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1.
Magn Reson Med ; 69(6): 1603-10, 2013 Jun.
Article in English | MEDLINE | ID: mdl-22821404

ABSTRACT

In vivo magnetic resonance imaging (MRI) assessment of neuronal tissue is prone to artifacts such as movement, pulsatile flow, and tissue susceptibility. Furthermore, stable in vivo scans of over 3 h are difficult to achieve, experimental design is therefore limited. Using isolated tissue maintained in a viable physiological state can mitigate many of these in vivo issues. This work describes the fabrication and validation of an MRI compatible viable isolated tissue maintenance chamber. Parameters measured from maintained rat optic nerves did not change significantly over 10 h: (i) mean axon radius [electron microscopy--0 h: 0.75±0.46; 5 h: 0.74±0.35; 10 h: 0.76±0.35 µm (P>>0.05, t-test], (ii) action potentials [grease-gap electrophysiology--4.89±0.16 mv, (P>>0.05, Pearson test], and (iii) diffusion tensor imaging parameters [fractional anisotropy: 0.86±0.02 (P>>0.05, Pearson test), mean diffusivity: 1.48E-06±9.74E-08 cm2/s, (P>>0.05, Pearson test)]. In addition, a thorough diffusion-weighted MR protocol demonstrated the comparable stability of viable isolated and chemically fixed rat optic nerve. This MRI compatible viable isolated tissue system allows researchers to probe neuronal physiology in a controlled environment by limiting in vivo artifacts and allowing extended MRI acquisitions.


Subject(s)
Magnetic Resonance Imaging/instrumentation , Optic Nerve/anatomy & histology , Organ Culture Techniques/instrumentation , Perfusion/instrumentation , Rheology/instrumentation , Animals , Environment, Controlled , Equipment Design , Equipment Failure Analysis , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity
2.
Br J Pharmacol ; 151(6): 870-6, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17502847

ABSTRACT

BACKGROUND AND PURPOSE: Specific and selective inhibitors for mGlu1 receptors are presently inadequate. A new generation of non-competitive mGlu1 antagonists with low nanomolar potencies is emerging. We evaluated two new compounds, YM-298198 and JNJ16259685, for effectiveness, potency and specificity for the first time in a brain slice preparation. EXPERIMENTAL APPROACH: Patch-clamp recording of Purkinje neurones in cerebellar slices were obtained. The slow mGlu1-mediated EPSP was used to establish a concentration-response curve. Fast excitatory synaptic inputs were tested for non-specific effects. KEY RESULTS: YM-298198 and JNJ16259685 inhibited the synaptic activation of mGlu1 in a concentration-dependent manner (IC(50) values of 24 nM and 19 nM, respectively). The antagonists were slow to inhibit and to reverse on washout, probably due to their lipophilic nature. There were no non-specific effects on fast AMPA receptor-mediated synaptic transmission in the cerebellum. CONCLUSIONS AND IMPLICATIONS: These compounds are more than a thousand-fold more potent than previously available compounds. Their selectivity and specificity will be very useful for studying the role of mGlu1 receptors both in vitro and in vivo.


Subject(s)
Benzimidazoles/pharmacology , Purkinje Cells/drug effects , Quinolines/pharmacology , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Synaptic Transmission/drug effects , Thiazoles/pharmacology , Animals , Benzimidazoles/administration & dosage , Dose-Response Relationship, Drug , Electrophysiology , Excitatory Postsynaptic Potentials , Hydrophobic and Hydrophilic Interactions , In Vitro Techniques , Inhibitory Concentration 50 , Purkinje Cells/physiology , Quinolines/administration & dosage , Rats , Rats, Sprague-Dawley , Receptors, AMPA/metabolism , Thiazoles/administration & dosage
3.
Eur J Neurosci ; 21(8): 2127-35, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15869509

