Altered responses of dopamine D3 receptor null mice to excitotoxic or anxiogenic stimuli: Possible involvement of the endocannabinoid and endovanilloid systems

Dopamine and the endocannabinoids, anandamide and 2-arachidonoylglycerol, interact at several levels in the brain, with the involvement of both cannabinoid CB₁ receptors and transient receptor potential vanilloid type-1 (TRPV1) channels (which are alternative anandamide receptors). Using pharmacological, immunohistochemical and analytical approaches, we investigated the response of dopamine D₃ receptor null (D3R^(−/−)) mice in models of epilepsy and anxiety, in relation to their brain endocannabinoid and endovanilloid tone. Compared to wild-type mice, D3R^(−/−) mice exhibited a delayed onset of clonic seizures, enhanced survival time, reduced mortality rate and more sensitivity to anticonvulsant effects of diazepam after intraperitoneal administration of picrotoxin (7 mg/kg), and a less anxious-like behaviour in the elevated plus maze test. D3R^(−/−) mice also exhibited different endocannabinoid and TRPV1, but not CB₁, levels in the hippocampus, nucleus accumbens, amygdala and striatum. Given the role played by CB₁ and TRPV1 in neuroprotection and anxiety, and based on data obtained here with pharmacological tools, we suggest that the alterations of endocannabinoid and endovanilloid tone found in D3R^(−/−) mice might account for part of their altered responses to excitotoxic and anxiogenic stimuli.     

The neuroprotective effect of cannabidiol in an in vitro model of newborn hypoxic–ischemic brain damage in mice is mediated by CB2 and adenosine receptors

To investigate the mechanisms involved in cannabidiol (CBD)-induced neuroprotection in hypoxic–ischemic (HI) immature brain, forebrain slices from newborn mice underwent oxygen and glucose deprivation in the presence of vehicle, or CBD alone or with selective antagonists of cannabinoid CB₁ and CB₂, and adenosine A₁ and A₂ receptors. CBD reduced acute (LDH efflux to the incubation medium) and apoptotic (caspase-9 concentration in tissue) HI brain damage by reducing glutamate and IL-6 concentration, and TNFα, COX-2, and iNOS expression. CBD effects were reversed by the CB₂ antagonist AM630 and by the A_2A antagonist SCH58261. The A_1A antagonist DPCPX only counteracted the CBD reduction of glutamate release, while the CB₁ antagonist SR141716 did not modify any effect of CBD. In conclusion, CBD induces robust neuroprotection in immature brain, by acting on some of the major mechanisms underlying HI cell death; these effects are mediated by CB₂ and adenosine, mainly A_2A, receptors.     

Potential antipsychotic properties of central cannabinoid (CB1) receptor antagonists

Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the principal psychoactive constituent of the Cannabis sativa plant, and other agonists at the central cannabinoid (CB₁) receptor may induce characteristic psychomotor effects, psychotic reactions and cognitive impairment resembling schizophrenia. These effects of Δ⁹-THC can be reduced in animal and human models of psychopathology by two exogenous cannabinoids, cannabidiol (CBD) and SR141716. CBD is the second most abundant constituent of Cannabis sativa that has weak partial antagonistic properties at the CB₁ receptor. CBD inhibits the reuptake and hydrolysis of anandamide, the most important endogenous CB₁ receptor agonist, and exhibits neuroprotective antioxidant activity. SR141716 is a potent and selective CB₁ receptor antagonist. Since both CBD and SR141716 can reverse many of the biochemical, physiological and behavioural effects of CB₁ receptor agonists, it has been proposed that both CBD and SR141716 have antipsychotic properties. Various experimental studies in animals, healthy human volunteers, and schizophrenic patients support this notion. Moreover, recent studies suggest that cannabinoids such as CBD and SR141716 have a pharmacological profile similar to that of atypical antipsychotic drugs. In this review, both preclinical and clinical studies investigating the potential antipsychotic effects of both CBD and SR141716 are presented together with the possible underlying mechanisms of action.     

