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.

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.

Proconvulsive effects of oxytocin in the generalized pentylenetetrazol mouse model are mediated by vasopressin 1a receptors.

The possible involvement of oxytocin (OT) in the generation of seizures has not received a lot of attention in the past, although generalized epileptic convulsions were observed in humans following intravenous OT infusion. We here aimed to investigate the effect of exogenous OT administration on seizure susceptibility in C57Bl/6 mice subjected to the pentylenetetrazol (PTZ) model. In addition, we studied via which receptor possible effects on seizure thresholds could be mediated since OT binds to both the OT receptor (OTR) and the vasopressin 1a receptor (V1aR). We showed that C57Bl/6 mice treated with 0.5 mg/kg OT had decreased PTZ thresholds for ear twitch, myoclonic twitch, tail twitch, forelimb clonus and falling. This pronconvulsive effect was reversed by the OTR antagonist L-368.899, however, it was not mimicked by the OTR agonist carbetocin (CBT). Nevertheless, CBT had antidepressant-like effects in the forced swim test that could be reversed by L-368.899. These experiments shed some doubt on the involvement of OTR in the observed effect of OT on seizure thresholds. Therefore, we investigated the role of the V1aR as a possible mediator of the proconvulsive effects of OT. We found that the proconvulsive effects of both arginine vasopressin and OT were reversed by the V1aR antagonist SR49059. In summary, OT has proconvulsive effects in our mouse model of generalized seizures that could not be mimicked by CBT. Our results suggest that the binding of OT to V1aRs is the most plausible explanation for the proconvulsive effects of OT.

Using microdialysis to analyse the passage of monovalent nanobodies through the blood-brain barrier.

BACKGROUND AND PURPOSE: Nanobodies are promising antigen-binding moieties for molecular imaging and therapeutic purposes because of their favourable pharmacological and pharmacokinetic properties. However, the capability of monovalent nanobodies to reach targets in the CNS remains to be demonstrated. EXPERIMENTAL APPROACH: We have assessed the blood-brain barrier permeability of Nb_An33, a nanobody against the Trypanosoma brucei brucei variant-specific surface glycoprotein (VSG). This analysis was performed in healthy rats and in rats that were in the encephalitic stage of African trypanosomiasis using intracerebral microdialysis, single photon emission computed tomography (SPECT) or a combination of both methodologies. This enabled the quantification of unlabelled and (99m) Tc-labelled nanobodies using, respectively, a sensitive VSG-based nanobody-detection elisa, radioactivity measurement in collected microdialysates and SPECT image analysis. KEY RESULTS: The combined read-out methodologies showed that Nb_An33 was detected in the brain of healthy rats following i.v. injection, inflammation-induced damage to the blood-brain barrier, as in the late encephalitic stage of trypanosomiasis, significantly increased the efficiency of passage of the nanobody through this barrier. Complementing SPECT analyses with intracerebral microdialysis improved analysis of brain disposition. There is clear value in assessing penetration of the blood-brain barrier by monovalent nanobodies in models of CNS inflammation. Our data also suggest that rapid clearance from blood might hamper efficient targeting of specific nanobodies to the CNS. CONCLUSIONS AND IMPLICATIONS: Nanobodies can enter the brain parenchyma from the systemic circulation, especially in pathological conditions where the blood-brain barrier integrity is compromised.

Deletion of insulin-regulated aminopeptidase in mice decreases susceptibility to pentylenetetrazol-induced generalized seizures.

The peptide angiotensin IV (Ang IV) influences seizure susceptibility in rat and mouse models. Indeed, Ang IV has been shown to protect rats from limbic seizures in the focal pilocarpine model. Moreover, both anticonvulsive and antiepileptogenic effects of Ang IV have been reported in the acute pentylenetetrazol (PTZ) and kindling model of generalized seizures in mice. It has been hypothesized that the latter effects on seizures could be established via a modulatory effect on dopamine receptors in the basal ganglia or via an indirect interaction between Ang IV and adenosine A1 receptors. However, a possible role for insulin-regulated aminopeptidase (IRAP), the high affinity binding site for Ang IV, has not been studied yet. To unequivocally unravel the involvement of IRAP in generalized seizure generation, we investigated the susceptibility of male IRAP wild-type (IRAP(+/+)) and knock-out (IRAP(-/-)) mice to PTZ-induced seizures. Challenging these mice intravenously with PTZ resulted in significantly increased thresholds for myoclonic twitch and generalized clonic seizures with loss of righting reflexes in IRAP(-/-) mice compared to their IRAP(+/+) littermates. These behavioural data were confirmed by video-electrocorticography monitoring. Our study shows that IRAP(-/-) mice are less sensitive to the development of PTZ-induced seizures and suggests that IRAP is involved in generalized seizure generation.

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.

Impact of efflux transporters and of seizures on the pharmacokinetics of oxcarbazepine metabolite in the rat brain.

