Super-resolution imaging of subcortical white matter using stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI).

AIMS: The spatial resolution of light microscopy is limited by the wavelength of visible light (the 'diffraction limit', approximately 250 nm). Resolution of sub-cellular structures, smaller than this limit, is possible with super resolution methods such as stochastic optical reconstruction microscopy (STORM) and super-resolution optical fluctuation imaging (SOFI). We aimed to resolve subcellular structures (axons, myelin sheaths and astrocytic processes) within intact white matter, using STORM and SOFI. METHODS: Standard cryostat-cut sections of subcortical white matter from donated human brain tissue and from adult rat and mouse brain were labelled, using standard immunohistochemical markers (neurofilament-H, myelin-associated glycoprotein, glial fibrillary acidic protein, GFAP). Image sequences were processed for STORM (effective pixel size 8-32 nm) and for SOFI (effective pixel size 80 nm). RESULTS: In human, rat and mouse, subcortical white matter high-quality images for axonal neurofilaments, myelin sheaths and filamentous astrocytic processes were obtained. In quantitative measurements, STORM consistently underestimated width of axons and astrocyte processes (compared with electron microscopy measurements). SOFI provided more accurate width measurements, though with somewhat lower spatial resolution than STORM. CONCLUSIONS: Super resolution imaging of intact cryo-cut human brain tissue is feasible. For quantitation, STORM can under-estimate diameters of thin fluorescent objects. SOFI is more robust. The greatest limitation for super-resolution imaging in brain sections is imposed by sample preparation. We anticipate that improved strategies to reduce autofluorescence and to enhance fluorophore performance will enable rapid expansion of this approach.

Transgene delivery to endothelial cultures derived from porcine carotid artery ex vivo.

Carotid artery disease is a widespread cause of morbidity and mortality. Porcine models of vascular disease are well established in vivo, but existing endothelial systems in vitro (e.g. human umbilical vein endothelial cells, rat aortic endothelial cultures) poorly reflect carotid endothelium. A reliable in vitro assay would improve design of in vivo experiments and allow reduction and refinement of animal use. This study aimed (1) to develop ex vivo endothelial cultures from porcine carotid and (2) to test whether these were suitable for lentivector-mediated transgene delivery. Surplus carotid arteries were harvested from young adult female Large White pigs within 10 min post-mortem. Small sectors of carotid artery wall (approximately 4 mm×4 mm squares) were immobilised in a stable gel matrix. Cultures were exposed to HIV-derived lentivector (LV) encoding a reporter transgene or the equivalent integration-deficient vector (IDLV). After 7-14 days in vitro, cultures were fixed and labelled histochemically. Thread-like multicellular outgrowths were observed that were positive for endothelial cell markers (CD31, VEGFR2, von Willebrand factor). A minority of cells co-labelled for smooth muscle markers. Sensitivity to cytotoxic agents (paclitaxel, cycloheximide, staurosporine) was comparable to that in cell cultures, indicating that the gel matrix permits diffusive access of small pharmacological molecules. Transgene-expressing cells were more abundant following exposure to LV than IDLV (4.7, 0.1% of cells, respectively). In conclusion, ex vivo adult porcine carotid artery produced endothelial cell outgrowths that were effectively transduced by LV. This system will facilitate translation of novel therapies to clinical trials, with reduction and refinement of in vivo experiments.

The cell survival kinase SGK1 and its targets FOXO3a and NDRG1 in aged human brain.

AIMS: Serum- and glucocorticoid-inducible kinase 1 (SGK1) protects neuronal cells from injury stimuli in vitro, and exerts anti-apoptotic effects via downstream targets including the forkhead-like transcription factor FOXO3a. SGK1 is a homolog of Akt, a related survival kinase that is up-regulated in Alzheimer's disease (AD). Here we aimed to examine the expression pattern of SGK1 and FOXO3a in aged human cerebral cortex. METHODS: Cortical tissue from aged donors without brain disease (aged controls, AC, n = 19) and from severe AD patients (Braak stage V-VI; n = 14) were examined by immunohistochemistry and immunoblot analysis. RESULTS: SGK1 was present in all samples (detected by immunohistochemistry and immunoblotting). Large cortical neuronal cells were strongly positive for SGK1, with predominantly nuclear labelling. Some astrocytes and oligodendrocytes were also labelled. SGK1 was not seen in nerve tracts (axons or myelin) and rarely seen in CD68-positive cells (microglia, perivascular macrophages) or vascular cells (myocytes or endothelia). The fraction of large cortical neurones with nuclear FOXO3a was lower in AD cases relative to AC (54%, 70%, respectively, P < 0.001). In immunoblots no difference in SGK1 abundance was detected between AC and AD tissues. Phosphorylation of NDRG1 (an SGK1-specific target) was greater in AD, relative to AC cases (approximately twofold, P = 0.023). CONCLUSIONS: Neuronal expression of SGK1 in aged human brain and its nuclear compartmentalization suggest a possible neuroprotective role. FOXO3a and NDRG1 data suggest augmented SGK1 activity (as reported for Akt) in severe AD. Increased intracellular SGK1 may complement enhanced Akt, with a distinct subcellular localization.

