The effect of neuropeptide FF in the amygdala kindling model.

OBJECTIVE: Neuropeptide FF (NPFF) and its receptors (NPFF1 R and NPFF2 R) are differentially distributed throughout the central nervous system. NPFF reduces cortical excitability in rats when administered intracerebroventricularly (i.c.v.), and both NPFF and NPFF1 R antagonists attenuate pilocarpine-induced limbic seizures. In this study, our aim was to determine whether NPFF exerts anticonvulsant or anti-epileptogenic effects in the rat amygdala kindling model for temporal lobe seizures. METHODS: Male Wistar rats were implanted with a recording/stimulation electrode in the right amygdala and a cannula in the left lateral ventricle. In a first group of animals, the afterdischarge threshold (ADT) was determined after a single i.c.v. infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; n = 10). Subsequently, daily infusion of saline (n = 8) or NPFF (1 nmol/h for 2 h; i.c.v.; n = 9) was performed, followed by a kindling stimulus (ADT+200 µA). Afterdischarge duration and seizure severity were evaluated after every kindling stimulus. A second group of rats (n = 7) were fully kindled, and the effect of saline or a high dose of NPFF (10 nmol/h for 2 h, i.c.v.) on ADT and the generalized seizure threshold (GST) was subsequently determined. RESULTS: In naive rats, NPFF significantly increased the ADT compared to control (435 ± 72 µA vs 131 ± 23 µA [P < 0.05]). When rats underwent daily stimulations above the ADT, NPFF did not delay or prevent kindling acquisition. Furthermore, a high dose of NPFF did not alter ADT or GST in fully kindled rats. CONCLUSIONS: I.c.v. administration of NPFF reduced excitability in the amygdala in naive, but not in fully kindled rats, and had no effect on kindling acquisition.

Dysfunctional astrocytic regulation of glutamate transmission in a rat model of depression.

Depression is usually associated with alterations in the monoaminergic system. However, new evidences suggest the involvement of the glutamatergic system in the aetiology of depression. Here we explored the glutamatergic system in a rat model of depression (i.e., the flinders sensitive line (FSL)) to reveal the mechanism underlying the emotional and cognitive aspects associated with the disease. We showed a dramatically elevated level of baseline glutamatergic synaptic transmission by whole-cell recordings as well as impairment in long-term potentiation induced by high-frequency stimulation in hippocampal slices from FSL rats compared with Sprague-Dawley rats. At behavioural level, FSL rats displayed recognition memory impairment in the novel object recognition test. Enantioselective chromatography analysis revealed lower levels of D-serine in the hippocampus of FSL rats and both synaptic plasticity and memory impairments were restored by administration of D-serine. We also observed dysfunctional astrocytic glutamate regulation including downregulation of the glia glutamate transporter GLAST as shown by western blot. One possibility is that the dysfunctional astrocytic glutamate reuptake triggers a succession of events, including the reduction of D-serine production as a safety mechanism to avoid NMDA receptor overactivation, which in turn causes the synaptic plasticity and memory impairments observed. These findings open up new brain targets for the development of more potent and efficient antidepressant drugs.

Comparison of the effects of intrathecal administration of levobupivacaine and lidocaine on the prostaglandin E2 and glutamate increases in cerebrospinal fluid: a microdialysis study in freely moving rats.