ABSTRACT

Excessive nitric oxide (NO) production from the inducible isoform of nitric oxide synthase (iNOS) has been invoked as a causative factor in many neurodegenerative disorders, including multiple sclerosis. This hypothesis has been supported by in vitro studies showing that glial iNOS expression results in toxic NO concentrations (near 1 microm). To investigate the relevance of such findings, experiments were carried out ex vivo on optic nerves from rats with exacerbated experimental allergic encephalomyelitis, a model of multiple sclerosis. The nerves displayed characteristic immunopathology and expression of iNOS in macrophages and/or microglia and there was overt axonal damage in localized regions of the optic chiasm. The resulting NO levels in the optic nerve were sufficient to cause activation of guanylyl cyclase-coupled NO receptors, resulting in marked cGMP accumulation in axons throughout the nerve. Nevertheless, calibration of cGMP levels against those evoked by exogenous NO indicated that the nerves were not compromised metabolically and that their ambient NO concentration was only approximately 1 nm. Consistent with this observation, electrophysiological tests indicated that there was no ongoing malfunctioning of the type that can be elicited by high exogenous NO concentrations. It is concluded that, with iNOS expressed in physiological locations and levels, the tissue levels of NO remain at concentrations far lower than those shown to have toxic effects, despite continuous NO synthesis. The fact that NO can rise to much higher levels in dispersed cultures in vitro may be attributable to a deficiency in NO inactivation in such preparations.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/pathology , Macrophages/enzymology , Nitric Oxide Synthase/metabolism , Optic Nerve/pathology , Ornithine/analogs & derivatives , 1-Methyl-3-isobutylxanthine/pharmacology , Action Potentials/drug effects , Animals , Arginine/pharmacology , Biomarkers/metabolism , CD11b Antigen/metabolism , CD2 Antigens/metabolism , Cyclic GMP/metabolism , DEET/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Electric Stimulation/methods , Encephalomyelitis, Autoimmune, Experimental/chemically induced , Female , Guanylate Cyclase/metabolism , Guinea Pigs , Hydrazines/pharmacology , Immunohistochemistry/methods , Macrophages/pathology , Microscopy, Electron, Transmission/methods , Myelin Basic Protein/metabolism , Nitric Oxide/pharmacology , Nitric Oxide Synthase Type II , Optic Chiasm/pathology , Optic Chiasm/ultrastructure , Optic Nerve/drug effects , Optic Nerve/enzymology , Optic Nerve/ultrastructure , Ornithine/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Rats , Rats, Inbred Lew , Time Factors
5.
Neuroscience ; 109(1): 145-55, 2002.
Article in English | MEDLINE | ID: mdl-11784706

ABSTRACT

Excessive nitric oxide formation may contribute to the pathology occurring in diseases affecting central white matter, such as multiple sclerosis. The rat isolated optic nerve preparation was used to investigate the potential toxicity of the molecule towards such tissue. The nerves were exposed to a range of concentrations of different classes of nitric oxide donor for up to 23 h, with or without a subsequent period of recovery, and the damage assessed by quantitative histological methods. Degeneration of axons and macroglia occurred in a time- and concentration-dependent manner, the order of susceptibility being: axons>oligodendrocytes>astrocytes. Use of NONOate donors differing in half-life indicated that nitric oxide delivered in an enduring manner at relatively low concentration was more toxic than the same amount supplied rapidly at high concentration. The mechanism by which nitric oxide affects axons was studied using a donor [3-(n-propylamino)propylamine/NO adduct, PAPA/NO] with an intermediate half-life that produced selective axonopathy after a 2-h exposure (plus 2 h recovery). Axon damage was abolished if, during the exposure, Na(+) or Ca(2+) was removed from the bathing medium or the sodium channel inhibitors tetrodotoxin or BW619C89 (sipatrigine) were added. In electrophysiological experiments, the donor elicited a biphasic depolarisation. The second, larger component (occurring after 7-10 min) was associated with a block of nerve conduction and could be inhibited by tetrodotoxin. Coincident with the secondary depolarisation was a reduction in ATP levels by about 50%, an effect that was also inhibited by tetrodotoxin. It is concluded that nitric oxide, in submicromolar concentrations, can kill axons and macroglia in white matter. The findings lend support to the hypothesis that nitric oxide may be of importance to white matter pathologies, particularly those in which inducible nitric oxide synthase is expressed. The axonopathy, at least when elicited over relatively short time intervals, is likely to be caused by metabolic inhibition. As in anoxia and anoxia/aglycaemia, nitric oxide-induced destruction of axons is likely to be caused by the Ca(2+) overload that follows a reduction in ATP levels in the face of continued influx of Na(+) through voltage-dependent channels.


Subject(s)
Central Nervous System/metabolism , Demyelinating Diseases/metabolism , Nerve Degeneration/metabolism , Nerve Fibers, Myelinated/metabolism , Neurotoxins/toxicity , Nitric Oxide Donors/toxicity , Nitric Oxide/metabolism , Adenosine Triphosphate/metabolism , Animals , Axons/drug effects , Axons/metabolism , Axons/pathology , Calcium/deficiency , Central Nervous System/drug effects , Central Nervous System/pathology , Demyelinating Diseases/pathology , Demyelinating Diseases/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Membrane Potentials/drug effects , Membrane Potentials/physiology , Nerve Degeneration/chemically induced , Nerve Degeneration/physiopathology , Nerve Fibers, Myelinated/drug effects , Nerve Fibers, Myelinated/pathology , Neuroglia/drug effects , Neuroglia/metabolism , Neuroglia/pathology , Optic Nerve/drug effects , Optic Nerve/metabolism , Optic Nerve/pathology , Organ Culture Techniques , Rats , Rats, Wistar , Sodium/deficiency , Tetrodotoxin/pharmacology
6.
Eur J Neurosci ; 14(8): 1313-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11703460