The cannabinoid WIN 55,212‐2‐mediated protection of dentate gyrus granule cells is driven by CB1 receptors and modulated by TRPA1 and Cav2.2 channels

Cannabinoids regulate numerous physiological and pathological events like inflammation or neurodegeneration via CB₁ and CB₂ receptors. The mechanisms behind cannabinoid effects show a high variability and may also involve transient receptor potential channels (TRP) and N‐type voltage‐gated Ca²⁺ channels (Ca_v2.2). In the present study we investigated the neuroprotective effects of the synthetic cannabinoid WIN 55,212–2 (WIN) on dentate gyrus (DG) granule cells and elucidated the involvement of TRP and Ca_v2.2 that are shown to participate in inflammatory processes. Organotypic hippocampal slice cultures were excitotoxically lesioned using NMDA and subsequently incubated with different WIN concentrations (0.001–10 μM). WIN showed neuroprotective properties in an inverse concentration‐dependent manner, most effectively at 0.01 μM. The CB₁ receptor antagonist AM251 blocked neuroprotection mediated by WIN whereas the CB₂ receptor antagonist AM630 showed no effects. Application of the TRPA1 blocker HC‐030031 enhanced the neuroprotective efficacy of high (10 μM) WIN concentrations and the number of degenerating neurons became equal to that seen after application of the most effective WIN dose (0.01 μM). In contrast, the application of TRPA1 agonist icilin or allyl isothiocyanate (AITC) led to a stronger neurodegeneration. The use of TRPV1 blocker 6‐iodo‐nordihydrocapsaicin did not affect WIN‐mediated neuroprotection. The selective Ca_v2.2 blocker ω‐conotoxin (GVIA) completely blocked neuroprotection shown by 10 μM WIN. GVIA and HC‐030031 exerted no effects at WIN concentrations lower than 10 μM. Our data show that WIN protects dentate gyrus granule cells in a concentration dependent manner by acting upon CB₁ receptors. At high (10 μM) concentrations WIN additionally activates TRPA1 and Ca_v2.2 within the hippocampal formation that both interfere with CB₁ receptor‐mediated neuroprotection. This leads to the conclusion that physiological and pharmacological effects of cannabinoids strongly depend on their concentration and the neuroprotective efficacy of cannabinoids may be determined by interaction of activated CB₁ receptor, TRPA1, and Ca_v2.2. © 2010 Wiley‐Liss, Inc.     

Brain Cannabinoid Systems as Targets for the Therapy of Neurological Disorders

Unprecedented developments in cannabinoid research within the past decade include discovery of a brain (CB₁) and peripheral (CB₂) receptor; endogenous ligands, anandamide, and 2-arachidonylglycerol; cannabinoid drug-induced partial and inverse agonism at CB₁receptors, antagonism of NMDA receptors and glutamate, and antioxidant activity; and preferential CB₁receptor localization in areas subserving spasticity, pain, abnormal involuntary movements, seizures, and amnesia. These endogenous structures and chemicals and mechanisms are potentially new pathophysiologic substrates, and targets for novel cannabinoid treatments, of several neurological disorders.     

Role of the Endogenous Cannabinoid System in the Regulation of Motor Activity

One of the prominent pharmacological features of drugs acting at the brain cannabinoid receptor (CB₁) is the induction of alterations in motor behavior. Catalepsy, immobility, ataxia, or the impairment of complex behavioral acts are observed after acute administration of either natural and synthetic cannabinoid receptor agonists or the endogenous CB₁ligand anandamide. The dense presence of CB₁receptors in the cerebellum and in the basal ganglia, especially at the outflow nuclei (substantia nigra and the internal segment of the globus pallidus), supports the existence of an endogenous cannabinoid system regulating motor activity. In the basal ganglia, the functionality of the anandamide–CB₁system is poorly understood. Dual effects are often observed after the administration of CB₁ligands in animal models of pharmacological manipulation of basal ganglia transmitter systems, indicating that the activity of the anandamide–CB₁system depends on the ongoing activation of the different elements of the basal ganglia. This finding is in agreement with the proposed activity-dependent release of anandamide from a plasmalemma precursor. Additionally, a potential state-dependent bidirectional coupling of the CB₁receptor to the adenylate cyclase transduction system has also been described. From this perspective, the endogenous cannabinoid system can be proposed as a local regulator of neurotransmission processes within the basal ganglia. This system may serve as a counterregulatory homeostatic mechanism preserving the functional role of basal ganglia circuits in coding the serial order of events that constitute movement.     