Neuropathologic evidence of endothelial changes in cerebral small vessel disease.

OBJECTIVES: Cerebral small vessel disease (SVD) is common in aged brains and causes lacunar stroke, diffuse white matter lesions (leukoaraiosis), and vascular cognitive impairment. The pathogenesis is unknown. Endothelial dysfunction is a possible causal factor, and circulating markers of endothelial activation (intercellular adhesion molecule-1, thrombomodulin) and inflammation (interleukin [IL]-6) are elevated in patients with SVD. In this case-control study, we tested whether brain endothelial ICAM1, thrombomodulin, and IL-6 are altered in SVD. METHODS: We examined small penetrating cerebral arteries of pathologically diagnosed SVD cases, aged controls without SVD, young control cases with no brain pathology, and cases with early-onset hereditary SVD (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [CADASIL]). All tissues had minimal cerebral amyloid angiopathy or other Alzheimer pathology. RESULTS: Thrombomodulin immunoreactivity was present in all aged SVD, aged control, and CADASIL cases, primarily in small artery endothelium. Thrombomodulin was augmented in aged SVD cases compared with aged controls (p = 0.012) and in vessels with higher sclerotic index (an indicator of SVD severity; p < 0.01). Thrombomodulin was sparse/absent in young controls. Endothelial ICAM1 and IL-6 were rarely seen, and were not related to SVD. CONCLUSIONS: Our data suggest that cerebral endothelial activation in deep penetrating arteries is not associated with SVD. Endothelial thrombomodulin increased with SVD severity, and CADASIL data suggest that this may be a cerebral response to SVD. Elevated thrombomodulin may be a protective agent in SVD. Our data confirm endothelial involvement in SVD.

Pharmacology of capsaicin-, anandamide-, and N-arachidonoyl-dopamine-evoked cell death in a homogeneous transient receptor potential vanilloid subtype 1 receptor population.

BACKGROUND: Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a primary pain-sensing relay at peripheral sensory nerve endings and is also widespread in the brain, where it is implicated in neurodegeneration. Previous studies of TRPV1 neurotoxicity have utilized heterogeneous receptor populations, non-selective ligands, or non-neuronal cell types. Here, we explored the pharmacology of TRPV1-induced cytotoxicity in a homogeneous, neurone-like cellular environment. METHODS: Cell death was examined in a human neurone-like cell line, stably expressing recombinant human TRPV1. Cytotoxicity was quantified in terms of nuclear morphology and mitochondrial complex II activity. Immunocytochemical markers of apoptotic cell death were also examined. RESULTS: The TRPV1-selective agonist capsaicin, and the endovanilloids anandamide and N-arachidonoyl-dopamine (NADA), induced TRPV1-dependent delayed cell death in a concentration- and time-dependent manner. Capsaicin exposure time was significantly correlated with potency (r(2)=0.91, P=0.01). Release of cytochrome c from mitochondria, activation of caspase-3, and condensed nuclear chromatin were evident 6 h after capsaicin exposure, but cytotoxicity was unaffected by a pan-caspase inhibitor (zVAD-fmk, 50 microM). CONCLUSIONS: We conclude that capsaicin, anandamide, and NADA can initiate TRPV1-dependent delayed cell death in neurone-like cells. This is an apoptosis-like process, but independent of caspase activity.

Death-associated protein kinase (DAPK1) in cerebral cortex of late-onset Alzheimer's disease patients and aged controls.