ABSTRACT

Metabotropic glutamate (mGlu) receptors are located pre- and postsynaptically at central synapses. Activation of the receptors by exogenous agonists usually results in a reversible depression of fast glutamatergic neurotransmission. Evidence that synaptically released glutamate has such an action, however, is scarce. Sharp microelectrode recordings were used to investigate the modulatory role of mGlu receptors at a well-studied glutamatergic synapse, the one between parallel fibres and Purkinje cells in rat cerebellar slices. Brief, tetanic stimulation of the parallel fibres caused a depression of subsequent fast EPSPs. This post-tetanic depression (PTD) reached its maximum 4.5 s after the tetanus. Measured at this point, PTD was frequency-dependent; 10 stimuli at 20 Hz produced no significant depression, whereas, at 100 Hz the same number of stimuli was maximally effective (approximately 50% depression). The nonselective mGlu antagonist, (S)-alpha-methyl-4-carboxyphenylglycine 1 mm or the GABAB antagonist, CGP35348 (1 mm), both decreased the magnitude of the PTD. In the presence of CGP35348 the mGlu1 antagonist, 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (300 microm), inhibited PTD further. A group II/III mGlu antagonist had no effect. These observations indicate that synaptically activated mGlu1 receptors not only generate a slow EPSP and induce Ca2+ mobilization in Purkinje cells, as reported previously, but also produce a transient depression of fast synaptic transmission. This short-term plasticity may be important for shaping the output of cerebellar circuits and/or it could provide a substrate for long-term depression when additional mechanisms are superimposed.


Subject(s)
Excitatory Postsynaptic Potentials/physiology , Glutamic Acid/metabolism , Neural Inhibition/physiology , Presynaptic Terminals/metabolism , Purkinje Cells/metabolism , Receptors, Metabotropic Glutamate/metabolism , Synaptic Transmission/physiology , Animals , Animals, Newborn , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , GABA-B Receptor Antagonists , Male , Neural Inhibition/drug effects , Organ Culture Techniques , Presynaptic Terminals/drug effects , Presynaptic Terminals/ultrastructure , Purkinje Cells/cytology , Purkinje Cells/drug effects , Rats , Rats, Wistar , Receptors, GABA-B/metabolism , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Synaptic Transmission/drug effects
7.
Neuropharmacology ; 41(1): 42-9, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11445184

ABSTRACT

The actions of reportedly group-selective metabotropic glutamate (mGlu) receptor agonists and antagonists on neurotransmission at parallel fibre-Purkinje cell synapses in the rat cerebellum have been characterised using sharp microelectrode recording and an in vitro slice preparation. Application of the group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG) or the group III selective agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed synaptic transmission in a reversible and concentration-dependent manner (EC(50)=18 and 5 microM, respectively). The depression produced by DHPG was unrelated to the depolarisation observed in some Purkinje cells. The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV, 1 microM) had no effect. The effects of DHPG were inhibited by the group I-selective antagonist 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), but not by the group II/III antagonist alpha-methyl-4-phosphonophenylglycine (MPPG). The effect of L-AP4 was inhibited by MPPG, but not by the group I/II antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG). By themselves, the antagonists did not affect the EPSPs, suggesting that neither receptor is activated during low frequency neurotransmission. It is concluded that, in addition to the excitatory role for group I receptors described previously, both group I and III (but not group II) mGlu receptors operate at this synapse to inhibit synaptic transmission. The specific receptor subtypes involved are likely to be mGlu1 and mGlu4.