BACKGROUND: Bupivacaine has a lower incidence of transient neurological symptoms than lidocaine after intrathecal (i.t.) injection. The increased toxic potential of lidocaine does not support its use in the clinical setting and could be related to augmented levels of spinal prostaglandin E(2) (PGE(2)). We tested whether levobupivacaine leads to lower PGE(2) levels than lidocaine. Moreover, we compared the release of PGE(2) and glutamate after i.t. injections of levobupivacaine or lidocaine. METHODS: Rats were anaesthetized for implantation of an i.t. dialysis catheter. This allowed sampling dialysates of cerebrospinal fluid (CSF) for measuring PGE(2) and glutamate levels. The microdialysis setting included baseline sampling and was followed by an i.t. injection of levobupivacaine 250 microg, 100 microg, or saline. PGE(2) and glutamate levels in CSF were analysed for 4 h. In addition, the residual effect of a second i.t. injection on, respectively, of PGE(2) and glutamate changes was compared after injection of either 250 or 100 microg levobupivacaine, 1000 or 400 microg lidocaine, or saline. RESULTS: Prolonged spinal PGE(2) increases lasting 50-120 min were observed after levobupivacaine injection. Higher PGE(2) concentrations were observed after the second lidocaine 1000 microg injection. Glutamate release after the second injection did not vary between the local anaesthetic groups. CONCLUSIONS: Spinal PGE(2) levels are similarly increased after i.t. levobupivacaine injection of 250 and 100 microg. A higher PGE(2) response was observed after a second i.t. injection in the animals receiving 1000 microg lidocaine than those receiving 400 mg lidocaine or either dose of levobupivacaine.

Effects of disrupted ghrelin receptor function on fear processing, anxiety and saccharin preference in mice.

BACKGROUND: Obesity is a risk factor for stress-related mental disorders such as post-traumatic stress disorder. The underlying mechanism through which obesity affects mental health remains poorly understood but dysregulation of the ghrelin system may be involved. Stress increases plasma ghrelin levels, which stimulates food intake as a potential stress-coping mechanism. However, diet-induced obesity induces ghrelin resistance which in turn may have deleterious effects on stress-coping. In our study, we explored whether disruption of ghrelin receptor function though high-fat diet or genetic ablation affects fear processing, anxiety-like behavior and saccharin preference in mice. METHODS: Adult male C57BL6/J mice were placed on a standard diet or high-fat diet for a total period of 8 weeks. We first established that high-fat diet exposure for 4 weeks elicits ghrelin resistance, evidenced by a blunted hyperphagic response following administration of a ghrelin receptor agonist. We then carried out an experiment in which we subjected mice to auditory fear conditioning after 4 weeks of diet exposure and evaluated effects on fear extinction, anxiety-like behavior and saccharin preference. To explore whether fear conditioning as such may influence the effect of diet exposure, we also subjected mice to auditory fear conditioning prior to diet onset and 4 weeks later we investigated auditory fear extinction, anxiety-like behavior and saccharin preference. In a final experiment, we further assessed lack of ghrelin receptor function by investigating auditory fear processing, anxiety-like behavior and saccharin preference in ghrelin receptor knockout mice and their wild-type littermates. RESULTS: High-fat diet exposure had no significant effect on auditory fear conditioning and its subsequent extinction or on anxiety-like behavior but significantly lowered saccharin preference. Similarly, ghrelin receptor knockout mice did not differ significantly from their wild-type littermates for auditory fear processing or anxiety-like behavior but showed significantly lower saccharin preference compared to wild-type littermates. CONCLUSION: Taken together, our data suggest that disruption of ghrelin receptor function per se does not affect fear or anxiety-like behavior but may decrease saccharin preference in mice.

Intrathecal lidocaine elevates prostaglandin E2 levels in cerebrospinal fluid: a microdialysis study in freely moving rats.

BACKGROUND: In this study, we have investigated whether intrathecal (i.t.) lidocaine administration is accompanied with changes of cerebrospinal fluid (CSF) prostaglandin E(2) (PGE(2)) levels. METHODS: Rats were anaesthetized for i.t. implantation of a triple-lumen spinal loop dialysis catheter. CSF changes in PGE(2) after i.t. injection of saline, 400, or 1000 microg of lidocaine were measured. The impact of i.t. pretreatment with 5 microg MK801 (N-methyl-D-aspartate glutamate antagonist) or 10 microg SC76309A (COX-2 inhibitor) was also investigated. CSF dialysates for measurement of PGE(2) were collected for 4 h. During the whole procedure, motor and sensory blocks were evaluated. A separate group receiving i.t. lidocaine 400 microg (without dialysate sampling) was assessed for mechanical (Von Frey) and radiant heat pain. RESULTS: PGE(2) levels increased to 400% of baseline and remained elevated for 90-120 min after i.t. lidocaine at both doses. Pretreatment with SC76309A and MK801 attenuated this increase. A 40 min period of enhanced pain response was observed after Von Frey filament stimulation during and after sensory and motor block recovery. CONCLUSIONS: I.T. lidocaine (400 or 1000 microg) increases PGE(2) levels in the CSF for 90-120 min along with a transient period of mechanical hyperalgesia after sensory and motor block recovery.