A role for endocannabinoids in viral-induced dyskinetic and convulsive phenomena

Dyskinesias and seizures are both medically refractory disorders for which cannabinoid-based treatments have shown early promise as primary or adjunctive therapy. Using the Borna disease (BD) virus rat, an animal model of viral encephalopathy with spontaneous hyperkinetic movements and seizure susceptibility, we identified a key role for endocannabinoids in the maintenance of a balanced tone of activity in extrapyramidal and limbic circuits. BD rats showed significant elevations of the endocannabinoid anandamide in subthalamic nucleus, a relay nucleus compromised in hyperkinetic disorders. While direct and indirect cannabinoid agonists had limited motor effects in BD rats, abrupt reductions of endocannabinoid tone by the CB₁ antagonist SR141716A (0.3 mg/kg, i.p.) caused seizures characterized by myoclonic jerks time-locked to periodic spike/sharp wave discharges on hippocampal electroencephalography. The general opiate antagonist naloxone (NLX) (1 mg/kg, s.c.), another pharmacologic treatment with potential efficacy in dyskinesias or L-DOPA motor complications, produced similar seizures. No changes in anandamide levels in hippocampus and amygdala were found in convulsing NLX-treated BD rats. In contrast, NLX significantly increased anandamide levels in the same areas of normal uninfected animals, possibly protecting against seizures. Pretreatment with the anandamide transport blocker AM404 (20 mg/kg, i.p.) prevented NLX-induced seizures. These findings are consistent with an anticonvulsant role for endocannabinoids, counteracting aberrant firing produced by convulsive agents, and with a functional or reciprocal relation between opioid and cannabinoid tone with respect to limbic convulsive phenomena.     

WAG/Rij rats show a reduced expression of CB1 receptors in thalamic nuclei and respond to the CB1 receptor agonist,R(+)WIN55,212‐2, with a reduced incidence of spike‐wave discharges

Purpose: Genetically epileptic WAG/Rij rats develop spontaneous absence‐like seizures after 3 months of age. We used WAG/Rij rats to examine whether absence seizures are associated with changes in the expression of type‐1 cannabinoid (CB₁) receptors. Methods: Receptor expression was examined by in situ hybridization and western blot analysis in various brain regions of “presymptomatic” 2‐month old and “symptomatic” 8‐month‐old WAG/Rij rats relative to age‐matched nonepileptic control rats. Furthermore, we examined whether pharmacologic activation of CB₁ receptor affects absence seizures. We recorded spontaneous spike‐wave discharges (SWDs) in 8‐month old WAG/Rij rats systemically injected with the potent CB₁ receptor agonist, R(+)WIN55,212‐2 (3–12 mg/kg, s.c.), given alone or combined with the CB₁ receptor antagonist/inverse agonist, AM251 (12 mg/kg, s.c.). Results: Data showed a reduction of CB₁ receptor mRNA and protein levels in the reticular thalamic nucleus, and a reduction in CB₁ receptor protein levels in ventral basal thalamic nuclei of 8‐month‐old WAG/Rij rats, as compared with age‐matched ACI control rats. In vivo, R(+)WIN55,212‐2 caused a dose‐dependent reduction in the frequency of SWDs in the first 3 h after the injection. This was followed by a late increase in the mean SWD duration, which suggests a biphasic modulation of SWDs by CB₁ receptor agonists. Both effects were reversed or attenuated when R(+)WIN55,212‐2 was combined with AM251. Discussion: These data indicate that the development of absence seizures is associated with plastic modifications of CB₁ receptors within the thalamic‐cortical‐thalamic network, and raise the interesting possibility that CB₁ receptors are targeted by novel antiabsence drugs.     