BACKGROUND AND PURPOSE: Accurate prediction of biophase pharmacokinetics (PK) is essential to optimize pharmacotherapy in epilepsy. Here, we characterized the PK of the active metabolite of oxcarbazepine, 10,11-dihydro-10-hydroxy-carbamazepine (MHD) in plasma and in the hippocampus. Simultaneously, the impact of acute seizures and efflux transport mechanisms on brain distribution was quantified. EXPERIMENTAL APPROACH: Rats received subtherapeutic and anticonvulsant doses of MHD in non-epileptic conditions and during focal pilocarpine-induced limbic seizures. To evaluate the effect of efflux transport blockade, a separate group received subtherapeutic doses combined with intrahippocampal perfusion of verapamil. Free plasma and extracellular hippocampal MHD concentrations were determined using microdialysis and liquid chromatography techniques. An integrated PK model describing simultaneously the PK of MHD in plasma and brain was developed using nonlinear mixed effects modelling. A bootstrap procedure and a visual predictive check were performed to assess model performance. KEY RESULTS: A compartmental model with combined zero- and first-order absorption, including lag time and biophase distribution best described the PK of MHD. A distributional process appeared to underlie the increased brain MHD concentrations observed following seizure activity and efflux transport inhibition, as reflected by changes in the volume of distribution of the biophase compartment. In contrast, no changes were observed in plasma PK. CONCLUSIONS AND IMPLICATIONS: Simultaneous PK modelling of plasma and brain concentrations has not been used previously in the evaluation of antiepileptic drugs (AEDs). Characterisation of biophase PK is critical to assess the impact of efflux transport mechanisms and acute seizures on brain disposition and, consequently, on AED effects.

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.

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.

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.

Formalin-induced spinal glutamate release in freely moving rats: comparison of two spinal microdialysis approaches.

Two different spinal microdialysis approaches using either a linear tissue probe (LM-3) or a loop probe were explored on freely-moving rats to investigate the basal and formalin-evoked release of glutamate (Glu) in the spinal dorsal horn or in the cerebrospinal fluid (CSF). Adult male Wistar rats were implanted either with a LM-3 probe transversely through the spinal dorsal horn or with a loop probe in the CSF. After 24 hours recovery, microdialysis was initiated with perfusion of modified Ringer's solution at a flow rate of 5 microliters/min and the basal Glu concentrations were sampled for 1 hour. The effects of altering the microdialysis flow rate and perfusion solution on basal Glu release were next investigated. Following the injection of 50 microliters of formalin 5% into the hind paw, 10-min samples were collected for 90 min. The baseline levels of Glu were 0.82 +/- 0.09 microM with LM-3 probes and 5.96 +/- 0.22 microM with the loop probes. Decreasing the flow rate from 5 to 2 microliters/min increased extracellular Glu concentrations by 222.7 +/- 7.3%, whereas perfusion with artificial CSF reduced baseline Glu by 61.5 +/- 9.5% with LM-3 probes. Injection of formalin induced a short-lasting but significant increase of Glu with a similar profile and time course when using either of the microdialysis approaches. In conclusion, microdialysis in the dorsal horn or in the CSF are both effective techniques to assess the alterations in Glu release following peripheral nociceptive input. The loop probe technique in CSF is more reproducible for routine investigation of drug effects, whereas the microdialysis of the dorsal horn provides a useful tool to precisely locate where the release of the neurotransmitters occurs.

Convulsant and subconvulsant doses of norfloxacin in the presence and absence of biphenylacetic acid alter extracellular hippocampal glutamate but not gamma-aminobutyric acid levels in conscious rats.

Fluoroquinolones are antibiotics with central excitatory side effects. These adverse effects presumably result from inhibition of gamma-aminobutyric acid (GABA) binding to GABA(A) receptors. This GABA antagonistic effect is greatly potentiated by the active metabolite of fenbufen, biphenylacetic acid (BPAA). Nevertheless, it remains questionable whether GABA receptor antagonism alone can explain the convulsant activity potentials of these antimicrobial agents. The present study was undertaken to investigate the possible effects of norfloxacin, both in the absence and in the presence of BPAA, on the extracellular hippocampal levels of GABA and glutamate, the main central inhibitory and excitatory amino acid neurotransmitters, respectively. This in vivo microdialysis approach with conscious rats allows monitoring of behavioral alterations and concomitant transmitter modulation in the hippocampus. Peroral administration of 100 mg of BPAA per kg of body weight had no effect on behavior and did not significantly alter extracellular GABA or glutamate concentrations. Intravenous perfusion of 300 mg of norfloxacin per kg did not change the rat's behavior or the concomitant neurotransmitter levels in about half of the experiments, while the remaining animals exhibited severe seizures. These norfloxacin-induced convulsions did not affect extracellular hippocampal GABA levels but were accompanied by enhanced glutamate concentrations. Half of the rats receiving both 100 mg of BPAA per kg and 50 mg of norfloxacin per kg displayed lethal seizures, while the remaining animals showed no seizure-related behavior. In the latter subgroup, again no significant alterations in extracellular GABA levels were observed, but glutamate overflow remained significantly elevated for at least 3 h. In conclusion, norfloxacin exerts convulsant activity in rats, accompanied by elevations of extracellular hippocampal glutamate levels but not GABA levels, even in the presence of BPAA.