Reduction of systemic exposure and toxicity of cisplatin by encapsulation in poly-lactide-co-glycolide.

The tissue distribution and normal tissue toxicity of cisplatin (cDDP) administered as poly-lactide-co-glycolide (PLAGA) microspheres, developed for loco-regional administration of cDDP to the liver, were studied in Wag/Rij rats. Venoportal administration of this formulation resulted in a reduction in total systemic and renal toxicity, which correlated with a decrease in normal tissue exposure to cDDP while maintaining high liver platinum levels. Liver-to-kidney platinum level ratios were 28 times higher after 4 h and 19 times higher after 24 h with PLAGA-cDDP microspheres than with free cDDP. Liver-to-blood platinum ratios at these times were 38 times and 36 times higher using PLAGA-cDDP. In a CC531 colon carcinoma liver micrometastases model, cytotoxicity of microsphere-released cDDP was confirmed in vivo by equal inhibition of tumor growth by PLAGA-cDDP and free cDDP over a period of 26 days. Free cDDP, however, caused significantly more histological renal damage and total body weight loss. The results were supported by the finding of higher plasma creatinine and urea concentrations 26 days after administration of free cDDP. Kidney platinum levels were 7 times lower when PLAGA-cDDP was used. These findings indicate a sparing effect on normal tissues when cDDP is targeted to the liver by formulation in PLAGA. PLAGA-cDDP microspheres may, therefore, be a useful and effective addition to current techniques of loco-regional chemotherapy for disseminated hepatic tumors.

Differential glutamatergic modulation of monoamine release in the limbic lobe by selective anticonvulsant ionotropic and metabotropic glutamate receptor ligands.

Several researchers are currently trying to unravel neurobiological relationships between epilepsy and depression. After all, these disorders often develop in the same vulnerable brain regions and the importance of comorbid depression and epilepsy is still underscored. Facilitation of central serotonin (5-HT), dopamine (DA) and noradrenaline (NAD) release seems to be associated with both anticonvulsant and antidepressant effects. We show that selective ionotropic and metabotropic glutamate receptor ligands with anticonvulsant properties differentially modulate NAD, DA and 5-HT in rat limbic lobe structures.

2-chloro-N(6)-cyclopentyladenosine-elicited attenuation of evoked glutamate release is not sufficient to give complete protection against pilocarpine-induced seizures in rats.

The effects of 2-chloro-N(6)-cyclopentyladenosine (CCPA) perfused intrahippocampally (1 microM) and injected intraperitoneally (0.5 mg/kg) were investigated in focally-evoked pilocarpine-induced (10 mM) seizures in freely moving rats. While the intrahippocampal perfusion of this highly selective adenosine A(1) receptor agonist gave complete protection against pilocarpine-induced seizures, systemic administration only partially protected the animals, as evaluated by concomitant behavioural and electrocorticographical (ECoG) observations and monitoring of the neurotransmitter alterations. However, pilocarpine-evoked elevation of hippocampal glutamate overflow was significantly attenuated by CCPA irrespective of the mode of administration. Acute pretreatment with systemic 8-cyclopentyl-1,3-dipropylxanthine, a selective A(1) antagonist, reversed both the partial protective effect and the attenuating effect on the extracellular glutamate elicited by systemic CCPA administration. Intrahippocampal CCPA markedly reduced basal hippocampal dopamine efflux but not GABA or glutamate and considerably attenuated the pilocarpine-evoked elevation in dopamine levels. Systemic CCPA appeared to have little influence on the overall pattern of dopamine elevation. The findings give evidence that CCPA-elicited abatement of the evoked glutamate release alone, cannot fully account for its anticonvulsant effect and may suggest that the effects mediated by adenosine on postsynaptic adenosine receptors could be more crucial for its anticonvulsant effect.