AIMS: Here our objective was to detect the pro-apoptotic serine/threonine kinase death-associated protein kinase (DAPK1) in aged human cerebral cortex and to test the hypothesis that DAPK1 abundance is associated with late-onset Alzheimer's disease (AD). METHODS: Using Western analysis and immunohistochemistry we evaluated post mortem frontal cerebral cortex from patients with severe AD (mean age 76 years, range 66-91, n = 11, all male), and from control cases without serious central nervous system illness (mean age 77 years, range 61-95, n = 12, all male). We also examined brains of Tg2576 transgenic mice (males, aged 16-21 months), a model for chronic amyloid-induced brain injury. RESULTS: Immunohistochemical labelling showed DAPK1 expression in cortical neurones of human cortex and axonal tracts within subcortical white matter, both in AD and in control brains. Western analysis confirmed DAPK1 expression in all samples, although expression was very low in some control cases. DAPK1 abundance in the AD group was not significantly different from that in controls (P = 0.07, Mann-Whitney test). In brains of Tg2576 mice DAPK1 abundance was very similar to that in wild-type littermates (P = 0.96, Mann-Whitney test). CONCLUSION: We found that DAPK1 was expressed in neurones of aged human frontal cortex, both in AD and in control cases.

Deep brain stimulation in Parkinson's disease patients: biochemical evidence.

Deep brain stimulation (DBS) of the subthalamic nucleus (STN) in Parkinson's disease (PD) patients augments STN-driven excitation of the internal globus pallidus (GPi). However, other DBS-induced changes are largely unknown. Here we report the biochemical effects of STN-DBS in two basal ganglia stations (putamen--PUT--and GPi) and in a thalamic relay nucleus, the anteroventral thalamus (VA). In six advanced PD patients undergoing surgery, microdialysis samples were collected from GPi, PUT and VA before, during and after one hour of STN-DBS. cGMP was measured in the GPi and PUT as an index of glutamatergic transmission, whereas GABA was measured in the VA. During clinically effective STN-DBS, we found a significant decrease in GABA extracellular concentrations in the VA (-25%). Simultaneously, cGMP extracellular concentrations were enhanced in the PUT (+200%) and GPi (+481%). DBS differentially affects fibers crossing the STN area: it activates the STN-GPi pathway while inhibiting the GPi-VA one. These findings support a thalamic dis-inhibition, as the main responsible for the clinical effect of STN-DBS. This, in turn, re-establishes a more physiological level of PUT activity.

Neuroprotective actions in vivo and electrophysiological actions in vitro of 202W92.

202W92 (R-(-)-2,4-diamino-6-(fluromethyl)-5-(2,3,5-trichlorophenyl)pyrimidine) is a novel compound in the same chemical series as the antiepileptic drug lamotrigine and the neuroprotective sipatrigine. Here 202W92 was quantitatively assessed as a neuroprotective agent in focal cerebral ischaemia, and as an inhibitor of sodium and calcium channels and of synaptic transmission. In the rat permanent middle cerebral artery occlusion (MCAO) model of acute focal ischaemia, 202W92 reduced infarct volume by 75% in cortex and by 80% in basal ganglia, with ED(50) approximately 2 mg/kg (single i.v. dose, 10 min post-occlusion). In whole-cell current recordings from single cells, 202W92 completely and reversibly inhibited voltage gated sodium channels (IC(50) 3 x 10(-6) M) in rat freshly-isolated cortical neurons and in the GH(3) pituitary cell line. 202W92 also inhibited a nifedipine-sensitive fraction (approximately 35%) of native high-voltage-activated (HVA) calcium current in rat cortical neurons (IC(50) 15 x 10(-6) M) and weakly inhibited low-voltage-activated (LVA) calcium currents of the recombinant alpha1I-mediated T-type (IC(50)>100 x 10(-6) M). The drug inhibited the amplitude and frequency of 4-aminopyridine-evoked glutamatergic excitatory post-synaptic currents (EPSCs). In conclusion, 202W92 is an effective neuroprotective agent when administered post-ischaemia and a potent sodium channel inhibitor in vitro.

Neuroprotective efficacy of AR-A008055, a clomethiazole analogue, in a global model of acute ischaemic stroke and its effect on ischaemia-induced glutamate and GABA efflux in vitro.