Cannabis,cannabidiol, and epilepsy — From receptors to clinical response

Recreational cannabis use in adults with epilepsy is widespread. The use of cannabis for medicinal purposes is also becoming more prevalent. For this purpose, various preparations of cannabis of varying strengths and content are being used. The recent changes in the legal environment have improved the availability of products with high cannabidiol (CBD) and low tetrahydrocannabinol (THC) concentrations. There is some anecdotal evidence of their potential efficacy, but the mechanisms of such action are not entirely clear. Some suspect an existence of synergy or “entourage effect” between CBD and THC. There is strong evidence that THC acts via the cannabinoid receptor CB₁. The mechanism of action of CBD is less clear but is likely polypharmacological. The scientific data support the role of the endocannabinoid system in seizure generation, maintenance, and control in animal models of epilepsy. There are clear data for the negative effects of cannabis on the developing and mature brain though these effects appear to be relatively mild in most cases. Further data from well-designed studies are needed regarding short- and long-term efficacy and side effects of CBD or high-CBD/low-THC products for the treatment of seizures and epilepsy in children and adults.     

The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. III. Pentylenetetrazole seizure models

Although seizure models using systemic administration of the chemoconvulsant pentylenetetrazol (PTZ) for induction of generalized clonic seizures in rodents are widely employed to identify potential anticonvulsants, the important role of diverse technical, biological and pharmacological factors in interpretation of results obtained with these models is often not recognized. The aim of this study was to delineate factors other than sex, age, diet, climate, and circadian rhythms, which are generally known. For this purpose, experiments with 8 clinically established antiepileptic drugs were undertaken in the following PTZ models: (1) the threshold for different types of PTZ seizures, i.e., initial myoclonic twitch, generalized clonus with loss of righting reflexes, and tonic backward extension of forelimbs (forelimb tonus), in mice; (2) the traditional PTZ seizure test with s.c. injection of the CD97 for generalized clonic seizures in mice; and (3) the s.c. PTZ seizure test in rats. In rats, in addition to evaluating drug effects on generalized clonic seizures, a ranking system was used to determine drug effects on other seizure types. When drugs were dissolved in vehicles which themselves did not exert effects on seizure susceptibility, the most important factors which influenced drug potencies were: (1) bishaped dose-response curves, i.e., a decline in anticonvulsant dose-response at high doses of some drugs, leading to misinterpretations of drug efficacy if only a single high drug dosage is tested; (2) effects of route of PTZ administration (i.v. infusion vs. s.c. injection) on estimation of anticonvulsant potency; (3) species differences in drug metabolism; (4) differences in drug potencies calculated on the basis of administered doses compared to potency calculations based on 'active' drug concentrations in plasma; (5) qualitative and quantitative species differences in drug actions; (6) endpoints used for PTZ tests; (7) misleading predictions from PTZ seizure models. Analysis of anticonvulsant drug actions indicated that myoclonic or clonic seizures induced by i.v. or s.c. PTZ might be suitable for predicting efficacy against myoclonic petit mal seizures in humans, but certainly not to predict efficacy against absence seizures. Tonic seizures induced by PTZ were blocked by drugs, such as ethosuximide, which exert no effect on tonic seizures in humans. In order to reduce the variability among estimates of anticonvulsant activity in PTZ seizure models, the various factors delineated in this study should be rigidly controlled in experimental situations involving assay of anticonvulsant agents.     

Modulatory effects of cannabinoids on brain neurotransmission

Recreational and chronic cannabis use has been associated with a range of acute and chronic effects including; anti‐nociceptive actions, anxiety, depression, psychotic symptoms and neurocognitive impairments. The mechanisms underlying cannabinoid‐based drugs effects are not fully known but given the neuro‐modulatory functions of the endocannabinoid system, it seems likely that agonistic activity at the cannabinoid type‐1 receptors (CB₁) might modulate the functions of other neurotransmitter systems. The present review has summarized the currently available pre‐clinical and clinical data on the interactions of CB₁ and cannabinoid type‐2 receptors (CB₂) with the central neurotransmitters; dopamine, serotonin, noradrenaline, GABA, glutamate and opioids. Acute and chronic exposures to cannabinoids exert pharmacological alterations in the mammalian brain that have profound implications for our understanding of the neuropharmacology of cannabinoid‐based drugs and their effects on mental health and the brain. A recent emergence uses of cannabis for medical purpose together with legalization and decriminalization of cannabis and increasing use of highly potent synthetic cannabinoids raise a growing concern over the effects of cannabinoids and their interaction with other neurotransmitters on physical and mental health.     

