RESUMEN
In vivo sampling of interstitial fluid by using microdialysis fibers has become a standard and accepted procedure. This sampling method is generally coupled to offline analysis of consecutive dialysate samples by high-performance liquid chromatography or capillary electrophoresis, but this combination is not the best approach for some applications, especially those which require high temporal resolution and rapid data collection. The purpose of this review is to provide information on enzyme-based online assays, i.e., continuous analysis of the dialysate as it emerges from the outlet of the sampling device. We have focused on methods developed specifically for the analysis of solutions perfused at a very slow flow rate, i.e., a feature of microdialysis and ultrafiltration techniques. These methods include flow enzyme-fluorescence assays, flow enzyme-amperometric assays, and sequential enzyme-amperometric detection. Each type of assay is discussed in terms of principle, applications, advantages, and limitations. We also comment on implantable biosensors, an obvious next step forward for in vivo monitoring of molecules in neuroscience.
Asunto(s)
Química Encefálica/fisiología , Encefalopatías/metabolismo , Pruebas Enzimáticas Clínicas/métodos , Microdiálisis/métodos , Monitoreo Fisiológico/métodos , Animales , Encefalopatías/diagnóstico , HumanosRESUMEN
Beneficial effects of glutamate-receptor antagonists in models of neurological disorders are often used to support the notion that endogenous excitotoxicity (i.e. resulting from extracellular accumulation of endogenous glutamate) is a major contributor to neuronal death associated with these conditions. However, this interpretation conflicts with a number of robust and important experimental evidence. Here, emphasis is placed on two key elements: (i) very high extracellular levels of glutamate must be reached to initiate neuronal death, far above those measured in models of neurological disorders; and (ii) changes in extracellular glutamate as measured by microdialysis are not related to changes in the synaptic cleft, i.e. the compartment where neurotransmitter glutamate interacts with its receptors. It has become clear that the diversity and complexity of glutamate-mediated processes allow for a wide range of potential abnormalities (e.g. loss of selectivity of glutamate-operated ion channels, abnormal modulation of glutamate receptors). In addition, as neuronal death subsequent to ischemia and other insults is likely to result from multifactorial processes that may be inter-related, inhibition of glutamate-mediated synaptic transmission may be neuroprotective by increasing the resistance of neurons to other deleterious mechanisms (e.g. inadequate energy supply) that are not directly related to glutamatergic transmission.
Asunto(s)
Ácido Glutámico/toxicidad , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Neurotoxinas/metabolismo , Animales , Muerte Celular/fisiologíaRESUMEN
A reduction in the apparent diffusion coefficient (ADC) of water measured by magnetic resonance imaging (MRI) has been shown to occur early after cerebrovascular occlusion. This change may be a useful indicator of brain tissue adversely affected by inadequate blood supply. The objective of this study was to test the hypothesis that loss of membrane ion homeostasis and depolarization can occur simultaneously with the drop in ADC. Also investigated was whether elevation of extracellular glutamate ([GLU]e) would occur before ADC changes. High-speed MRI of the trace of the diffusion tensor (15-second time resolution) was combined with simultaneous recording of the extracellular direct current (DC) potential and on-line [GLU]e from the striatum of the anesthetized rat. After a control period, data were acquired during remote middle cerebral artery occlusion for 60 minutes, followed by 30 minutes of reperfusion, and cardiac arrest-induced global ischemia. After either focal or global ischemia, the ADC was reduced by 10 to 25% before anoxic depolarization occurred. After either insult, the time for half the maximum change in ADC was significantly shorter than the corresponding DC potential parameter (P < 0.05). The [GLU]e remained at low levels during the entire period of varying ADC and DC potential and did not peak until much later after either ischemic insult. This study demonstrates that ADC changes can occur before membrane depolarization and that high [GLU]e has no involvement in the early rapid ADC decrease.