Anticonvulsant effect and neurotransmitter modulation of focal and systemic 2-chloroadenosine against the development of pilocarpine-induced seizures.

The present microdialysis study was aimed at evaluating the anticonvulsant effect of the adenosine A(1) receptor agonist 2-chloroadenosine (2-CADO) against pilocarpine-induced seizures in rats. The hippocampal neurotransmitter modulation on the action of 2-CADO and its possible activation of hippocampal adenosine A(2a) receptors was also assessed. Intrahippocampal perfusion of 2-CADO (100 microM) produced a sustained attenuation of baseline dopamine levels, while eliciting a delayed augmentation of both glutamate and GABA efflux. When co-perfused with pilocarpine (10 mM) or injected systemically (7.5 mg/kg), 2-CADO prevented the development of seizures as well as pilocarpine-evoked augmentation of the glutamate and dopamine levels. However, the delayed increase in glutamate overflow with intrahippocampal 2-CADO was still observed. Intraperitoneal injection of selective adenosine A(2a) receptor antagonist SCH 58261 reversed the 2-CADO-elicited attenuation of pilocarpine-induced increment in dopamine efflux and completely abolished the delayed augmentation of glutamate levels, irrespective of perfusion with pilocarpine. Intraperitoneal injection of 5 mg/kg 2-CADO mostly prevented the elevation of pilocarpine-induced glutamate efflux but could not confer adequate protection. We conclude that 2-CADO can prevent pilocarpine-induced seizures by both intrahippocampal perfusion and systemic administration. The attenuation of pilocarpine-induced dopamine efflux and the late elevations of glutamate are likely to be mediated by hippocampal A(2a) receptors. Inhibition of presynaptic glutamate release does not appear to be sufficient for the anticonvulsant action. Postsynaptic events could play a more important role.

NMDA receptor-mediated pilocarpine-induced seizures: characterization in freely moving rats by microdialysis.

1. Pilocarpine administration has been used as an animal model for temporal lobe epilepsy since it produces several morphological and synaptic features in common with human complex partial seizures. Little is known about changes in extracellular neurotransmitter concentrations during the seizures provoked by pilocarpine, a non-selective muscarinic agonist. 2. Focally evoked pilocarpine-induced seizures in freely moving rats were provoked by intrahippocampal pilocarpine (10 mM for 40 min at a flow rate of 2 microl min(-1)) administration via a microdialysis probe. Concomitant changes in extracellular hippocampal glutamate, gamma-aminobutyric acid (GABA) and dopamine levels were monitored and simultaneous electrocorticography was performed. The animal model was characterized by intrahippocampal perfusion with the muscarinic receptor antagonist atropine (20 mM), the sodium channel blocker tetrodotoxin (1 microM) and the N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine maleate, 100 microM). The effectiveness of locally (600 microM) or systemically (10 mg kg(-1) day(-1)) applied lamotrigine against the pilocarpine-induced convulsions was evaluated. 3. Pilocarpine initially decreased extracellular hippocampal glutamate and GABA levels. During the subsequent pilocarpine-induced limbic convulsions extracellular glutamate, GABA and dopamine concentrations in hippocampus were significantly increased. Atropine blocked all changes in extracellular transmitter levels during and after co-administration of pilocarpine. All pilocarpine-induced increases were completely prevented by simultaneous tetrodotoxin perfusion. Intrahippocampal administration of MK-801 and lamotrigine resulted in an elevation of hippocampal dopamine levels and protected the rats from the pilocarpine-induced seizures. Pilocarpine-induced convulsions developed in the rats which received lamotrigine perorally. 4. Pilocarpine-induced seizures are initiated via muscarinic receptors and further mediated via NMDA receptors. Sustained increases in extracellular glutamate levels after pilocarpine perfusion are related to the limbic seizures. These are arguments in favour of earlier described NMDA receptor-mediated excitotoxicity. Hippocampal dopamine release may be functionally important in epileptogenesis and may participate in the anticonvulsant effects of MK-801 and lamotrigine. The pilocarpine-stimulated hippocampal GABA, glutamate and dopamine levels reflect neuronal vesicular release.