Therapeutic effects of cannabinoids in animal models of seizures,epilepsy, epileptogenesis,and epilepsy-related neuroprotection

The isolation and identification of the discrete plant cannabinoids in marijuana revived interest in analyzing historical therapeutic claims made for cannabis in clinical case studies and anecdotes. In particular, sources as old as the 11th and 15th centuries claimed efficacy for crude marijuana extracts in the treatment of convulsive disorders, prompting a particularly active area of preclinical research into the therapeutic potential of plant cannabinoids in epilepsy. Since that time, a large body of literature has accumulated describing the effects of several of the > 100 individual plant cannabinoids in preclinical models of seizures, epilepsy, epileptogenesis, and epilepsy-related neuroprotection.We surveyed the literature for relevant reports of such plant cannabinoid effects and critically reviewed their findings. We found that acute CB₁R agonism in simple models of acute seizures in rodents typically produces anti-convulsant effects whereas CB₁R antagonists exert converse effects in the same models. However, when the effects of such ligands are examined in more complex models of epilepsy, epileptogenesis and neuroprotection, a less simplistic narrative emerges. Here, the complex interactions between (i) brain regions involved in a given model, (ii) relative contributions of endocannabinoid signaling to modulation of synaptic transmission in such areas, (iii) multi-target effects, (iv) cannabinoid type 1 and type 2 receptor signaling interactions and, (v) timing, (vi) duration and (vii) localization of ligand administration suggest that there is both anti-epileptic therapeutic potential and a pro-epileptic risk in up- and down-regulation of endocannabinoid signaling in the central nervous system. Factors such receptor desensitization and specific pharmacology of ligands used (e.g. full vs partial agonists and neutral antagonists vs inverse agonists) also appear to play an important role in the effects reported. Furthermore, the effects of several plant cannabinoids, most notably cannabidiol (CBD) and cannabidavarin (CBDV), in models of seizures, epilepsy, epileptogenesis, and neuroprotection are less ambiguous, and consistent with reports of therapeutically beneficial effects of these compounds in clinical studies. However, continued paucity of firm information regarding the therapeutic molecular mechanism of CBD/CBDV highlights the continued need for research in this area in order to identify as yet under-exploited targets for drug development and raise our understanding of treatment-resistant epilepsies.The recent reporting of positive results for cannabidiol treatment in two Phase III clinical trials in treatment-resistant epilepsies provides pivotal evidence of clinical efficacy for one plant cannabinoid in epilepsy. Moreover, risks and/or benefits associated with the use of unlicensed Δ⁹-THC containing marijuana extracts in pediatric epilepsies remain poorly understood. Therefore, in light of these paradigm-changing clinical events, the present review's findings aim to drive future drug development for newly-identified targets and indications, identify important limitations of animal models in the investigation of plant cannabinoid effects in the epilepsies, and focuses future research in this area on specific, unanswered questions regarding the complexities of endocannabinoid signaling in epilepsy.This article is part of a Special Issue titled Cannabinoids and Epilepsy.     