Asunto(s)
Isquemia Encefálica/metabolismo , Ácido Glutámico/metabolismo , Hipoxia/fisiopatología , Imagen por Resonancia Magnética/métodos , Animales , Isquemia Encefálica/diagnóstico , Difusión , Electrofisiología , Hipoxia/diagnóstico , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/diagnósticoRESUMEN
Long-term potentiation (LTP) of excitatory transmission is a likely candidate for the encoding and storage of information in the mammalian brain. There is a general agreement that LTP involves an increase in synaptic strength, but the mechanisms underlying this persistent change are unclear and controversial. Synaptic efficacy may be enhanced because more transmitter glutamate is released or because postsynaptic responsiveness increases or both. The purpose of this study was to examine whether increased extracellular glutamate concentration was associated with the robust and well-characterized LTP that can be induced in the rat dentate gyrus. To favor the detection of any putative change in extracellular glutamate associated with LTP, our experimental strategy included the following features. 1) Two separate series of experiments were carried out with animals under pentobarbital or urethan anesthesia; 2) changes in extracellular concentration of glutamate were monitored continuously by microdialysis coupled to enzyme amperometry; and 3) dialysate glutamate levels and changes in the slope of excitatory postsynaptic potential evoked by activation of the perforant path were recorded precisely at the same site. Tetanic stimulation of the perforant path increased persistently test-evoked responses in the dentate gyrus (by 19 and 14% in barbiturate and urethan group, respectively), but there was no glutamate change either during or after LTP induction and no indication of increased glutamate efflux when low-frequency stimulation was applied. The results do not rule out a possible contribution of enhanced glutamate exocytosis to LTP induction and/or maintenance because such a presynaptic change may not be detectable extracellularly. However, our findings and other data supporting the notion that neurotransmitter glutamate may hardly leak out of the synaptic cleft conflict with the hypothesis that LTP could also involve a broad synaptic spillover of glutamate.
Asunto(s)
Giro Dentado/fisiología , Ácido Glutámico/metabolismo , Potenciación a Largo Plazo/fisiología , Anestésicos Intravenosos/farmacología , Animales , Barbitúricos/farmacología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Espacio Extracelular/metabolismo , Masculino , Microdiálisis , Vía Perforante/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Uretano/farmacologíaRESUMEN
It has been proposed that deficient glutamate uptake, by increasing the extracellular concentration of this excitatory neurotransmitter, may contribute to the pathophysiology of cerebral ischaemia. This study aimed to examine whether pharmacological inhibition of glutamate uptake altered the kinetics of ischaemia-induced glutamate efflux, and precipitated anoxic depolarisation. Microdialysis was used for application of the glutamate-uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), recording of the EEG and extracellular direct current (DC) potential with an electrode within the probe, and continuous monitoring of changes in extracellular glutamate. L-trans-PDC was applied locally from 8 min prior to cardiac arrest to the end of the recording period. L-trans-PDC (2.5 mM) barely altered the time course of postmortem glutamate efflux in the cortex. Only the maximum rate of efflux during the first exocytotic phase, and the concentration reached at the end of this phase, appeared slightly increased. L-trans-PDC (5 mM) reduced significantly the delay between EEG silence and anoxic depolarization in the cerebral cortex (59.2 +/- 9.2 s vs. 79.7 +/- 11.5 s; n = 6), but not in the striatum and hippocampus. These effects contrast with the marked increase in dialysate glutamate that L-trans-PDC produces in all these three brain regions. Together, these data do not support the hypothesis that inhibition of glutamate uptake plays a critical role, early in cerebral ischaemia. However, a contribution of reversed glutamate uptake to the secondary Ca2+-independent phase of ischaemia-induced glutamate efflux cannot be ruled out.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/efectos de los fármacos , Ácidos Dicarboxílicos/farmacología , Ácido Glutámico/metabolismo , Hipoxia/metabolismo , Inhibidores de la Captación de Neurotransmisores/farmacología , Pirrolidinas/farmacología , Encéfalo/metabolismo , MicrodiálisisRESUMEN
1. It is generally considered that glutamate-mediated transmission can be altered from a physiological to neurotoxic action when extracellular glutamate levels become excessive subsequent to impaired uptake and/or excessive release. However, high extracellular glutamate does not consistently correlate with neuronal dysfunction and death in vivo. The purpose of this study was to examine in situ the local depolarizations, as indicated by negative shifts of the extracellular field (d.c.) potential, produced by local inhibition of high-affinity glutamate uptake, with or without co-application of exogenous glutamate, in three brain regions of anaesthetized rats. 2. Microdialysis probes incorporating an electrode were used to apply exogenous glutamate and/or its uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), and to monitor the resulting changes in extracellular glutamate and d.c. potential at the sites of application within the cortex, striatum and hippocampus. 