceptor mechanisms can be effective in protecting neurons from excitotoxicity at delayed time points.

Transcatheter closure of patent foramen ovale associated with paradoxical embolism using the amplatzer PFO occluder: initial and intermediate-term results of the U.S. multicenter clinical trial.

Closure of patent foramen ovale (PFO) has been proposed as an alternative to anticoagulation in patients with presumed paradoxical emboli. We report the immediate and mid‐term results of the phase 1 U.S. Multicenter Clinical Trial of patients who underwent transcatheter PFO closure for paradoxical embolism using the new Amplatzer PFO device. Fifty patients (28 male/22 female) underwent catheter closure of their PFOs at a mean age of 41 ± 11 years. Thirty‐six patients had ischemic stroke, 10 had transient ischemic attack, and 4 had peripheral embolism. Seventeen patients had atrial septal aneurysm. The implantation procedure was successful in 49/49 patients; one patient did not have a PFO. Complete closure was seen immediately after the procedure in 26/49 patients; 17 had minimal residual shunt, 4 had moderate and 2 had large residual shunts. The median fluoroscopy time was 10.5 min (2.8–43 min). There were no complications related to the device. One patient developed an arteriovenous fistula at the catheter site requiring surgical repair. At a mean follow‐up interval of 16.5 ± 7.2 months, there were no deaths or recurrent neurological or peripheral embolic events. Eight patients reported an episode of dizziness or palpitations (four of them within 18 days of the procedure). No episodes of atrial dysrhythmias were noted. Contrast bubble study at last follow‐up documented complete closure in 45/48 patients; one patient had minimal, one had moderate residual shunt, and one had a large shunt. One patient was lost to follow‐up. We conclude that catheter closure of PFO associated with stroke/transient ischemic attack or peripheral embolism using the new Amplatzer PFO device is a safe and effective method in preventing recurrence of such episodes. Randomized clinical trials comparing device closure versus continued medical therapy are underway. Catheter Cardiovasc Interv 2003;60:524–528.

Selective Expression of Heteromeric AMPA Receptors Driven by Flip–Flop Differences

Initial models of AMPA receptor assembly postulated the unrestricted stochastic association of individual subunits. The low Ca2+ permeability and nonrectified current–voltage relationship of most native AMPA receptors were ascribed to dominant effects of the glutamate receptor 2 (GluR2) subunit. A recent model, however, proposes instead the preferred assembly of GluR1 and GluR2 subunits into tetrameric complexes as pairs of identical heteromeric dimers. To compare unrestricted versus selective models of GluR1 and GluR2 assembly, these subunits, in both flip and flop isoforms, were expressed in varying ratios in human embryonic kidney 293 cells. Coexpression of pairs of wild-type subunits produced expression of a predominance of heteromeric over homomeric receptors. Only a single functional type of heteromeric receptor was observed, indicating a pattern of apparent dominance not only of GluR2 for ion selectivity, but also of the flip isoform for receptor desensitization. Expression of wild-type GluR1 flip, however, with a mutant form of the same subunit carrying an arginine residue at the glutamine/arginine site (GluR1R flip) demonstrated a lack of dominance of GluR1R in determination of ion selectivity, whereas expression of GluR1R flip with GluR1 flop reproduced the pattern of apparent complete dominance. Together, the data support the selective expression of heteromeric receptors and are compatible with an equilibrium model of assembly of tetramers as pairs of identical heteromeric dimers. Expression of co-assemblies of the flip and flop isoforms, like that of the GluR1 and GluR2 subunits, is strongly favored over that of homomeric assemblies.

Septic encephalopathy: inflammation in man and mouse.

Septic encephalopathy is a frequent complication of the sepsis syndrome, with no therapies available that can prevent the associated neurological dysfunction in humans. It is caused by a number of processes and networks going awry, the exact cellular and molecular mechanisms of which remain an enigma. Several mediators of inflammation have been assigned a key role in sepsis, including cytokines, chemokines and complement cascade. With the observations that brain dysfunction in a sepsis setting can be alleviated by regulation of the cytokines and complement proteins in various species of animals, optimism is building for a possible therapy of sepsis-damaged brain. This article reviewed the advances in the understanding of the underlying mechanisms causing pathology in SE, with an emphasis on the inflammatory and excitatory mediators such as the cytokines, complement proteins and neurotransmitters, investigating their potential as possible therapeutic targets.

Transcatheter closure of patent foramen ovale in patients with paradoxical embolism: intermediate-term risk of recurrent neurological events.

Closure of patent foramen ovale (PFO) has been proposed as an alternative to anticoagulation in patients with presumed paradoxical emboli. We report our preliminary intermediate results of patients who underwent transcatheter PFO closure for paradoxical embolism using DAS‐Angel Wings occluder or Amplatzer devices. Eighteen patients (8 male/10 female) underwent catheter closure of their PFOs at a median age of 42 years. The complete closure rate was 67% immediately after the procedure and 100% at a mean follow‐up interval of 2.2 ± 1.8 years. The mean fluoroscopy time and procedure time in the Amplatzer group were 8.5 ± 3.2 min and 65 ± 21 min, respectively, which were significantly shorter than those of DAS‐Angel Wings group (18.9 ± 4.7 min and 137 ± 28 min, respectively). There were no recurrent embolic neurological events following device placement in this subset of patients. No complications were encountered either during or after the closure procedure. In conclusion, transcatheter closure of PFO seems to be an effective alternative therapy in the prevention of presumed paradoxical emboli. Further study is needed to identify patients most likely to benefit from this intervention. Cathet Cardiovasc Intervent 2002;55:189–194.