Involvement of transient receptor potential vanilloid type 1 channels in the pro-convulsant effect of anandamide in pentylenetetrazole-induced seizures

Anandamide, an endogenous agonist of CB(1) receptors, also activates TRPV1 but at a higher concentration. Studies demonstrate the anticonvulsant activity of anandamide via CB(1) receptors, while its action through TRPV1 is still ambiguous. Thus, the present study investigated the influence of anandamide on pentylenetetrazole-induced seizures in mice pretreated with TRPV1 or CB(1) receptor antagonists. Acute intracerebroventricular administration of low doses of anandamide (10, 20, or 40μg/mouse) produced anticonvulsant effect, while the pro-convulsant effect was evident at high doses (80 or 100μg/mouse). Interestingly, AM251 (2μg/mouse), a CB(1) antagonist pretreatment blocked the anticonvulsant effect, but augmented the pro-convulsant effect. Conversely, in the presence of inactive dose of capsazepine (1μg/mouse), a TRPV1 antagonist, anandamide exhibited significant anticonvulsant effect even at high doses with no change in its anticonvulsant effect. Moreover, mice treated with capsaicin, a TRPV1 agonist (10, or 100μg/mouse) exhibited pro-convulsant activity that was blocked by capsazepine pretreatment. However, capsazepine, per se at doses 10 or 100μg/mouse exhibited anticonvulsant effect. Like anandamide, the agents (AM404 and URB597), which increase its synaptic concentrations produced similar biphasic effects. Thus, these results indicate that anandamide exhibits both pro- and anticonvulsant activities by activating TRPV1 and CB(1) receptor respectively.     

Endocannabinoid modulation of jejunal afferent responses to LPS

Background Endocannabinoids influence immune function and nociceptive signaling. This study examines cannabinoid modulation of sensory signaling from the GI tract following an acute inflammatory response triggered by systemic administration of bacterial lipopolysaccharide (LPS). Methods A segment of proximal jejunum was intubated, to measure intraluminal pressure, in anesthetized rats. Afferent impulse traffic was recorded from a single isolated paravascular nerve bundle supplying the jejunal loop. Drugs and LPS were administered intravenously and changes in afferent firing were determined. Key Results The non‐selective cannabinoid agonist, WIN55,212‐2 (1 mg kg^?1 i.v.) and the anandamide transport inhibitor, VDM11 (1 mg kg^?1 i.v.) but not the fatty acid amide hydrolase (FAAH) inhibitor, URB597 (0.3 mg kg^?1) caused a significant increase in afferent activity. The WIN55,212‐2‐induced afferent response was mediated by activation of CB₁ receptors whereas the VDM11 response was mediated by both CB₁ and CB₂ receptor mechanisms. LPS (10 mg kg^?1) evoked an increase in afferent activity which was significantly reduced in the presence of WIN55,212‐2 and VDM11 but not URB597. The inhibitory effect of WIN55,212‐2 was prevented by CB₁ but not CB₂ receptor antagonism. In contrast, the inhibitory effect of VDM11 remained unaltered after CB₁ or CB₂ receptor blockade. Conclusions & Inferences Endocannabinoids play a role in modulating afferent signaling and may represent a target for the treatment of visceral hypersensitivity. In contrast to the effects of blocking endocannabinoid uptake (VDM11), inhibiting breakdown of endocannabinoids (URB597) had no effect on baseline or LPS induced afferent firing. Therefore, uptake of cannabinoids rather than breakdown via FAAH terminates their action in the GI tract.     

Differential regulation of NMDAR and NMDAR‐mediated metaplasticity by anandamide and 2‐AG in the hippocampus

Endocannabinoids (eCBs), including AEA and 2‐AG, are endogenous signaling mediators involved in many physiological and pathological events. The G protein‐coupled cannabinoid receptor 1 (CB₁R) is an important target for eCBs, however, additional non‐CB₁ receptor targets have also been identified. Although recent evidence suggests that NMDA receptor function may be regulated by eCBs, the underlying mechanisms remain poorly characterized. Using acutely isolated CA1 neurons and slices from the hippocampus, we found that both AEA and 2‐AG potentiate NMDAR‐mediated currents independently of CB₁ receptors (CB₁Rs) and via distinct signaling pathways. Potentiation by AEA requires the activation of TRPV1 channels. In contrast, potentiation by 2‐AG requires the sequential activation of PKC and Src. Additionally, in hippocampal slices, we found that both AEA and 2‐AG induce NMDAR‐mediated metaplasticity and facilitate the induction of subsequent LTD independently of CB₁Rs. Enhanced LTD by AEA, but not 2‐AG, was dependent on TRPV1 channels. Our findings reveal previously unrecognized non‐CB₁R‐dependent signaling cascades through which the two major eCBs regulate NMDA receptor function and consequently synaptic plasticity. © 2014 Wiley Periodicals, Inc.     