3. Perfusion of 1 to 10 mM L-trans-PDC markedly and concentration-dependently increased extracellular glutamate levels (by up to 1700% of basal level in the parietal cortex). Despite their large magnitude, glutamate changes were associated with minor negative shifts of the d.c. potential (< 2 mV), which were not suppressed by the N-methyl-D-aspartate (NMDA)-channel blocker, dizocilpine (MK-801, 2 mg kg-1, i.v.), or the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/ kainate-receptor antagonist, 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX, 30 mg kg-1, i.p.). L-trans-PDC had virtually identical concentration-dependent effects on dialysate glutamate in the hippocampus and striatum, but those induced in the cortex were around 40% larger (P < 0.002). In contrast, the associated depolarizations were around twice as large in the striatum and cortex as in the hippocampus (P < 0.002). Finally, co-application of L-trans-PDC did not enhance the d.c. potential changes evoked by perfusion of 5 or 20 mM glutamate. 4. As the neurotoxic potency of glutamate agonists is considered to be linked to excessive opening of glutamate-operated ion channels, these results challenge the notion that high extracellular glutamate levels may be the key to excitotoxicity in neurological disorders. In particular, they do not support the hypothesis that high extracellular glutamate causes the sudden negative shifts of the d.c. potential associated with ischaemia (i.e. anoxic depolarization), traumatic brain injury and spreading depression. Impaired uptake and excessive release of glutamate may well lead to excitotoxicity, but only at the synaptic level, not by spreading through the interstitial fluid.
Asunto(s)
Encéfalo/fisiología , Ácido Glutámico/fisiología , Animales , Encéfalo/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/fisiología , Ácidos Dicarboxílicos/farmacología , Electrofisiología , Ácido Glutámico/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Masculino , Microdiálisis , Inhibidores de la Captación de Neurotransmisores/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Spreading depression (SD) is a wave of cellular depolarization which contributes to neuronal damage in experimental focal ischaemia, and may also underlie the migraine aura. The purpose of this study was to examine the effects of probenecid, an inhibitor of organic anion transport, on K+-evoked SD in vivo. Microdialysis electrodes were implanted in the rat striatum, and recurrent SD elicited by perfusion of artificial cerebrospinal fluid containing 160 mM K+ for 20 min. Probenecid was administered either directly through the microdialysis probe, starting 50 min before application of high K+, or intravenously. SD was markedly reduced by perfusion of 5 mM probenecid through the microdialysis probe. In contrast, a high intravenous dose of probenecid (250 mg/kg) only slightly inhibited SD elicitation 90 min after treatment, despite clear changes in the amplitude and spectrum of the electroencephalogram, as early as 10 min after drug administration, confirming that probenecid readily penetrated the central nervous system. As SD is inhibited by hypercapnia, we have examined the possibility that probenecid may inhibit SD through extracellular acidification subsequent to blockade of lactate transport. Perfusion of 1-20 mM probenecid increased dose-dependently the dialysate levels of lactate, but without extracellular acidosis since the dialysate pH was not significantly reduced. How probenecid inhibits SD deserves further investigation because it may help identify novel strategies to suppress this phenomenon, now recognized deleterious to neuronal function and survival.
Asunto(s)
Depresión de Propagación Cortical/efectos de los fármacos , Neostriado/efectos de los fármacos , Probenecid/farmacología , Uricosúricos/farmacología , Animales , Electroencefalografía/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/metabolismo , Concentración de Iones de Hidrógeno , Ácido Quinurénico/metabolismo , Ácido Láctico/metabolismo , Masculino , Microdiálisis , Potasio/antagonistas & inhibidores , Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/agonistasRESUMEN
L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy) phenyl-2-(1H)-quinolone) is a novel, orally active antagonist at the N-methyl-D-aspartate (NMDA) receptor glycine site. As NMDA receptor antagonism is generally associated with anaesthetic effects, we have examined the electroencephalographic alterations produced by doses of L-701,324 that effectively reduce NMDA-evoked responses in vivo. Microdialysis probes incorporating an electrode were implanted in the striatum of rats and perfused with artificial cerebrospinal fluid (ACSF). Under light halothane anaesthesia, 12 consecutive depolarizations were elicited by switching to ACSF containing 200 microM NMDA for 2 or 3 min, every 20 min. NMDA-evoked depolarizations and EEG were recorded with the microdialysis electrode. L-701,324 (5 or 10 mg kg-1 i.v.) or vehicle were administered 5 min after the 3rd NMDA stimulus. L-701,324 dose-dependently inhibited NMDA-evoked depolarizations, with 10 mg kg-1 reducing these responses by 50% for at least 3 h. The average amplitude of the EEG in the window 0.25-6 Hz (low frequencies) and 6-21 Hz (high frequencies) did not change in the control group. At the higher dose of 10 mg kg-1 L-701,324 transiently increased the amplitude of low frequencies by around 20%. In contrast, both 5 and 10 mg kg-1 significantly reduced the high frequencies to around 70% of control, and this action was sustained with the higher dose. Analysis of the relative EEG power spectra confirmed a small, but persistent shift from high to low EEG frequencies. Our results suggest that L-701,324 slightly strengthened halothane anaesthesia at doses inhibiting effectively NMDA receptor function. Accordingly, the resulting anticonvulsant and neuroprotective actions of L-701,324 may not be associated with marked anaesthesia-like side-effects.