We have investigated the neuroprotective properties of AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl-1-phenyl-methylamine], a novel compound structurally related to clomethiazole. Administration (i.p.) of (+/-)-AR-A008055 60 min after 5 min of global cerebral ischaemia in gerbils produced a dose-dependent protection of the hippocampus from damage. Both enantiomers [(R)-(+)-AR-A008055 and (S)-(-)- AR-A008055] at 600 micromol/kg produced similar protection to that following clomethiazole (600& micromol/kg) and both produced similar and sustained neuroprotection, at 4, 7 and 21 days post-insult. When infused intravenously over a 2-h period, both enantiomers produced concentration-dependent neuroprotection, with the enantiomers providing similar protection at every plasma concentration (50-200 nmol/ml). The efficacy of (S)-(-)-AR-A008055 was similar to clomethiazole, but it was slightly less potent. Ischaemia-induced glutamate efflux from rat brain cortical prisms in vitro was inhibited by both isomers (100 microM). The inhibitory effect of (R)-(+)-AR-A008055 was blocked by bicuculline (10 microM) and picrotoxin (100 microM), while the effect of (S)-(-)-AR-A008055 was only antagonised by picrotoxin. This indicated that (S)-(-)-AR-A008055, like clomethiazole, is able to open the GABA(A)-chloride channel in the absence of endogenous GABA. (R)-(+)-AR-A008055 was more potent than (S)-(-)-AR-A008055 in enhancing the concentration of GABA in the medium following 30 min exposure of tissue to the ischaemic conditions, suggesting that it is an effective GABA uptake inhibitor. This action may explain both its effect on glutamate efflux in vitro and its neuroprotective effect in vivo.

The interaction of AR-A008055 and its enantiomers with the GABA(A) receptor complex and their sedative, muscle relaxant and anticonvulsant activity.

AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl)-1-phenylmethylamine] is structurally related to clomethiazole and has been used to probe the mechanism of the neuroprotective effect of clomethiazole. Clomethiazole, (+/-)-AR-A008055 and (S)-(-)-AR-A008055 all displaced [35S]-t-butyl-bicyclophosphorothionate ([35S]TBPS) from rat cerebral cortex tissue (IC50 values: GABA, 8.1+/-0.04 microM; clomethiazole, 130+/-30 microM; (+/-)-AR-A008055, 494+/-7 microM; (S)-(-)-AR-A008055, 221+/-14 microM. (R)-(+)-AR-A008055 was without significant effect (IC50>1000 microM). None of the compounds interacted with NMDA or AMPA receptors or with sodium or calcium (N, P/Q) channels. Brain penetration of both enantiomers following their i.p. administration was excellent, with brain and plasma concentrations being similar. Clomethiazole dose-dependently inhibited spontaneous locomotor activity in rats and was approximately 10 times more sedative than either enantiomer of AR-A008055. Clomethiazole was more potent than (S)-(-)-AR-A008055 in the "pull-up" test (muscle relaxation) and in producing loss of righting reflex, while (R)-(+)-AR-A008055 had little effect. The time animals remained on a Rota-rod was of the order: clomethiazole<(S)-(-)-AR-A008055<(R)-(+)-AR-A008055. (S)-(-)-AR-A008055 (210 micromol/kg) raised seizure threshold to pentylenetetrazole (i.v.) by 119+/-21%. The (R)-(+)- enantiomer was not anticonvulsant. Overall, (S)-(-)-AR-A008055 exhibited a similar pharmacology to clomethiazole. However, its sedative and muscle relaxant effects were substantially less than clomethiazole, emphasising that these properties are not directly related to neuroprotective efficacy. The current data suggest that the proposed GABA uptake inhibitory property of (R)-(+)-AR-A008055 fails to produce significant sedative, myorelaxant or anticonvulsant activity.

Effects of extracellular pH on the interaction of sipatrigine and lamotrigine with high-voltage-activated (HVA) calcium channels in dissociated neurones of rat cortex.

Acidic extracellular pH reduced high-voltage-activated (HVA) currents in freshly isolated cortical pyramidal neurones of adult rats, shifting activation to more positive voltages (V(1/2)=-18 mV at pH 7.4, -11 mV at pH 6.4). Sipatrigine inhibited HVA currents, with decreasing potency at acidic pH (IC(50) 8 microM at pH 7.4, 19 microM at pH 6.4) but the degree of maximal inhibition was >80% in all cases (pH 6.4-8.0). Sipatrigine has two basic groups (pK(A) values 4.2, 7.7) and at pH 7.4 is 68% in monovalent cationic form and 32% uncharged. From simple binding theory, the pH dependence of sipatrigine inhibition indicates a protonated group with pK(A) 6.6. Sipatrigine (50 microM) shifted the voltage dependence of channel activation at pH 7.4 (-7.6 mV shift) but not at pH 6.4. Lamotrigine has one basic site (pK(A) 5.5) and inhibited 34% of the HVA current, with similar potency over the pH range 6.4--7.4 (IC(50) 7.5--9 microM). These data suggest that the sipatrigine binding site on HVA calcium channels binds both cationic and neutral forms of sipatrigine, interacts with a group with pK(A)=6.6 and with the channel activation process, and differs from that for lamotrigine.