Subject(s)
Cerebellum/drug effects , Methoxyhydroxyphenylglycol/analogs & derivatives , Nerve Fibers/drug effects , Purkinje Cells/drug effects , Receptors, Metabotropic Glutamate/drug effects , Synapses/drug effects , Synaptic Transmission/drug effects , Aminobutyrates/pharmacology , Animals , Anticonvulsants/pharmacology , Cyclopropanes/pharmacology , Glycine/analogs & derivatives , Glycine/pharmacology , Male , Methoxyhydroxyphenylglycol/pharmacology , Rats , Rats, Wistar
8.
Intensive Care Med ; 26(4): 426-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10872135

ABSTRACT

OBJECTIVES: To determine the incidence of sleep-related breathing disorders and nocturnal hypoxaemia in patients discharged from ICU following prolonged mechanical ventilation. DESIGN: Prospective, consecutive patient observational study. SETTING: The medical and surgical wards of a University Hospital. PATIENTS AND PARTICIPANTS: Fifteen consecutive, adult patients discharged from the ICU who had received more than 48 h of mechanical ventilation were studied. Ten healthy volunteers acted as controls. MEASUREMENTS AND RESULTS: Overnight, multi-channel pneumographic studies were performed on all patients and controls. Chest and abdominal wall movement, air flow, oxygen saturation and snoring were continuously recorded. Data was analysed by both visual inspection of the traces and by computer-based algorithms. An apnoea/hypopnoea index was calculated for each patient and volunteer. Volunteers had an apnoea/hypopnoea index of less than 5 and had no episodes of nocturnal oxygen desaturation (SaO2 < 90%). Despite oxygen therapy 13/15 patients had episodes of desaturation and 9/15 spent more than 2 h with an SaO2 < 90%. Eleven patients had an abnormal apnoea/hypopnoea index (range 5-34 events/h). Four patients had predominantly obstructive events while 7 primarily had hypopnoeas. CONCLUSIONS: Significant overnight oxygen desaturation is common in patients discharged from ICU who have received prolonged mechanical ventilation. This group also has a significant incidence of sleep-related breathing disorders and this mechanism is likely to be important in the pathogenesis of the hypoxaemia.


Subject(s)
Respiration, Artificial/adverse effects , Sleep Apnea Syndromes/etiology , Adult , Aged , Case-Control Studies , Female , Humans , Hypoxia/epidemiology , Hypoxia/etiology , Hypoxia/physiopathology , Incidence , Intensive Care Units , Male , Middle Aged , Prospective Studies , Respiratory Function Tests , Sleep Apnea Syndromes/epidemiology , Sleep Apnea Syndromes/physiopathology
9.
J Physiol ; 522 Pt 2: 231-46, 2000 Jan 15.
Article in English | MEDLINE | ID: mdl-10639100

ABSTRACT

1. Whole-cell recordings were made from rat CA1 neurones in brain slices. When electrodes contained diazo-2 (2 mM) or dibromo BAPTA (1 mM) a large steady-state outward current (hundreds of picoamps) developed within 5 min of breakthrough at a VH of -60 mV. BAPTA itself (1 mM) caused qualitatively similar but smaller effects. 2. The outward current was accompanied by increased conductance with a null potential close to the calculated K+ equilibrium potential (EK) of -110 mV. Development of outward current occurred concurrently with progressive loss of slow AHP tail current (IsAHP) evoked by brief depolarizations. The peak latency of IsAHP increased during the onset of chelator action. 3. The persistent outward current was reversibly inhibited by noradrenaline (10 microM) or isoprenaline (2-5 microM), and completely prevented by 8-bromoadenosine 3',5' cyclic monophosphate (8-Br cAMP; 100 microM) or QX-314 (10 mM) in recording electrodes. After development of outward current, diazo-2 photolysis caused inward current and decreased conductance. Both flash- and noradrenergic-sensitive responses were inwardly rectifying outward currents with null potentials close to EK. 4. The outward current induced by dibromo BAPTA was not blocked by internal EGTA (10 mM). However, experiments incorporating Ca2+ influx or Ca2+ loading of the buffer indicate that Ca2+ facilitated the outward current. 5. The outward currents induced by dibromo BAPTA or diazo-2 were not associated with significant changes in resting [Ca2+]i. Regions of the cell contributing to the outward current were deduced from measurements of fura-2 diffusion. These were compared with regions of [Ca2+]i elevation during IsAHP. 6. These results are consistent with the hypothesis that the BAPTA series Ca2+ buffers can activate those Ca2+-activated K+ channels that underlie the slow AHP, without the predicted elevation of bulk [Ca2+]i. Therefore these results cannot be interpreted solely in terms of Ca2+ concentration changes, although the observations illustrate a novel, investigative role for these compounds in the study of Ca2+-dependent processes.