Asunto(s)
Electroencefalografía/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/antagonistas & inhibidores , Quinolonas/farmacología , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Glicina/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Kynurenic acid is an endogenous, competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor glycine site. Accordingly, increasing the brain extracellular concentration of this metabolite may be a suitable alternative to administration of exogenous NMDA antagonists for the treatment of neurological disorders involving excessive NMDA-receptor activation. As competitive inhibition of organic anion transport by probenecid increased brain extracellular levels of kynurenic acid, the purpose of this study was to examine whether intracerebral application of probenecid reduced depolarizations evoked at the same tissue site by NMDA. Microdialysis probes incorporating an electrode were implanted into the striatum of rats and perfused with artificial cerebrospinal fluid. Local depolarizations were produced by perfusing 200 microM NMDA for 2 min, either alone, or co-applied with 1, 5 or 20 mM probenecid. The lowest concentration of probenecid had no effect. At 5 mM, probenecid abolished the hyperpolarization which consistently followed NMDA-responses, but the slight decrease in depolarization amplitude did not reach significance. Inhibition of post-depolarization hyperpolarization suggests that sustained, high extracellular concentrations of probenecid reduce the capacity of the tissue to recover from a depolarizing stimulus, presumably because intensive transport of probenecid imposes a heavy load on Na+, K(+)-ATPase. At 20 mM, probenecid inhibited NMDA-evoked depolarization by approximately 60% (from 4.7 +/- 0.7 mV to 2.1 +/- 0.2 mV; n = 6, P < 0.005). This effect was more marked 30 min after returning to perfusion with normal artificial cerebrospinal fluid, suggesting that high concentrations of probenecid may be toxic to nerve cells, or initiate long-lasting effects linked to inhibition of the transport of important organic anions. These data suggest that inhibition of organic anion transport is not, by itself, sufficient to protect against neurological disorders involving excessive NMDA-receptor activation. However, results from other studies suggest that it may be a valid strategy for enhancing the neuroprotective actions of treatments which stimulate kynurenic acid synthesis, or those of exogenous glutamate receptor antagonists.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , N-Metilaspartato/farmacología , Probenecid/farmacología , Animales , Cuerpo Estriado/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
We have examined how various challenges to brain acid-base homeostasis, resulting in extracellular acidosis, alter N-methyl-D-aspartate (NMDA)-evoked depolarizations in vivo. Repeated stimuli were produced by perfusion of 200 microM NMDA for 2 min through a microdialysis probe implanted into the striatum of halothane anesthetized rats. Hypercapnia reduced NMDA-evoked responses in a concentration-dependent manner, with 7.5 and 15 % CO2 in the breathing mixture reducing the depolarization amplitude to 74 % and 64 % of that of the initial stimuli, respectively. Application of 50 mM NH4+ progressively reduced dialysate pH, and a further acidification was observed when NH4+ was discontinued. Perfusion of NMDA after NH4+ application evoked smaller depolarizations (56 % of the corresponding control, 5 min after NH4+ removal), and this effect persisted for over 1 h. Perfusion of acidic ACSF did not alter the amplitude of NMDA-evoked depolarization, despite changes in dialysate pH confirming that exchange/buffering of acid equivalents took place between the perfusion medium and the surrounding tissue. This negative result probably reflected the remarkable capacity of the brain to buffer H+. Together, these results demonstrate that extracellular acidosis, such as that associated with excessive neuronal activation or ischemia, inhibits NMDA-evoked responses in vivo.
