Pentylenetetrazole Seizure Models Research Articles

Acute administration of docosahexaenoic acid increases resistance to pentylenetetrazol-induced seizures in rats

Objective Docosahexaenoic acid (DHA), an omega-3 fatty acid, has been reported to raise seizure thresholds. The purpose of the present study was to test the acute anticonvulsant effects of unesterified DHA in rats, using the maximal pentylenetetrazol (PTZ) seizure model, and also to examine DHA incorporation and distribution into blood serum total lipids and brain phospholipids and unesterified fatty acids. Sedation was measured to monitor for the potential toxicity of DHA. Methods Male Wistar rats received subcutaneous injections of saline, oleic acid (OA), or DHA. An initial pilot study (Experiment 1) established 400 mg/kg as an effective dose of DHA in the maximal PTZ seizure test. A subsequent time–response study, using 400 mg/kg (Experiment 2), established 1 hour as an effective postinjection interval for administering DHA subcutaneously. A final study (Experiment 3) comprised two different groups. The first group (“seizure-tested rats”) received saline, OA, or DHA (400 mg/kg) subcutaneously, and were seizure tested in the maximal PTZ test 1 hour later to confirm the seizure latency measurements at that time. The second group (“assay rats”) received identical subcutaneous injections of saline, OA, or DHA (400 mg/kg). One hour postinjection, however, they were sacrificed for assay rather than being seizure tested. Assays involved the analysis of serum and brain DHA. Sedation was measured in both Experiment 3 groups during the 1-hour period prior to seizure testing or sacrifice. Results As noted above, 400 mg/kg proved to be an effective subcutaneous dose of DHA (Experiment 1), and 1 hour proved to be the most effective injection–test interval (Experiment 2). In Experiment 3, in the seizure-tested animals, subcutaneous administration of 400 mg/kg of DHA significantly increased latency to PTZ seizure onset 1 hour postinjection relative to the saline- and OA-injected controls, which did not differ significantly from each other ( P > 0.05). In the assay animals, no significant effects of treatment on blood serum total lipids or on brain phospholipid or unesterified fatty acid profiles ( P > 0.05) were observed. There were also no differences in sedation among the three groups ( P > 0.05). Conclusion DHA increases resistance to PTZ-induced seizures without altering measures of sedation and, apparently, without changing DHA concentrations in serum or brain.

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 effects of the BK‐channel antagonist paxilline

Mutations that enhance currents through the Ca(2+)- and voltage-gated K(+) channel BK (Slo, maxiK, KCNMA1) have been associated with seizure disorders in both rodent models and humans. Previously we have found that seizures themselves induce a gain-of-function in BK channels that is associated with elevated excitability in neocortical neurons. In this study, we sought to examine whether administration of BK-channel antagonists possess anticonvulsant activity in vivo. Seizures were induced in animals by intraperitoneal (i.p.) injection of the gamma-aminobutyric acid (GABA)(A) antagonists picrotoxin or pentylenetetrazole. Twenty-four hours following induction of the initial seizure episode, animals were reinjected with chemoconvulsant in the presence of the BK-channel antagonist paxilline or saline. The presence and duration of tonic-clonic seizures were evaluated. Intraperitoneal injection of paxilline was sufficient to eliminate tonic-clonic seizures in picrotoxin-treated animals. Paxilline reduced seizure duration and intensity in pentylenetetrazole-injected animals. The BK-channel antagonist paxilline possesses significant anticonvulsant activity in both picrotoxin and pentylenetetrazole seizure models, an effect that may be related to the seizure-dependent gain-of-function in BK channel previously observed in neocortical neurons in vitro.

Developing Novel Antiepileptic Drugs: Characterization of NAX 5055, a Systemically-Active Galanin Analog, in Epilepsy Models

The endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs, NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active after intravenous, intraperitoneal, and subcutaneous administration, and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved antiepileptic drugs.

Dose‐dependent anticonvulsant effects of linoleic and α‐linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats

Linoleic and alpha-linolenic polyunsaturated fatty acids, derived from plant oils, have been reported to reduce neuronal excitability ex vivo and in cell culture. The evidence derived from animal seizure models, however, has been contradictory. The goal of the present study was to assess the dose-dependent anticonvulsant effects of a fatty acid mixture containing linoleic and alpha-linolenic acids in a 4 to 1 ratio (the "SR-3" compound). The maximal pentylenetetrazol seizure model and Long-Evans hooded rats were used. Daily intraperitoneal injection of SR-3 for 21 consecutive days raised omega-3 polyunsaturated fatty acid (n-3 PUFA) composition in the unesterified fatty acid fraction of brain lipids (p < 0.05), and increased latency to seizure onset when administered at 200 mg/kg (p < 0.05), but not at 40 mg/kg (p > 0.05). There were no significant effects of SR-3 on seizure occurrence or on seizure severity (p > 0.05). A toxic effect of the SR-3 compound on peristalsis was observed at a dose of 400 mg/kg and above. Linoleic and alpha-linolenic polyunsaturated fatty acids in a 4 to 1 ratio raises n-3 PUFA composition of unesterified fatty acids in the brain and increases resistance to pentylenetetrazol-induced seizures.