The analysis of excitatory, inhibitory and other amino acids in rat brain microdialysates using microbore liquid chromatography.

Three microbore liquid chromatography (LC) assays for determination of amino acids in rat brain dialysates are described: one for separation of amino acids by gradient elution and electrochemical detection, one for analysis of GABA by isocratic elution and electrochemical detection, and one for fast measurement of glutamate and aspartate by gradient elution and fluorescence detection. The assays are reliable, reproducible and sensitive. In comparison with conventional LC, a 5-fold increase in sensitivity was obtained for GABA. Optimization of the derivatization chemistry and the microbore LC system are discussed, as well as important practical aspects.

Polymer-coated albumin microspheres as carriers for intravascular tumour targeting of cisplatin.

We used a poly-lactide-co-glycolide polymer (PLAGA 50:50) to formulate cisplatin (cDDP) into microspheres designed for intravascular administration. Two systems were developed. PLAGA-coated albumin microspheres and microspheres consisting of PLAGA only. PLAGA-coated microspheres displayed a mean diameter of 31.8 +/- 0.9 microns and a payload of 7.5% cDDP (w/w). Solid PLAGA microspheres exhibited a mean diameter of 19.4 +/- 0.6 microns and a payload of 20% cDDP. Release characteristics and in vitro effects on L1210 leukemia and B16 melanoma cell lines were investigated. Both types of microsphere overcame the initial rapid release of cDDP (burst effect), and PLAGA-coated albumin microspheres also showed a lag phase of approximately 30 min before cDDP release began. PLAGA-coated albumin microspheres released most of their payload through diffusion, and the coating eventually cracked after 7 days' incubation in saline supplemented with 0.1% Tween at 37 degrees C, enabling the release of any cDDP remaining. Effects of platinum, pre-released from PLAGA-coated albumin microspheres on the in vitro growth of L1210 cells were comparable with those of standard formulations (dissolved) of cDDP. Material released from non-drug-loaded PLAGA microspheres had no effect on L1210 cell growth, suggesting the absence of cytotoxic compounds in the matrix. The colony-forming ability of B16 cells was also equally inhibited by standard cDDP and pre-released drug. These studies show that formulation of cDDP in PLAGA-based microspheres prevents the rapid burst effect of cDDP seen in previous preparations and offers an improved system of administration for hepatic artery infusion or adjuvant therapy, enabling better clinical handling and the promise of a higher ratio of tumour tissue to normal tissue.

Dopaminergic regulation of serotonin release in the substantia nigra of the freely moving rat using microdialysis.

The functional regulation by dopamine (DA) receptors of serotonin (5-HT) release from the rat substantia nigra (SN) was investigated using in vivo microdialysis. A D1- and D2-receptor-mediated inhibition of nigral 5-HT release was demonstrated in this study. Continuous administration of the D1-receptor agonist CY 208243 (10 microM) through the probe did not alter extracellular DA nor 5-HT from the SN, whereas intranigral administration of the D1-receptor antagonist SCH-23390 HCl (10 microM) significantly increased both DA (to 214%) and 5-HT release (to 168%) from the SN. Co-perfusion of the D1-receptor agonist and antagonist did not change nigral DA nor 5-HT release compared to perfusion of the antagonist alone. The continuous intranigral perfusion of the D2-receptor agonist, (-)-quinpirole HCl (1 microM) significantly decreased both DA ad 5-HT release to 71% and 78%, respectively. These decreases were abolished when the D2-receptor antagonist S(-)-sulpiride (10 microM) and the D2-receptor agonist (-)-quinpirole HCl (1 microM) were co-perfused. In contrast, the intranigral perfusion of the DA precursor, L-DOPA (5 microM; 1 h), significantly increased nigral and striatal 5-HT release to 202% and 155%, respectively. This enhanced nigral 5-HT release might not be receptor-mediated. The results of the present study suggest a D1 and D2 regulation of nigral 5-HT release, either directly mediated by DA receptors on nigral 5-HT terminals or indirectly by nigral GABA, Glu or Asp. Alternatively, the observed DA-5HT-interaction in the SN might not reflect a local interaction but might involve an interaction at the level of the serotonin cell body region, the dorsal raphe nuclei (DRN).

Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia.

Generalized neurotransmitter overflow into the extracellular space, after cerebral ischemia, has been suggested to contribute to subsequent neuronal death. This study aims to investigate the striatal release of the neurotransmitters dopamine (DA), glutamate (Glu) and gamma-aminobutyric acid (GABA) by means of microdialysis, in a rat model for focal transient cerebral ischemia. Ischemia was induced by the application of 120 pmol endothelin-1 (Et-1), adjacent to the middle cerebral artery (MCA) in freely moving rats. Ischemia produced a large increase in extracellular striatal DA concentrations (2400%), Glu (5500%) and GABA (800%) concentrations. Laser Doppler flowmetry in anaesthetized rats, indicated that the blood flow within the striatum decreased by 75+/-11%. The period of sustained drop of blood flow, was dose-dependently related to the concentration Et-1 injected. Histological analysis of brain slices, taken from anaesthetized and conscious animals, indicated a 500 pmol dose of Et-1 was required to produce a similar infarct in anaesthetized rats to a 120 pmol dose of Et-1 in freely moving rats. The immediate drop in striatal blood flow, and the prompt increase of extracellular DA, after the micro-application of Et-1, were quite striking. This suggests that the DA release, rather than the Glu overflow may be the primary event initiating the cascade of processes ultimately leading to cell death and neurological deficits.

Postischemic mild hypothermia reduces neurotransmitter release and astroglial cell proliferation during reperfusion after asphyxial cardiac arrest in rats.

The present study investigated whether postischemic mild hypothermia attenuates the ischemia-induced striatal glutamate (GLU) and dopamine (DA) release, as well as astroglial cell proliferation in the brain. Anesthetized rats were exposed to 8 min of asphyxiation, including 5 min of cardiac arrest. The cardiac arrest was reversed to restoration of spontaneous circulation (ROSC), by brief external heart massage and ventilation within a period of 2 min. After the insult and during reperfusion, the extracellular glutamate and dopamine overflow increased to, respectively, 3000% and 5000% compared with the baseline values in the normothermic group and resulted in brain damage, ischemic neurons and gliosis. However, when hypothermia was induced for a period of 60 min after the insult and restoration of spontaneous circulation, the glutamate and dopamine overflows were not significantly different from that in the sham group. Histological analysis of the brain showed that postischemic mild hypothermia reduced brain damage, ischemic neurons, as well as astroglial cell proliferation. Thus, postischemic mild hypothermia reduces the excitotoxic process, brain damage, as well as astroglial cell proliferation during reperfusion. Moreover, these results emphasize the trigger effect of dopamine on the excitotoxic pathway.

Tonic GABA-ergic modulation of striatal dopamine release studied by in vivo microdialysis in the freely moving rat.

GABAA and GABAB receptor agonists and antagonists were administered locally in the striatum of intact and kainic acid lesioned rats. (+/-)-Baclofen, a GABAB receptor agonist, significantly decreased the level of extracellular dopamine in the striatum of intact rats. (+/-)-Phaclofen, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats and to a lesser extent in the striatum after kainic acid lesion. Pregnanolone (5 beta-pregnan-3 alpha-ol-20-one), a positive allosteric modulator of the GABAA receptor, significantly decreased the level of extracellular dopamine in intact rats. (-)-Bicuculline, a GABAB receptor antagonist, increased the level of extracellular dopamine in the striatum of intact rats, but failed to increase the level of extracellular dopamine after kainic acid lesion. The release of extracellular dopamine, due to infusion of phaclofen or bicuculline, was totally suppressed by tetrodotoxin. These results support a direct influence of GABA on the dopaminergic terminals via presynaptic GABAB receptors, while the effects via the GABAA receptor seem to be postsynaptic and mediated by striatal interneurons or the striatonigral feedback loop.

Hippocampal and cerebellar extracellular amino acids during pilocarpine-induced seizures in freely moving rats.