Asunto(s)
Equilibrio Ácido-Base , Acidosis/metabolismo , N-Metilaspartato/farmacología , Neostriado/efectos de los fármacos , Equilibrio Ácido-Base/efectos de los fármacos , Animales , Análisis de los Gases de la Sangre , Electrofisiología , Hipercapnia/metabolismo , Masculino , Microdiálisis , Neostriado/fisiología , Compuestos de Amonio Cuaternario/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
This study ascertains whether high extracellular glutamate contributes to the initiation of spreading depression (SD) by K+. Two microdialysis probes, each incorporating an electrode to record the extracellular direct current (DC) potential at the elicitation site, were implanted symmetrically in the cortex of anesthetized rats. Recurrent SD was triggered by perfusion of 130 mM K+ through the microdialysis probe for 20 min. On one side, this medium was supplemented with increasing concentrations of glutamate (0.1-1 mM) or of the selective glutamate uptake inhibitor 1-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC: 1-10 mM). The effects of L-trans-PDC on extracellular glutamate and basal DC potential were studied in separate experiments. Application of K+ for 20 min consistently elicited five to seven waves of SD. Increasing the concentration of glutamate in the perfusion medium did not alter SD elicitation. Application of L-trans-PDC concentration dependently increased the dialysate levels of glutamate (by approximately 19-fold with 10 mM L-trans-PDC) but, unexpectedly, reduced SD elicitation. These data do not support the hypothesis that SD is elicited because high extracellular glutamate resulting from exocytosis and/or reversal of glutamate uptake depolarizes adjacent neurons. As SD elicitation requires activation of N-methyl-D-aspartate (NMDA) receptors, these results also illustrate that sensitivity of a pathological or experimental event to NMDA receptor antagonists does not necessarily imply involvement of increased extracellular glutamate. This does not rule out a selective action of glutamate, transiently released from presynaptic vesicles, on immediately juxtaposed postsynaptic receptors.
Asunto(s)
Depresión de Propagación Cortical/efectos de los fármacos , Ácido Glutámico/farmacología , Potasio/farmacología , Animales , Corteza Cerebral/fisiología , Ácidos Dicarboxílicos/farmacología , Interacciones Farmacológicas , Conductividad Eléctrica , Espacio Extracelular/metabolismo , Ácido Glutámico/metabolismo , Masculino , Microdiálisis , Inhibidores de la Captación de Neurotransmisores/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
As seizures in experimental models can be induced by the activation and suppressed by the inhibition of glutamate receptors, it is often proposed that a high extracellular glutamate level subsequent to excessive presynaptic release and/or altered glutamate uptake is epileptogenic. The purpose of this study was to ascertain the link between seizure activity and high extracellular glutamate. To assist the detection of any putative rise in extracellular glutamate during seizures, microdialysis was coupled to enzyme-amperometric detection of glutamate, which provides maximal sensitivity and time resolution. Electrical activity and field potential were also recorded through the dialysis membrane to confirm that epileptic activity was present at the sampling site. No increase in dialysate glutamate content was detected during picrotoxin-induced seizures, even when the K+ concentration in the perfusion medium was raised to 50% above that measured previously during paroxysmal activity. In addition, sustained inhibition of glutamate uptake by L-trans-pyrrolidine-2,4-dicarboxylate increased the extracellular glutamate level > 20-fold but did not produce electrophysiological changes indicative of excessive excitation. These findings indicate that seizures are not necessarily accompanied by an increased extracellular glutamate level and that increased glutamatergic excitation in epilepsy may result from other abnormalities such as increased density of glutamate receptors, enhanced activation subsequent to reduced modulation, or sprouting of glutamatergic synapses.