Nociceptin/orphanin FQ inhibits ischaemia-induced glutamate efflux from rat cerebrocortical slices.

Nociceptin/orphanin FQ (NC), the endogenous ligand for the G-protein coupled nociceptin receptor (NCR), has a modulatory role in various physiological processes including neurotransmitter release. We have examined the effects of NC, the analogues NC(1-13)NH2 and [F/G]NC(1-13)NH2 and the competitive antagonist [Nphe1]NC(1-13)NH2 (Nphe1) on glutamate efflux during an acute simulated ischaemic challenge in rat cerebrocortical slices. The increase in glutamate efflux seen with ischaemia was inhibited by NC (EC50 250 nM). At micromolar concentrations, the analogues were found to have a similar effect on glutamate efflux compared to NC. In all cases, inhibition of glutamate efflux was abolished by Nphe1 (30 microM). These results suggest a neuroprotective action for NC.

On the regulation of ischaemia-induced glutamate efflux from rat cortex by GABA; in vitro studies with GABA, clomethiazole and pentobarbitone.

Prisms of adult rat cortex were maintained in vitro in either aerobic conditions (control) or conditions simulating an acute ischaemic challenge (hypoxia with no added glucose). Endogenous glutamate efflux increased with time in ischaemic conditions, being 2.7 fold higher than control efflux at 45 min. Returning prisms to control solution after 20 min of simulated ischaemia resulted in glutamate efflux returning to near-control values. Endogenous GABA efflux in ischaemic conditions also increased, being 4.5 fold higher than control efflux at 45 min. Ischaemia-induced glutamate efflux was not accompanied by increased lactate dehydrogenase efflux and was unaltered by omitting calcium from the extra-cellular solution and adding EGTA (0.1 mM). Both GABA and the GABA-mimetic clomethiazole inhibited ischaemia-induced glutamate efflux, with IC(50) values of 26 and 24 microM respectively. The maximum inhibition by either drug was 60 - 70%. Bicuculline (10 microM) abolished the inhibitory effect of GABA (100 microM) but not clomethiazole (100 microM). Picrotoxin (100 microM) abolished the action of both GABA and clomethiazole. Pentobarbitone inhibited glutamate efflux at 100 - 300 microM (maximal inhibition: 39%). Bicuculline (10 microM) abolished this effect. These data suggest that ischaemia-induced glutamate efflux from rat cerebral cortex is calcium-independent and not due to cell damage up to 45 min. The inhibitory effect of GABA, clomethiazole and pentobarbitone on ischaemia-induced glutamate efflux appears to be mediated by GABA(A) receptors. The results suggest that clomethiazole, unlike pentobarbitone, is able to activate the GABAA receptor-linked chloride channel directly and not merely potentiate the effect of endogenous GABA.

GABA potentiation: a logical pharmacological approach for the treatment of acute ischaemic stroke.

It has been shown that enhancing the function of the major inhibitory neurotransmitter GABA decreases glutamatergic activity in the brain. Since increased glutamatergic activity is the major primary event that results in cell death following an acute hypoxic-ischaemic stroke, GABAmimetic drugs might therefore be expected to be neuroprotective. This review examines the evidence that GABAergic function is acutely depressed following an ischaemic insult, and also reviews the data that suggest that increasing cerebral GABA concentration has a neuroprotective effect, as does the administration of some (but not all) GABAmimetic agents. The GABA uptake inhibitor CI-966, the GABA(A) agonist muscimol and the GABA(A)mimetic clomethiazole have all been shown to be neuroprotective in animal models of stroke when given after the ischaemic insult. In contrast, benzodiazepines and particularly barbiturates, although potent GABA(A) potentiators, have shown little promise as neuroprotectants. The diversity of GABA(A) receptor subtypes and the in vivo efficacy of certain GABA(A) receptor ligands in animal models of stroke suggests that GABAmimetic drugs are an undervalued approach to stroke therapy.

Inhibition of recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine).

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.