Limbic seizures were provoked in freely moving rats by intrahippocampal administration of the muscarinic receptor agonist pilocarpine via a microdialysis probe (10 mM for 40 min at 2 microliters/min). Changes in extracellular hippocampal and cerebellar glutamate, aspartate and gamma-aminobutyric acid (GABA) levels were monitored during and after pilocarpine administration. Effects of systemic or local administration of anticonvulsants on the seizures and concomitant changes in amino-acid concentrations, were investigated. Pilocarpine-induced seizures were completely abolished after intraperitoneal premedication for 7 days with phenobarbital (15 mg/kg per day) and after intrahippocampal administration of 10 mM phenobarbital and 1 mM carbamazepine (180 min at 2 microliters/min). Rats premedicated with carbamazepine (5 mg/kg per day) still developed seizures. The changes in extracellular hippocampal amino-acid levels suggest that glutamate, aspartate and GABA are not involved in seizure onset, but may play a role in seizure maintenance and/or spread in the pilocarpine animal model of epilepsy. The increases in extracellular amino acids in ipsi- and contralateral cerebellum following limbic seizures provoked in the hippocampus, probably play a role in the 'reversed' diaschisis phenomenon.

Flumazenil prevents diazepam-elicited anticonvulsant action and concomitant attenuation of glutamate overflow.

Systemic administration of diazepam (5 mg/kg, i.p.) produced a prompt anticonvulsant effect in pilocarpine-induced seizures in freely moving rats. The anticonvulsant effect was associated with significant attenuation of pilocarpine-evoked increases in extracellular hippocampal glutamate levels to below the baseline levels. The purpose of the present microdialysis study, therefore, was to investigate if the effect of diazepam on glutamate release was mediated at the level of the benzodiazepine gamma-aminobutyric acid(A) (GABA(A)) receptor complex to preclude any non-GABAergic mechanisms. Systemic administration of the specific benzodiazepine-receptor antagonist flumazenil (10 mg/kg, i.p. )-elicited complete reversal of diazepam-evoked anticonvulsant action and concomitant attenuation of extracellular glutamate efflux below the baseline levels. This provides evidence that under the given experimental conditions, diazepam-evoked alterations in glutamate overflow associated with the anticonvulsant action were indeed mediated at the level of benzodiazepine-GABA(A) receptor complex, possibly involving the modulation of both pre- and post-synaptic sites of the receptor complex.

Effects of diazepam on extracellular brain neurotransmitters in pilocarpine-induced seizures in rats.

The present study was undertaken to gain insights into the mechanism of action of diazepam in focally-evoked pilocarpine-induced seizures by concomitantly assessing the changes produced in the extracellular levels of glutamate, GABA (gamma-aminobutyric acid) and dopamine. In vivo microdialysis, coupled to continuous monitoring of electrocorticographic (ECoG) recordings, was performed in freely moving rats. Intrahippocampal perfusion with 10 mM pilocarpine (40 min, 2 microl/min) produced limbic seizures. A single dose of intraperitoneal diazepam (5 mg/kg) was administered 2 h after pilocarpine perfusion was started. Dialysates were sampled both from hippocampus and cerebellum and analysed by microbore liquid chromatography. Diazepam produced instant inhibition of behavioural and ECoG seizure activity. Pilocarpine-induced increases in the extracellular levels of glutamate and dopamine in hippocampus were promptly reduced by diazepam. No concurrent alterations in pilocarpine-induced increases in the extracellular levels of GABA in either hippocampus or cerebellum were seen. Pilocarpine enhanced cerebellar glutamate levels only transiently and levels returned to baseline before diazepam administration. No further changes in cerebellar glutamate levels were observed with diazepam. Our findings suggest that the anti-convulsant action of diazepam against pilocarpine-induced seizures is associated with a prompt attenuation of extracellular hippocampal glutamate overflow without concurrent alteration of pilocarpine-induced increases in endogenous GABA levels. Diazepam also significantly decreased pilocarpine-induced increases in extracellular dopamine levels within the hippocampus. No immediate alterations of the basal levels of the neurotransmitters monitored were observed with diazepam.