Asunto(s)
Ácido Glutámico/metabolismo , Convulsiones/inducido químicamente , Animales , Ácidos Dicarboxílicos/farmacología , Electroencefalografía , Electrofisiología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Microdiálisis , Inhibidores de la Captación de Neurotransmisores/farmacología , Picrotoxina/farmacología , Pirrolidinas/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
1. Spreading depression (SD) is a propagating transient suppression of electrical activity, associated with cellular depolarization, which probably underlies the migraine aura and may contribute to neuronal damage in focal ischaemia. The purpose of this study was to examine whether L-701,324 (7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2-(1H)-quinolone), a high affinity antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex, inhibits the initiation and propagation of K(+)-induced SD in the rat cerebral cortex in vivo. 2. Microdialysis probes incorporating a recording electrode were implanted in the cerebral cortex of anaesthetized rats and perfused with artificial cerebrospinal fluid (ACSF). Five episodes of repetitive SD were elicited by switching to a medium containing 130 mM K+ for 20 min, each separated by 40 min of recovery (i.e. perfusion with normal ACSF). The brief negative shifts of the extracellular direct current (d.c.) potential, characteristic of SD elicitation, were recorded with the microdialysis electrode and a reference electrode placed under the scalp. Propagation of SD was examined using glass capillary electrodes inserted about 3 mm posterior to the microdialysis electrode. L-701,324 (5 or 10 mg kg-1) or its vehicle were administered i.v. 10 min after the end of the second K(+)-stimulus. The effects of L-701,324 were compared to those of dizocilpine (MK-801; 1 mg kg-1 i.v.), a NMDA-channel blocker known to potently block SD elicitation. 3. Potassium-induced SD initiation was inhibited by 10 mg kg-1 (but not by 5 mg kg-1) of L-701,324. Thirty minutes after administration of 10 mg kg-1 L-701,324, the cumulative area of SD peaks elicited during 20 min was 15.3 +/- 2.1 mV min, versus 23.2 +/- 1.1 mV min in animals which received only the drug vehicle (P < 0.02; n = 6). The delay between application of 130 mM K+ and occurrence of the first SD was also significantly increased. It was approximately doubled in animals treated with 10 mg kg-1 of L-701,324. 4. SD propagation was more sensitive than SD elicitation to L-701,324, as both 5 and 10 mg kg-1 produced an effective inhibition. Even at the lower dose of 5 mg kg-1, L-701,324 completely blocked the propagation of SD elicited 30 min after drug administration. This differential sensitivity of SD elicitation and propagation is not specific to L-701,324 since it was previously observed with other drugs. At doses effective against SD, L-701,324 did not produce any marked alterations of the electroencephalogram. 5. L-701,324 (10 mg kg-1) and MK-801 (1 mg kg-1) had identical effects on the d.c. potential when administered during the recovery which followed the second K+ stimulus. Both drugs produced a positive shift of around 4.5 mV within 10 min of i.v. drug administration, indicating rapid drug penetration into the CNS. Paradoxically, L-701,324 (10 mg kg-1) was markedly less effective than MK-801 (1 mg kg-1) in blocking SD, since this dose of MK-801 was sufficient virtually to abolish SD initiation and completely block its propagation. The higher potency of MK-801 against SD may reflect its use-dependency, i.e. binding of MK-801 and channel blockade are enhanced when the NMDA-receptor ionophore is open. 6. Taken together, these data demonstrate that L-701,324 has an inhibitory effect on both SD initiation and propagation. This action may be beneficial in focal ischaemia, and possibly also against migraine, especially as this drug was shown to be active when administered orally.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Depresión de Propagación Cortical/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Animales , Presión Sanguínea/efectos de los fármacos , Maleato de Dizocilpina/farmacología , Electroencefalografía/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
It is accepted that the ionic composition of the medium perfused through a microdialysis probe should match that of the extracellular fluid (ECF) under physiological conditions. In contrast, the possibility that control artificial cerebrospinal fluid may influence the experimental or pathological conditions under study, by buffering changes in the ECF composition, has been neglected. Spreading depression (SD) is a propagating transient suppression of electrical activity due to cellular depolarization which may contribute to neuronal damage in focal ischaemia, and underlie the migraine aura Here we report that microdialysis markedly inhibits SD propagation, by buffering the sudden increase in extracellular K+ associated with this event. This effect is independent of the microdialysis flow rate and does not result from tissue injury following probe implantation. This finding clearly illustrates that microdialysis can influence the pathological conditions under investigation.