Subject(s)
Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Hippocampus/metabolism , Neurons/metabolism , Potassium Channels/drug effects , Adrenergic alpha-Agonists/pharmacology , Animals , Calcium/metabolism , Cyclic AMP/metabolism , Diazonium Compounds , Egtazic Acid/pharmacology , Electric Stimulation , Electrophysiology , Fluorescent Dyes , Fura-2 , Hippocampus/cytology , Hippocampus/drug effects , In Vitro Techniques , Membrane Potentials/drug effects , Membrane Potentials/physiology , Membrane Potentials/radiation effects , Neurons/drug effects , Neurons/ultrastructure , Norepinephrine/pharmacology , Patch-Clamp Techniques , Phenoxyacetates , Rats , Ultraviolet Rays
10.
Intensive Care Med ; 25(10): 1061-5, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10551959

ABSTRACT

OBJECTIVE: To examine the effect of high levels of pre-intensive care unit (ICU) discharge care, as assessed by the Therapeutic Intervention Scoring System (TISS), on subsequent hospital mortality. DESIGN: A 1-year prospective, observational study. SETTING: The ICU and wards of a university teaching hospital with no high dependency facility (HDU). PATIENTS: A total of 283 patients were discharged to hospital wards between July 1997 and June 1998. ++ RESULTS: 11 % of all ICU discharges subsequently died in hospital. Patients discharged with a TISS of 20 or greater had a 21.4 % mortality compared to 3.7 % for those with a TISS of less than 10. Increasing age, Acute Physiology Score (APS) on admission and male sex were also significantly associated with post-discharge death. CONCLUSIONS: In a hospital without HDU facilities, patients who are receiving HDU levels of care on discharge from the ICU have a high in-hospital mortality.


Subject(s)
Critical Care/standards , Hospital Mortality , Patient Discharge/standards , Severity of Illness Index , Workload , APACHE , Adolescent , Adult , Age Distribution , Aged , Aged, 80 and over , Bed Occupancy/statistics & numerical data , Female , Humans , Length of Stay/statistics & numerical data , Logistic Models , Male , Middle Aged , Outcome Assessment, Health Care , Predictive Value of Tests , Prognosis , Prospective Studies , Reproducibility of Results , Sex Distribution , Workforce
11.
Neuropharmacology ; 38(6): 805-15, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10465684

ABSTRACT

The role of metabotropic glutamate receptors (mGluRs) in the mossy fibre-granule cell pathway in rat cerebellum was studied using slice preparations and electrophysiological techniques. Application of the group I selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) evoked, in a concentration-dependent manner (EC50 = 33 microM), a depolarising/hyperpolarising complex response from granule cells which was preferentially inhibited by the group I selective antagonist (S)-4-carboxyphenylglycine (4CPG). The group III selective agonist L-amino-4-phosphonobutyrate (AP4) evoked a hyperpolarising response (EC50 = 10 microM) which was inhibited by the group II/III selective antagonist (S)-alpha-methyl-4-phosphonophenylglycine (MPPG). The group II agonist (2S,2'R,3'R)-2-(2',3'-dicarboxylcyclopropyl)glycine (DCG-IV) elicited no measurable voltage change. The amplitude of the synaptically-mediated mossy fibre response in granule cells was unaffected during application of AP4, was reduced by DHPG and was enhanced by DCG-IV (EC50 = 80 nM). These effects were inhibited by the group selective antagonists 4CPG and (2S,1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG-4), respectively. Further investigation using patch-clamp recording revealed that DCG-IV potently inhibited spontaneous GABAergic currents. We conclude that group I and III (but not group II) mGluRs are functionally expressed by granule cells, whereas unexpectedly group II or III mGluRs do not appear to be present presynaptically on mossy fibre terminals. Group II mGluRs are located on Golgi cell terminals; when activated these receptors cause disinhibition, a function which may be important for gating information transfer from the mossy fibres to the granule cells.


Subject(s)
Nerve Fibers/physiology , Receptors, Metabotropic Glutamate/physiology , Synaptic Transmission/physiology , Animals , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , In Vitro Techniques , Nerve Fibers/drug effects , Patch-Clamp Techniques , Rats , Rats, Wistar , Receptors, GABA-A/physiology , Receptors, Metabotropic Glutamate/drug effects , Synaptic Transmission/drug effects
12.
Neuroscience ; 94(4): 1219-30, 1999.
Article in English | MEDLINE | ID: mdl-10625062