Effects of dietary sucrose on hippocampal serotonin release: a microdialysis study in the freely-moving rat.

The effects of dietary supplementation with either sucrose or starch (50 g/kg regular food for 2 weeks) on central 5-hydroxytryptamine (5HT; serotonin) release were investigated in freely-moving rats. It has been suggested that the amount of transmitter that serotoninergic neurons release might be altered by food intake. We monitored the effects of sucrose and starch on concentrations of extracellular 5HT, its metabolite 5-hydroxyindoleacetic acid (5HIAA), gamma-aminobutyric acid (GABA) and dopamine in the hippocampus, using in vivo microdialysis. The major finding was that baseline levels of extracellular hippocampal 5HT in rats with ad libitum access to food supplemented with sucrose were significantly higher compared with the starch control group. We then verified that sucrose supplementation affected the potency of S(+)fenfluramine to increase hippocampal 5HT levels. In both groups of rats, acute intraperitoneal injection (1 mg/kg) of this anorectic drug induced a response curve of the extracellular hippocampal 5HT levels, with a shape that corresponded with earlier data for different brain areas often using up to 10-fold higher doses of S(+)fenfluramine. Nevertheless, we showed that throughout the experiment the absolute values of the sucrose response curve remained higher than in the starch group. On the other hand, S(+)fenfluramine exerted longer lasting effects in the starch group, as compared with the sucrose group. Significant decreases in levels of extracellular hippocampal 5HIAA levels following S(+)fenfluramine administration were simultaneously observed. A practical implication of the present findings is that dietary sucrose may bias the results of studies investigating brain serotoninergic mechanisms and the effects of (anorectic) drugs interacting with 5HT systems in the hippocampus.

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.

The effects of LY393613, nimodipine and verapamil, in focal cerebral ischaemia.

This study evaluates the effects of N-(2-[bis (4-fluorophenyl)methoxy]ethyl)-1-butanamine hydrochloride (LY393613), a novel neuronal (N/P/Q-type) Ca(2+) channel blocker, in ischaemia. For comparison, two commonly used L-type Ca(2+) channel blockers; nimodipine and verapamil were also evaluated. Ischaemia was induced in freely moving rats by micro-injection of endothelin-1 near the middle cerebral artery. In vivo microdialysis, laser Doppler flowmetry and histology were used to monitor ischaemia. Administration of LY393613, before and after the insult, attenuated the ischaemia-induced glutamate release, but not the dopamine release. Both nimodipine and verapamil failed to affect transmitter releases significantly, when administered post-occlusion. None of the compounds tested, produced any significant change in striatal blood flow. Histology showed that ischaemic damage was significantly less in LY393613 pre-treated rats. In conclusion, LY393613, a neuronal N/P/Q-Ca(2+) channel blocker, can attenuate ischaemic brain damage. The protective mechanism appears to be mainly the attenuation of the ischaemia-induced glutamate release, rather than its effect on cerebral hemodynamics.

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.

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.

In vivo study of the effect of valpromide and valnoctamide in the pilocarpine rat model of focal epilepsy.

PURPOSE: We evaluated the effectiveness of the commonly used antiepileptic drug sodium valproate (400 mg/kg) and two of its amide derivatives, valpromide and valnoctamide (both 100 mg/kg), in an in vivo rat model of focal epilepsy. Our main interest was to get insight into possible changes in extracellular amino acid neurotransmitter levels following administration of the drugs, both in control and in epileptic conditions. METHODS: Seizures were evoked in freely moving rats by intrahippocampal administration of pilocarpine via a microdialysis probe (10 mM for 40 min at 2 microl/min). Microdialysis was also used as in vivo sampling technique and alterations in extracellular hippocampal glutamate and GABA levels were monitored. Electrophysiological evidence for the presence or absence of seizures was simultaneously recorded with electrocorticography. RESULTS: The focally evoked pilocarpine-induced seizures were completely prevented by acute intraperitoneal pretreatment with each of the three drugs in the respective doses. Effective protection was reflected in the electrocorticographic recordings and in the lack of sustained elevations of the extracellular glutamate levels after pilocarpine perfusion. Little effects were seen on the basal extracellular amino acid levels after systemic administration of each of the compounds, nor after the intrahippocampal administration of sodium valproate. CONCLUSIONS: Valnoctamide and valpromide (100 mg/kg) proved to be at least as effective as their parent compound sodium valproate (400 mg/kg) against pilocarpine-induced seizures. All three compounds however failed to induce significant initial alterations in extracellular hippocampal GABA release. This questions the enhancement of GABA-mediated inhibition as being one of their mechanisms of action.