Inhibitor effect of dexketoprofen in rat model of pentylenetetrazol-induced seizures

Objectives: The relationship between epilepsy and inflammation is known, and it has been reported that there is an increase in cyclooxygenase (COX) levels in epilepsy. We aim to reveal the anticonvulsant effects of dexketoprofen in pentylenetetrazol (PTZ)-induced seizures in rats.

Materials and Methods: Forty-eight male Sprague-Dawley rats, 24 of them for EEG recording and 24 of them are for behavioral studies, were randomly divided in two groups: Group A for EEG recordings and Group B for behavioral assessment. A weight of 70 mg/kg PTZ was used for behavioral studies after dexketoprofen administration. Thirty-five milligrams per kilogram PTZ were used for EEG recording after dexketoprofen administration. The electrodes were implanted on dura over the left frontal cortex and the reference electrode was implanted over the cerebellum for EEG recording. The Racine convulsion scale (RCS), first myoclonic jerk (FMJ) onset time, and spike percentages were evaluated between the two groups.

Results: There was a significant (P< 0·05) difference between the RCS, FMJ onset time (P< 0·001), and spike percentage (P< 0·05) between the groups (Group 2 compared with Groups 3 and 4).

Conclusion: Dexketoprofen has an antiepileptic feature and this effect increases as the dosage increases, however it is currently unknown through which mechanism this drug shows its anticonvulsant effect. Dexketoprofen, in the group of NSAIDs, shows an anticonvulsant effect on PTZ-induced epilepsy model. This study suggests that dexketoprofen can preferably be used with NSAIDs for epileptic patients in clinical practice.     

Postnatal caffeine treatment affects differently two pentylenetetrazol seizure models in rats

Effects of repeated postnatal administration of caffeine (10 and 20 mg/kg s.c. daily from P7 to P11) were studied in two models of epileptic seizures characterized by spike-and-wave EEG rhythm in 18- and 25-day-old rats. Rhythmic metrazol activity (RMA, model of human absences) was induced by low dose of pentylenetetrazol (PTZ, 20 mg/kg or 40 mg/kg, i.p.) and minimal clonic seizures (model of human myoclonic seizures) by two successive doses of PTZ (20 and 40 mg/kg i.p.). Early postnatal caffeine treatment resulted in significant changes of RMA only in 18-day-old rats. Anticonvulsant effects were observed in RMA episodes elicited by the 20-mg/kg dose of PTZ in both caffeine groups whereas latency of RMA episodes induced by the 40-mg/kg dose was shortened and their duration was prolonged. No changes were found in 25-day-old animals. Incidence, EEG and motor pattern of minimal clonic seizures were not changed. Some animals in both control age groups exhibited transition to generalized tonic–clonic seizures. This type of seizures never appeared in caffeine-treated 25-day-old animals. Mixed effects of postnatal caffeine exposure were demonstrated; these predominantly anticonvulsant effects are age- and model-specific.     

Anticonvulsant and antiepileptogenic effects of GABAA receptor ligands in pentylenetetrazole-kindled mice