ABSTRACT

The mechanism of ischaemic injury to white matter axons was studied by transiently depriving rat optic nerves in vitro of oxygen and glucose. Light and electron microscopic analysis showed that increasing periods of oxygen/glucose deprivation (up to 1 h) caused, after a 90-min recovery period, the appearance of increasing numbers of swollen axons whose ultrastructure indicated that they were irreversibly damaged. This conclusion was supported by experiments showing that the damage persisted after a longer recovery period (3 h). To quantify the axonal pathology, an automated morphometric method, based on measurement of the density of swollen axons, was developed. Omission of Ca2+ from the incubation solution during 1 h of oxygen/glucose deprivation (and for 15 min either side) completely prevented the axonopathy (assessed following 90 min recovery). Omission of Na+ was also effective, though less so (70% protection). The classical Na+ channel blocker, tetrodotoxin (1 microM), provided 92% protection. In view of this evidence implicating Na+ channels in the pathogenesis of the axonal damage, the effects of three different Na+ channel inhibitors, with known neuroprotective properties towards gray matter in in vivo models of cerebral ischaemia, were tested. The compounds used were lamotrigine and the structurally-related molecules, BW619C89 and BW1003C87. All three compounds protected the axons to varying degrees, the maximal efficacies (observed at 30 to 100 microM) being in the order: BW619C89 (>95% protection) > BW1003C87 (70%) > lamotrigine (50%). At a concentration affording near complete protection (100 microM), BW619C89 had no significant effect on the optic nerve compound action potential. Experiments in which BW619C89 was added at different times indicated that its effects were exerted during two distinct phases, one (accounting for about 50% protection) was during the early stage of oxygen/glucose deprivation itself and the other (also about 50%) during the first 15 min of recovery in normal incubation solution. The results are consistent with a pathophysiological mechanism in which Na+ entry through tetrodotoxin-sensitive Na+ channels contributes to Na+ loading of the axoplasm which then results in a lethal Ca2+ overload through reversed Na(+)-Ca2+ exchange. The identification of BW619C89 as a compound able to prevent oxygen/glucose deprivation-induced injury to white matter axons without affecting normal nerve function opens the way to testing the importance of this pathway in white matter injury in vivo.


Subject(s)
Axons/pathology , Ischemia/pathology , Optic Nerve/blood supply , Optic Nerve/pathology , Animals , Glucose/deficiency , Hypoxia/pathology , Hypoxia/physiopathology , In Vitro Techniques , Lamotrigine , Microscopy, Electron , Neuroprotective Agents/pharmacology , Optic Nerve/drug effects , Optic Nerve/physiopathology , Piperazines/pharmacology , Pyrimidines/pharmacology , Rats , Rats, Wistar , Sodium Channel Blockers , Triazines/pharmacology
13.
Neuropharmacology ; 36(3): 401-3, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9175620

ABSTRACT

The mGluR-mediated EPSP at parallel fibre-Purkinje cell synapses in the cerebellum was blocked concentration-dependently and reversibly by antagonists acting selectively on group-I mGluRs but not by an inhibitor of group-III receptors. The results provide pharmacological evidence that the receptor type responsible for the mGluR-EPSP is mGluR1.


Subject(s)
Benzoates/pharmacology , Cerebellum/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Glycine/analogs & derivatives , Receptors, Metabotropic Glutamate/drug effects , Synaptic Transmission/drug effects , Animals , Dose-Response Relationship, Drug , Glycine/pharmacology , Rats
14.
Nature ; 385(6611): 74-7, 1997 Jan 02.
Article in English | MEDLINE | ID: mdl-8985249

ABSTRACT

In the classical view, a central neuron integrates incoming synaptic information by simple algebraic summation of the resultant bioelectrical signals that coincide in time. The voltage dependence of the NMDA (N-methyl-D-aspartate) type of ionotropic glutamate receptor endows neurons with an additional tool that allows one synaptic input to influence another, providing, again, that the two are active simultaneously. Here we identify a new mechanism by which non-coincident signals generated by different synaptic inputs are integrated. The device serves to regulate neuronal excitation through G-protein-coupled, metabotropic glutamate receptors (mGluRs) in a powerful and specific manner. We show that, in cerebellar Purkinje cells, a single activation of the climbing-fibre input markedly potentiates mGluR-mediated excitation at parallel-fibre synapses. The potentiation results from a transient rise in cytosolic Ca2+ which is 'memorized' in such a way that it promotes excitation through mGluRs for about two minutes. A Ca2+-transient is also effective if imposed up to two seconds after parallel-fibre stimulation. By allowing temporally and spatially dispersed synaptic signals to be assimilated, this mechanism adds a new element to the computational power of central neurons.