Electrophysiology of sipatrigine: a lamotrigine derivative exhibiting neuroprotective effects.

Sipatrigine (BW619C89), a derivative of the antiepileptic agent lamotrigine, has potent neuroprotective properties in animal models of cerebral ischemia and head injury. In the present study we investigated the electrophysiological effects of sipatrigine utilizing intracellular current-clamp recordings obtained from striatal spiny neurons in rat corticostriatal slices and whole-cell patch-clamp recordings in isolated striatal neurons. The number of action potentials produced in response to a depolarizing current pulse in the recorded neurons was reduced by sipatrigine (EC(50) 4.5 microM). Although this drug preferentially blocked action potentials in the last part of the depolarizing current pulse, it also decreased the frequency of the first action potentials. Sipatrigine also inhibited tetrodotoxin-sensitive sodium (Na(+)) current recorded from isolated striatal neurons. The EC(50) for this inhibitory action was 7 microM at the holding potential (V(h)) of -65 mV, but 16 microM at V(h) = -105, suggesting a dependence of this pharmacological effect on the membrane potential. Moreover, although the inhibitory action of sipatrigine on Na(+) currents was maximal during high-frequency activation (20 Hz), it could also be detected at low frequencies. The amplitude of excitatory postsynaptic potentials (EPSPs), recorded following stimulation of the corticostriatal pathway, was depressed by sipatrigine (EC(50) 2 microM). This inhibitory action, however, was incomplete; in fact maximal concentrations of this drug reduced EPSP amplitude by only 45%. Sipatrigine produced no increase in paired-pulse facilitation, suggesting that the modulation of a postsynaptic site was the main pharmacological effect of this agent. The inhibition of voltage-dependent Na(+) channels exerted by sipatrigine might account for its depressant effects on both repetitive firing discharge and corticostriatal excitatory transmission. The modulation of Na(+) channels described here, as well as the previously observed inhibition of high-voltage-activated calcium currents, might contribute to the neuroprotective efficacy exerted by this compound in experimental models of in vitro and in vivo ischemia.

Is pharmacological neuroprotection dependent on reduced glutamate release?

BACKGROUND AND PURPOSE: The aim of this study was to determinate the possible role of the ionotropic glutamate receptor in the expression of irreversible electrophysiological changes induced by in vitro ischemia and to test whether the neuroprotective action of various neurotransmitter agonists and drugs of clinical interest is related to a presynaptic inhibitory action at glutamatergic synapses. METHODS: Intracellular and extracellular recordings have been performed in a rat corticostriatal slice preparation. Different pharmacological compounds have been tested on corticostriatal glutamatergic transmission in control conditions and in an in vitro model of ischemia (oxygen and glucose deprivation). RESULTS: In vitro ischemia lasting 10 minutes produced an irreversible loss of the field potential recorded from striatal slices after cortical stimulation. Preincubation of the slices with 3 micromol/L 6-cyano-7-nitroquinoxaline-2,3-dione (an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid [AMPA] receptor antagonist) allowed a significant recovery of the field potential amplitude (P<0.05, n=6), whereas incubation with 30 micromol/L aminophosphonovaleric acid (an N-methyl-D-aspartate receptor antagonist) did not produce a significant recovery after 10 minutes of ischemia (P>0.05, n=7). Bath application of 3 mmol/L glutamate for 5 minutes produced a complete but reversible inhibition of the field potential amplitude. When a similar application was coupled with a brief period of ischemia (5 minutes), which produced, per se, only a transient inhibition of the field potential, it caused an irreversible loss of this parameter. We also tested the possible neuroprotective effect of neurotransmitter agonists reducing the release of glutamate from corticostriatal terminals. Agonists acting on purinergic (adenosine), muscarinic (oxotremorine), and metabotropic glutamate receptors (L-serine o-phosphate [L-SOP]) significantly (P<0.001, n=8 for each agonist) reduced glutamatergic synaptic potentials, with each showing different potencies. The EC(50) was 26.4 micromol/L for adenosine, 0. 08 micromol/L for oxotremorine, and 0.89 micromol/L for L-SOP. Concentrations of these agonists producing the maximal inhibition of the synaptic potential were tested on the ischemia-induced irreversible loss of field potential. Adenosine (P<0.05, n=9) and oxotremorine (P<0.05, n=8) showed significant neuroprotective action, whereas L-SOP was ineffective (P>0.05, n=10). Similarly, putative neuroprotective drugs significantly (P<0.001, n=10 for each drug) reduced the amplitude of corticostriatal potential, with different EC(50) values (phenytoin, 33.5 micromol/L; gabapentin, 96.8 micromol/L; lamotrigine, 26.7 micromol/L; riluzole, 6 micromol/L; and sipatrigine, 2 micromol/L). Concentration of these drugs producing maximal inhibition of the amplitude of corticostriatal potentials showed a differential neuroprotective action on the ischemic electrical damage. Phenytoin (P<0.05, n=10), lamotrigine (P<0.05, n=10), riluzole (P<0.05, n=9), and sipatrigine (P<0.001, n=10) produced a significant neuroprotection, whereas gabapentin (P>0.05, n=11) was ineffective. The neuroprotective action of transmitter agonists and clinical drugs was not related to their ability in decreasing glutamate release, as detected by changes in the paired-pulse facilitation protocol. CONCLUSIONS: Ionotropic glutamate receptors, and particularly AMPA-like receptors, play a role in the irreversible loss of field potential amplitude induced by ischemia in the striatum. Drugs acting by reducing glutamatergic corticostriatal transmission may show a neuroprotective effect. However, their efficacy does not seem to be directly related to their capability to decrease glutamate release from corticostriatal terminals. We suggest that additional modulatory actions on voltage-dependent conductances and on ischemia-induced ion distribution at the postsynaptic site may also exert a crucial role.