Although animal models based on pentylenetetrazole (PTZ) are widely used, the mechanism by which PTZ elicits its action is not very well understood. At the molecular level, a generally accepted mechanism of PTZ is noncompetitive antagonism of the gamma-aminobutyric acid (GABA)(A) receptor complex. By a systematic pharmacological investigation of various GABA(A) receptor ligands, our aim was to gain a better understanding of the GABAergic mechanisms involved in different PTZ-induced seizures. We investigated anticonvulsant effects of various specific GABA(A) receptor ligands, which are believed to bind to different binding sites on the GABA(A) receptor complex, on PTZ-induced clonic seizures in drug naive and PTZ-kindled mice as well as their effects on the development of PTZ kindling. Diazepam and alphaxalone produced potent anticonvulsant effects and completely suppressed the development of kindling. In contrast, the antagonists bicuculline and dehydroepiandrosterone sulfate (DHEAS) displayed neither anticonvulsant nor antiepileptogenic effects. Flumazenil, often used as a reference antagonist at the GABA(A) receptor benzodiazepine (BZ) binding site, lacked anticonvulsant effects but surprisingly inhibited the development of PTZ-kindled seizures. The agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP) was devoid of both anticonvulsant and antiepileptogenic effects. Marked differences in drug sensitivity were observed between models based on single and chronic administration of PTZ showing that the two sets of models are fundamentally different. These results describe the pharmacology of a set of ligands believed to bind to different sites at the GABA(A) receptor complex in animal models based on PTZ and demonstrate that a drug's action in these models cannot be readily explained by agonistic or antagonistic properties at the receptor level.     

Anticonvulsant activity of BmK AS, a sodium channel site 4-specific modulator

The anticonvulsant activity of BmK AS, a sodium channel site 4-selective modulator purified from scorpion venom (Buthus martensi Karsch), was investigated in unanesthetized rats with acute pentylenetetrazole (PTZ)- and pilocarpine-induced seizures. Rats were microinjected in the CA1 region with either saline or BmK AS, followed by epileptogenic doses of PTZ or pilocarpine 30 minutes later. The anticonvulsant efficacy of BmK AS in PTZ- or pilocarpine-evoked seizure-like behavior and cortical epileptiform EEG activity was assessed. Intrahippocampal injections of BmK AS (0.05-1 μg in 1 μL) produced dose-dependent anticonvulsant activity in the PTZ model, suppressing seizure-associated behavior and reducing both the number and duration of high-amplitude, high-frequency discharges (HAFDs) on the EEG. In contrast, BmK AS did not affect the epileptiform EEG in the pilocarpine model over the same dose range, although it did increase the latency to status epilepticus onset and slightly, but significantly, reduced the seizure score. In summary, our results demonstrate that the sodium channel site 4-selective modulator BmK AS is an effective inhibitor of PTZ- but not pilocarpine-induced acute seizures. These results indicate that BmK AS may serve as a novel probe in exploring the role of different sodium channel subtypes in an epileptogenic setting and as a potential lead in developing antiepileptic drugs specifically for the therapy of sodium channel site 4-related epilepsy.     

EP2 receptor agonist ONO-AE1-259-01 attenuates pentylenetetrazole- and pilocarpine-induced seizures but causes hippocampal neurotoxicity

Epilepsy is a common and devastating neurological disease affecting more than 50 million people worldwide. Accumulating experimental and clinical evidence suggests that inflammatory pathways contribute to the development of seizures in various forms of epilepsy. In this context, while the activation of the PGE₂ EP2 receptor causes early neuroprotective and late neurotoxic effects, the role of EP2 receptor in seizures remains unclear. We investigated whether the systemic administration of the highly selective EP2 agonist ONO-AE1-259-01 prevented acute pentylenetetrazole (PTZ)- and pilocarpine-induced seizures. The effect of ONO-AE1-259-01 on cell death in the hippocampal formation of adult male mice seven days after pilocarpine-induced status epilepticus (SE) was also evaluated. ONO-AE1-259-01 (10 μg/kg, s.c.) attenuated PTZ- and pilocarpine-induced seizures, evidenced by the increased latency to seizures, decreased number and duration of seizures episodes and decreased mean amplitude of electrographic seizures. ONO-AE1-259-01 and pilocarpine alone significantly increased the number of pyknotic cells per se in all hippocampal subfields. The EP2 agonist also additively increased pilocarpine-induced pyknosis in the pyramidal cell layer of CA1 but reduced pilocarpine-induced pyknosis in the granule cell layer of the dentate gyrus (DG). Although the systemic administration of ONO-AE1-259-01 caused a significant anticonvulsant effect in our assays, this EP2 agonist caused extensive cell death. These findings limit the likelihood of EP2 receptor agonists being considered as novel potential anticonvulsant drugs.