Subject(s)
Receptors, Metabotropic Glutamate/metabolism , Synapses/metabolism , Action Potentials , Animals , Calcium/metabolism , Cerebellum/cytology , In Vitro Techniques , Nerve Fibers/metabolism , Purkinje Cells/metabolism , Rats
15.
Neuroreport ; 7(12): 1949-52, 1996 Aug 12.
Article in English | MEDLINE | ID: mdl-8905700

ABSTRACT

Brief tetanic stimulation of parallel fibres can evoke a slow excitatory postsynaptic potential (EPSP) in cerebellar Purkinje cells that is mediated by metabotropic glutamate receptors (mGluRs). It is likely that the receptor subtype involved is mGluR1, which couples to the production of diacylglycerol and inositol-1,4,5-trisphosphate (IP3). We therefore examined whether the mGluR-EPSP is associated with an increase in cytosolic free calcium [Ca2+]i using simultaneous Ca2+ imaging and electrophysiological recordings. An mGluR-EPSP could be evoked in all nine Purkinje cells tested. In all but one this potential was not associated with measurable changes in [Ca2+]i whereas single calcium spikes produced large Ca2+ transients. In the one Purkinje cell where [Ca2+]i was elevated, the rise was estimated to be roughly 20-fold smaller than that produced by a single Ca2+ spike.


Subject(s)
Calcium/pharmacology , Cerebellum/metabolism , Purkinje Cells/metabolism , Receptors, Metabotropic Glutamate/drug effects , Synaptic Transmission/physiology , Animals , Calcium/metabolism , Rats
16.
J Neurosci Methods ; 64(2): 189-97, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8699880

ABSTRACT

Unlike certain other brain areas, notably the hippocampal formation, synaptic transmission in the cerebellum, a 3-dimensional anatomical structure, is no ideally studied in a 2-dimensional slice preparation. We describe a method for cutting a cerebellar slice that incorporates two planes of section: one sagittal (in the plane of the Purkinje cell dendrites and of afferent and efferent fibres running in the white matter), and the other in the plane of the parallel fibres (granule cell axons). Details are given of how neurotransmission in the parallel fibre-to-Purkinje cell pathway and in the mossy fibre-to-granule cell pathway can be studied by applying a specially designed grease-gap recording technique to the biplanar slice. We further demonstrate the utility of the slice for intracellular recording.


Subject(s)
Cerebellum/physiology , Neurophysiology/methods , Synaptic Transmission/physiology , Afferent Pathways/physiology , Animals , Axons/physiology , Cerebellum/cytology , Cerebellum/ultrastructure , Dendrites/physiology , Efferent Pathways/physiology , In Vitro Techniques , Nerve Fibers/physiology , Neurophysiology/instrumentation , Purkinje Cells/physiology , Rats
17.
Neuroscience ; 63(4): 911-5, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7535396

ABSTRACT

Glutamate, the major excitatory neurotransmitter in the central nervous system, acts through two broad classes of receptors: ion channel-linked (ionotropic) receptors, which include N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and metabotropic receptors which couple via G-proteins to intracellular messenger cascades. Seven subtypes of mGluR are known to exist but their roles in synaptic physiology are poorly understood. In cerebellar Purkinje cells, application of the mGluR agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid, or the active enantiomer, 1S,3R-ACPD, results in a depolarization associated with an inward current and an elevation of intracellular Ca2+ (for review see Ref. 29). Moreover, using an extracellular (grease-gap) technique that monitors population responses, we have previously discovered that, in Purkinje cells of adult rat cerebellum, brief tetanic stimulation of the glutamatergic parallel fibre input gives rise to a slow depolarising synaptic potential that is resistant to ionotropic glutamate receptor blockers and to antagonists acting at GABA receptors. It was suggested that this novel potential is mediated by metabotropic receptors. The advent of antagonists for metabotropic receptors has allowed us to test this hypothesis. We find that the S-enantiomer of alpha-methyl-4-carboxyphenylglycine stereoselectively antagonizes the slow synaptic potential recorded using the grease-gap method. The results were confirmed by intracellular recording from Purkinje cells. To our knowledge this is the first direct evidence of an mGluR-mediated EPSP in intact brain tissue.


Subject(s)
Cerebellum/metabolism , Nerve Fibers/physiology , Purkinje Cells/physiology , Receptors, Metabotropic Glutamate/physiology , Synapses/physiology , Animals , Benzoates/pharmacology , Cerebellum/cytology , Cerebellum/drug effects , Electric Stimulation , Glycine/analogs & derivatives , Glycine/pharmacology , In Vitro Techniques , Nerve Fibers/drug effects , Neural Pathways/drug effects , Neural Pathways/physiology , Purkinje Cells/drug effects , Rats , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/drug effects , Synapses/drug effects , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/pharmacology
18.
Anaesth Intensive Care ; 22(2): 179-83, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8210022