Asunto(s)
Encéfalo/metabolismo , Depresión de Propagación Cortical , Microdiálisis , Animales , Tampones (Química) , Líquido Cefalorraquídeo/fisiología , Electrofisiología , Espacio Extracelular/metabolismo , Perfusión , Potasio/metabolismo , RatasRESUMEN
An enzyme-amperometric detector cell is described for flow analysis of glutamate in dialysate emerging from an implanted microdialysis probe. Its small size allows it to be placed within a few centimetres of the animal preparation, reducing the delay for data acquisition to around 2 min. The selectivity is provided by glutamate oxidase, immobilised with glutaraldehyde on surfaces adjacent to the 3-electrode system. A film of 1,2-diaminobenzene, electropolymerized on the platinum working electrode, eliminates interference from ascorbic acid and other endogenous electroactive compounds. The high sensitivity (< 0.5 mumol/l) and fast response time of the cell (90% of maximum response in 30 s) make it particularly suitable for investigating conditions that produce rapid changes in brain extracellular glutamate. This is illustrated by monitoring changes in extracellular glutamate subsequent to cardiac arrest, and K(+)-induced local depolarization.
Asunto(s)
Química Encefálica/fisiología , Espacio Extracelular/metabolismo , Ácido Glutámico/metabolismo , Aminoácido Oxidorreductasas , Animales , Técnicas Biosensibles , Química Encefálica/efectos de los fármacos , Enzimas Inmovilizadas , Espacio Extracelular/efectos de los fármacos , Concentración de Iones de Hidrógeno , Masculino , Microdiálisis , Oxidación-Reducción , Consumo de Oxígeno/fisiología , Cloruro de Potasio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
1. Cortical spreading depression (SD) is a propagating transient suppression of electrical activity associated with depolarization, which may contribute to the pathophysiology of important neurological disorders, including cerebral ischemia and migraine. The purpose of this study is to ascertain whether SD propagation depends on local accumulation of extracellular K+ or glutamate. 2. Propagating SD recorded through microdialysis probes perfused with artificial cerebrospinal fluid (ACSF) was much smaller than that recorded with conventional glass microelectrodes, presumably because some SD-induced transient changes in the extracellular fluid composition were buffered by ACSF. We have exploited this effect to determine whether perfusion with a medium containing increasing amounts of K+ and/or glutamate favors SD propagation. 3. Increasing the concentration of K+ (15-60 mmol/l) in the perfusion medium dose-dependently restored SD propagation, whereas application of 100-250 mumol/l glutamate through the microdialysis probe had no effect. Superimposing 200 mumol/l glutamate onto 15 and 30 mmol/l K+ did not further improve the restoration of SD propagation by K+. 4. Because potent uptake mechanisms may efficiently clear exogenous glutamate from the extracellular space, the effect of local inhibition of high-affinity glutamate uptake was also studied. Perfusion of the recording microdialysis probe with 1 mmol/l L-trans-pyrrolidine-2,4-dicarboxylate (L-trans-PDC), either alone or together with 200 mumol/l glutamate, had no effect. In addition, L-trans-PDC did not potentiate the positive effect of 30 mmol/l K+ on SD propagation. 5. These results strongly suggest that high extracellular K+, and not extracellular glutamate, is the driving force sustaining SD propagation.(ABSTRACT TRUNCATED AT 250 WORDS)
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Potenciales de Acción/fisiología , Depresión de Propagación Cortical/fisiología , Ácido Glutámico/fisiología , Potasio/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Isquemia Encefálica/fisiopatología , Depresión de Propagación Cortical/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Espacio Extracelular/fisiología , Ácido Glutámico/farmacología , Masculino , Microdiálisis , Trastornos Migrañosos/fisiopatología , Potasio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Using microdialysis probes incorporating an electrode for the recording of extracellular field potentials, we have found that microdialysis markedly inhibited the propagation of spreading depression. This effect was independent of the microdialysis flow rate and did not result from tissue injury following probe implantation. Increasing the K+ concentration in the perfused artificial CSF dose-dependently restored the propagation of spreading depression and revealed a large, synchronous transient increase in extracellular glutamate. These findings clearly illustrate that microdialysis can influence the experimental or pathological conditions under study, by buffering transient changes in the extracellular fluid composition. Epileptic seizures and ischaemia are two important conditions that may be prone to such a detrimental interaction.