Inhibition of C5a receptor alleviates experimental CNS lupus

To investigate the role of C5a generated on complement activation in brain, the lupus model, MRL/lpr mice were treated with C5a receptor(R) antagonist (ant). Neutrophil infiltration, ICAM, TNF-alpha and iNOS mRNA expression, neuronal apoptosis and the expression of p-JNK, pSTAT1 and p-Erk were reduced and p-Akt increased on C5aR inhibition in MRL/lpr brains. MRL/lpr serum caused increased apoptosis in neurons showing that lupus had a direct effect on these cells. C5aRant pretreatment prevented the lupus serum induced loss of neuronal cells. Our findings demonstrate for the first time that C5a/C5aR signaling plays an important role in the pathogenesis of CNS lupus.

Subcellular localization of calcium-permeable AMPA receptors in spinal motoneurons

Activation of Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptors has been linked to potent effects on survival and dendritic outgrowth of spinal motoneurons. Ca2+ permeability of AMPA receptors is controlled by the GluR2 subunit. Whole‐cell electrophysiological studies have suggested that GluR2‐containing and GluR2‐lacking AMPA receptors may coexist in individual motoneurons. However, there has not been a direct demonstration of heterogeneity in AMPA receptor subunit composition in single motoneurons, nor of distinct subcellular distributions of GluR2‐containing and GluR2‐lacking receptors. In the present study, we have used confocal microscopy, immunocytochemistry and Ca2+ imaging to characterize the subcellular localization of AMPA receptors in cultured rat spinal motoneurons. Immunoreactivity for GluR2 and GluR4 was concentrated in clusters, the vast majority of which were found in dendrites at synapses. Double‐labelling for GluR2 and GluR4 revealed variability in relative expression of GluR2 and GluR4 between clusters within individual motoneurons; most AMPA receptor clusters were immunoreactive for both GluR2 and GluR4, but a significant minority of clusters were immunoreactive for GluR2 only or for GluR4 only. The majority of GluR2‐immunonegative AMPA receptor clusters was present in dendrites, but the relative proportion of GluR2‐immunonegative and GluR2‐immunopositive clusters was similar in dendrites and soma. Imaging of [Ca2+]i rises triggered by AMPA receptor activation confirmed Ca2+ influx in motoneuron dendrites. These findings strongly support a model in which GluR2‐containing and GluR2‐lacking AMPA receptors coexist in motoneurons, clustered at synapses, and mixed in a relative proportion that varies considerably between cell membrane microdomains.

C3aR inhibition reduces neurodegeneration in experimental lupus

Complement activation is an important aspect of systemic lupus erythematosus. In this study we investigated the role of C3a/C3a receptor (R) signaling in brains of the lupus model, MRL/lpr mice, by treating the mice with C3aR antagonist (a) from 13 to 19 weeks of age. C3aR mRNA (0.2 ± 0.027 versus 0.56 ± 0.19) and protein (0.16 ± 0.09 versus 0.63 ± 0.19) expression was increased in MRL/lpr brains compared with MRL+/+ controls. Apoptosis, a key feature in lupus brain, was significantly reduced by C3aRa treatment, as assessed by DNA laddering, TUNEL staining and caspase3 activity (48% of MRL/lpr mice). mRNA expression of proinflammatory molecules that cause apoptosis, TNFα (0.33 ± 0.07 versus 0.15 ± 0.1), MIP2 (3.8 ± 1.3 versus 1.7 ± 0.6), and INFγ (4.8 ± 1.0 versus 2.07 ± 1.28) are reduced in MRL/lpr brains with C3aRa treatment. In line with these results, Western blotting demonstrates the significant increase in phosphorylation of survival molecules Akt and Erk, decrease in PTEN and reduced iNOS expression. INFγ receptor (R) and AMPA-GluR1 co-localized, and concomitant with reduced INFγR expression, AMPAGluR1 expression was also decreased by C3aR antagonist. All of these variables that modulate neuronal excitability and regulate synaptic plasticity are C3aR dependent in the MRL/lpr brains and suggest a potential therapeutic role for C3aR inhibition in CNS lupus. Lupus (2010) 19, 73—82.

Physiological hypoxia promotes survival of cultured cortical neurons

Physiological oxygen (O2) tensions in brain tissues vary widely, from ≈ 5 to 40 Torr (1–6%), encompassing levels of moderate hypoxia that have often been considered neurotoxic in vitro. The effects of such hypoxia were examined in embryonic murine cortical neurons cultured continuously from plating in an atmosphere of 1% O2. Remarkably, cortical neurons thrived in 1% O2, with survival at 7–14 days significantly greater than that of neurons cultured in ambient conditions (20% O2). Immunostaining for microtubule‐associated protein‐2 (MAP‐2) and NeuN confirmed the neuronal identity of surviving cells, and demonstrated robust development of dendritic structures and MAP‐2 expression in hypoxia. Survival of neurons in 20% O2 could be promoted by transfer of medium conditioned by neurons in 1% O2, or by pharmacological induction of hypoxia‐inducible factor‐1α (HIF‐1α), suggesting a possible role for secreted factors under transcriptional regulation by HIF‐1 in the trophic effects of hypoxia. Vascular endothelial growth factor (VEGF), a factor regulated by HIF‐1, was strongly stimulated in neurons cultured in 1% O2. Treatment of neurons with exogenous VEGF partially improved survival in 20% O2, and inhibitors of VEGF action reduced survival of neurons in 1% O2. These data point to the dynamic role played by hypoxia, associated with HIF‐1 up‐regulation, in promoting survival of cortical neurons, in part through stimulation of VEGF expression and release.