ABSTRACT

A prospective analysis of 100 successive intubations with Robertshaw tubes in patients submitted for routine thoracic surgery is presented. Clinical guidelines for intubation were used as the protocol. Tube position was judged on clinical assessment only and not confirmed with fibreoptic bronchoscopy. In approximately 60% of cases the tubes were judged to be correctly placed on initial introduction. In 20-30% of cases, minor and simple adjustments were required to achieve suitable conditions for one lung anaesthesia. In no case did it prove impossible to achieve conditions adequate for surgery. Common problems related to the endobronchial portion of tubes entering the wrong bronchus, being inserted too far, or not far enough. Two of six episodes of hypoxaemia related to minor problems with tubes and were easily corrected. The experience is compared with that of other workers. The pertinence and implications of the experience and the data to the training of personnel in the techniques of one-lung ventilation and the future of Robertshaw tubes are discussed. It is concluded that the good practical results achieved are specifically related to the Robertshaw design and the material of manufacture.


Subject(s)
Intubation, Intratracheal/instrumentation , Adolescent , Adult , Aged , Aged, 80 and over , Anesthesia, Endotracheal/adverse effects , Anesthesia, Endotracheal/instrumentation , Anesthesia, Endotracheal/methods , Auscultation , Bronchoscopy , Equipment Design , Female , Humans , Hypoxia/blood , Hypoxia/etiology , Intubation, Intratracheal/adverse effects , Intubation, Intratracheal/methods , Laryngoscopy , Male , Middle Aged , Nitrous Oxide , Oxygen/blood , Pneumonectomy , Prospective Studies , Respiration, Artificial/instrumentation , Surface Properties , Thoracotomy
19.
Neuropharmacology ; 32(1): 11-20, 1993 Jan.
Article in English | MEDLINE | ID: mdl-7679208

ABSTRACT

Glutamate receptors of both the ionotropic (ion channel-linked) and metabotropic (enzyme-linked) categories are abundantly expressed by Purkinje cells in the cerebellum but the functional significance of the latter receptors is unknown. We have tested the possibility that they are activated by the parallel fibre input by recording from Purkinje cells within a biplanar cerebellar slice preparation using the grease-gap technique. Under conditions where ionotropic (NMDA and non-NMDA) glutamate and GABA receptors were blocked pharmacologically, electrical stimulation of parallel fibres gave rise to two very slow potentials. The first peaked about 400 msec from the start of stimulation and was depolarising. It was not evident with single stimuli but reached maximum amplitude after 6 shocks delivered at 50 Hz. The wave was abolished when the slices were perfused with Ca(2+)-free solution or with drugs that inhibit synaptic transmission, but it was resistant to blockade of GABAB receptors, acetylcholine receptors and adrenergic receptors. Next came a slow hyperpolarising potential that peaked about 30 sec after stimulation and which was also Ca(2+)-dependent. The sequence of potentials was replicated by perfusion of an exogenous agonist acting selectively on metabotropic glutamate receptors. We conclude that parallel fibre-to-Purkinje cell synaptic transmission involves not only fast signals generated through ionotropic non-NMDA receptors but also much slower potentials that are likely to be mediated by metabotropic glutamate receptors. These potentials are likely to be significant both for shorter-term (seconds to minutes) Purkinje cell excitability as well as for the induction of longer-term synaptic plasticity.


Subject(s)
Purkinje Cells/physiology , Receptors, Glutamate/physiology , Synapses/physiology , 2-Amino-5-phosphonovalerate/pharmacology , Adenosine/pharmacology , Animals , Baclofen/pharmacology , Bicuculline/pharmacology , Calcium/physiology , Cerebellum/drug effects , Electric Stimulation , In Vitro Techniques , Interneurons/drug effects , Interneurons/physiology , Ion Channels/metabolism , Nerve Fibers/drug effects , Quinoxalines/pharmacology , Rats , Synaptic Transmission/drug effects
20.
Br J Anaesth ; 69(3): 325-7, 1992 Sep.
Article in English | MEDLINE | ID: mdl-1389854

ABSTRACT

A patient with a carcinoid tumour and a history suggestive of carcinoid syndrome, but with no biochemical evidence in support, had a cardiovascular collapse during an anaesthetic with propofol and suxamethonium. Subsequent investigations suggested an anaphylactoid reaction to suxamethonium, but there were features in common with a carcinoid crisis. The necessity for a second anaesthetic soon afterwards posed a dilemma. In the event of a similar reaction during another anaesthetic, a management plan beforehand should include ready availability of appropriate drugs and the use of sympathomimetic drugs that are less likely to exacerbate the situation.


Subject(s)
Anaphylaxis/complications , Hypotension/etiology , Intraoperative Complications/etiology , Malignant Carcinoid Syndrome/complications , Succinylcholine/adverse effects , Acute Disease , Adult , Female , Humans
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