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Encéfalo/metabolismo , Encéfalo/fisiología , Microdiálisis , Animales , Encéfalo/efectos de los fármacos , Depresión de Propagación Cortical/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Electrofisiología , Masculino , Perfusión , Potasio/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
1. The purpose of this study was to examine whether depolarizations evoked by excitatory amino acids can be recorded quantitatively, in vivo, with a microelectrode incorporated within a microdialysis probe. 2. Microdialysis probes incorporating a chlorided silver wire were implanted in the striatum of anaesthetized rats and perfused with artificial cerebrospinal fluid (ACSF). Increasing concentrations of excitatory amino acids were applied for 2 min via the microdialysis probe, and the extracellular direct current (d.c.) potential was recorded between the microdialysis electrode and a reference electrode placed under the scalp. 3. N-methyl-D-aspartate (NMDA, 25-500 microM), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA, 5-1000 microM), kainate (5-500 microM), and glutamate (0.25-100 mM) evoked concentration-dependent depolarizations with maxima ranging from 7 to 10 mV, i.e. 3 to 10 times larger than those recorded from brain slices in vitro. Depolarizations evoked by glutamate receptor agonists applied by microdialysis shared several features with those recorded from brain slices. The most characteristic were: steep onset and recovery of NMDA and glutamate responses; marked post-depolarization hyperpolarization with NMDA; and very slow recovery after kainate application. At high concentrations (500 microM), NMDA occasionally initiated spreading depression. The relative potency of glutamate and NMDA was of the same order of magnitude to that obtained with the cortical wedge and hippocampal slices, glutamate being 100 to 400 times less potent than NMDA. 4. Two consecutive series of NMDA-stimuli within the same procedure evoked comparable depolarizations, indicating that reliable quantitative analysis of drug action can be performed, with each animal serving as its own control. This is relevant to the study of drugs acting on glutamate receptors especially antagonists. The remarkable inter-animal reproducibility is also a valuable feature.5. Pretreatment with dizocilpine maleate (MK-801, 2mgkg'1, i.p.) reduced by 65% the responses evoked by NMDA (500 fM). The non-NMDA antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX,100 1M) applied via the microdialysis probe reduced by around 78% the responses to AMPA and kainate (250 micro M). The fact that drugs, especially antagonists, can be administered either systemically, or directly through the dialysis probe to by-pass the blood-brain barrier or avoid peripheral effects, is especially relevant for neuropharmacological studies.6. Intracerebral microdialysis combined with in vivo recording of extracellular field potential is a novel and valuable method for the quantitative analysis of the action of drugs acting on glutamate receptors.This method should prove especially useful for comparing the sensitivity of specific brain structures to selective glutamate receptor agonists under normal conditions and when the neuronal micro environment is altered. It should also be useful for investigating the action of other depolarizing agents, such as veratridine, and their antagonists.
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Polaridad Celular/efectos de los fármacos , Aminoácidos Excitadores/farmacología , Espacio Extracelular/fisiología , Microdiálisis/instrumentación , Neostriado/fisiología , Animales , Electroencefalografía/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Ácido Glutámico/farmacología , Ácido Kaínico/antagonistas & inhibidores , Ácido Kaínico/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , N-Metilaspartato/antagonistas & inhibidores , N-Metilaspartato/farmacología , Neostriado/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/antagonistas & inhibidores , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Stable xenon-enhanced X-ray computed tomography (XeCT) was used to measure the regional cerebral blood flow (rCBF) of 12 patients with drug resistant partial epilepsy and a marked unilateral focus on electroencephalography (EEG). Interictal mean rCBF of fixed regions of interest (ROIs) was reduced by 25% in the cortex of the epileptogenic cerebral lobe compared with the same regions on the contralateral side (p less than 0.02). Six control scans showed a mean side to side cortical difference in rCBF of 14%, whereas the epileptogenic focus was associated with a reduction in the cortical rCBF of greater than 30% in six out of the 12 patients. In an additional patient with partial epilepsy XeCT demonstrated significant focal hypoperfusion when interictal EEGs and conventional CT scans showed no abnormalities.
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Circulación Cerebrovascular , Epilepsias Parciales/fisiopatología , Adulto , Electroencefalografía , Epilepsias Parciales/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Tomografía Computarizada por Rayos X/métodos , XenónRESUMEN
Patients with intracranial abscesses may be exposed to high doses of radiation through repeated pre-operative and follow-up CT studies, combined with conventional radiographic techniques. In a series of 30 patients, the calculated doses did not, however, exceed a theoretically cataractogenic dose of 200 cGy (rad). Ways in which the radiation exposure may be kept to a minimum are discussed.