On the inhibition of voltage activated calcium currents in rat cortical neurones by the neuroprotective agent 619C89.

1. The lamotrigine analogue 619C89, utilised to reduce postischaemic and posttraumatic neuronal injury, has been shown to inhibit sodium channels and cloned N-type calcium channels. To verify whether this neuroprotective agent also blocked native calcium channels, we have tested its action in cortical pyramidal neurones, acutely isolated from the adult rat brain. 2. 619C89 inhibited more than 90% of the high voltage-activated calcium currents recorded in the whole-cell configuration. The response was relatively slow in onset (30-60 s), recovered incompletely (96%), but showed no consistent desensitization. 3. This inhibitory effect was not selective for any calcium channel subtype, being largely unaffected by omega-conotoxin-GVIA, omega-agatoxin-IVA, omega-conotoxin-MVIIC and dihydropyridine antagonists. 4. Saturating responses to 619C89 were detected for concentrations > or = 50 microM. Dose-response curves revealed that 619C89 have an approximately 8 microM binding site. 5. The effect of 619C89 was dependent on the divalent concentrations in that its potency was reduced on increase of the charge carrier up to 20 mM barium. Since the lamotrigine analogue shifted to the right the dose-dependence of the cadmium block, the 619C89-mediated inhibition of calcium currents seemed to rely on a direct interaction with the channel pore. Functional implications are discussed.

Acute effects of interleukin-1 beta on noradrenaline release from the human neuroblastoma cell line SH-SY5Y.

Interleukins are potent intercellular messenger peptides, initially found in cells of the immune system and best known for producing chronic, genomic effects in target cells. Here, interleukin-1beta (IL-1beta) was tested for acute effects on neurotransmitter release. The human neuroblastoma-derived cell-line SH-SY5Y is a model for mature post-ganglionic sympathetic neurones and release of tritiated noradrenaline from these cells was measured, in response to stimulation with either elevated extracellular K+ concentration (100 K+) orveratridine. Pre-incubation for 15-25 min with 60 pM (but not 0.06 pM) IL-1beta significantly reduced 100 K+-evoked release (by approximately 75%). The interleukin was without effect on basal or veratridine-evoked noradrenaline release. The present data suggest two distinct stimulatory pathways: one that is activated by 100 K+ and veratridine and is unaffected by IL-1beta and another that is activated by 100 K+ but not veratridine and is inhibited by IL-1beta. The acute depression of 100 K+-evoked transmitter release may be involved in immune system-nervous system interactions.

Inhibition of human N-type voltage-gated Ca2+ channels by the neuroprotective agent BW619C89.

Human N-type Ca2+ channels were rapidly and reversibly inhibited by 5-100 microM BW619C89 (IC50 = 16.4 microM at Vtest = + 10 mV and Vhold = - 90 mV). In the presence of 20 microM BW619C89, activation kinetics were significantly faster. The degree of inhibition observed was affected by both test and holding potential, indicating state-dependent interactions with the N-type Ca2+ channel.