Deep brain stimulation of the posterior hypothalamus activates the histaminergic system to exert antiepileptic effect in rat pentylenetetrazol model

Deep brain stimulation (DBS) is a promising therapy for intractable epilepsy, yet the optimum target and underlying mechanism remain controversial. We used the rat pentylenetetrazol (PTZ) seizure model to evaluate the effectiveness of DBS to three targets: two known to be critical for arousal, the histaminergic tuberomammillary nucleus (TMN) and the orexin/hypocretinergic perifornical area (PFN), and the anterior thalamic nuclei (ATH) now in clinical trial. TMN stimulation provided the strong protection against the seizure, and PFN stimulation elicited a moderate effect yet accompanying abnormal behavior in 25% subjects, while ATH stimulation aggravated the seizure. Power density analysis showed EEG desynchronization after DBS on TMN and PFN, while DBS on ATH caused no effect with the same stimulation intensity. EEG desynchronization after TMN stimulation was inhibited in a dose-dependent manner by pyrilamine, a histamine H 1 receptor selective antagonist, while the effect of PFN stimulation was inhibited even at a low dose. In parallel, in vivo microdialysis revealed a prominent increase of histamine release in the frontal cortex after TMN stimulation, a moderate level with PFN and none with ATH. Furthermore, antiepileptic effect of DBS to TMN was also blocked by an H 1 receptor antagonist. This study clearly indicates that EEG desynchronization and the activation of the histaminergic system contributed to the antiepileptic effects caused by DBS to the posterior hypothalamus.

Lack of benefit of linoleic and α-linolenic polyunsaturated fatty acids on seizure latency, duration, severity or incidence in rats

Background Polyunsaturated fatty acids have been reported to increase seizure threshold and to reduce seizure duration and severity in rats. Objective The purpose of the present study was to test the anticonvulsant effects of an essential fatty acid mixture containing linoleic and α-linolenic acids at a 4:1 ratio (SR-3 compound), using the pentylenetetrazol seizure model in Long-Evans hooded rats. Results There were no significant effects of SR-3 on seizure latency, duration or severity ( P > 0.05). There were also no significant differences in the incidence of myoclonic jerks, forelimb and hindlimb clonus, forelimb and hindlimb tonus or running fits in rats that received SR-3, as compared to control rats ( P > 0.05). Conclusion Linoleic and α-linolenic polyunsaturated fatty acids have no beneficial effects on seizure latency, duration, average severity or incidence.

TPA023 [7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine], an Agonist Selective for α2- and α3-Containing GABAAReceptors, Is a Nonsedating Anxiolytic in Rodents and Primates

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has partial agonist efficacy at the alpha2 and alpha3 subtypes and essentially antagonist efficacy at the alpha1 and alpha5 subtypes. In rats, TPA023 gave time- and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-beta-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel alpha2/alpha3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.

The antihyperalgesic effects of the T-type calcium channel blockers ethosuximide, trimethadione, and mibefradil

The purpose of the present study was to explore the analgesic effects of the low voltage-activated T-type Ca 2+ channel blockers ethosuximide, trimethadione, and mibefradil in persistent and acute nociceptive tests. The anticonvulsant effects of the compounds were also determined in the intravenous pentylenetetrazol seizure model. Following intraperitoneal administration, ethosuximide and trimethadione dose-dependently reversed capsaicin-induced mechanical hyperalgesia. Similarly, the highest dose of mibefradil tested (30 μg, intracisternal) reversed capsaicin-induced mechanical hyperalgesia. Ethosuximide and mibefradil produced statistically significant analgesic effects in both early and late phase formalin-induced behaviors and trimethadione reduced late phase behaviors. Additionally, ethosuximide and trimethadione produced antinociceptive effects in the rat-tail flick reflex test. In contrast, following intracisternal administration, mibefradil had no effect in the tail flick reflex test. In addition, the anticonvulsants ethosuximide and trimethadione increased the doses of pentylenetetrazol required to produce both first twitch and clonic seizures. In contrast however, mibefradil had no anticonvulsant effect. The present results demonstrate that the clinically used anticonvulsants ethosuximide and trimethadione provide analgesic effects at doses, which are anticonvulsant. Furthermore, the data further supports the idea that T-type Ca 2+ channels may be important targets for treating persistent pain syndromes.

Allopregnanolone analogs that positively modulate GABA receptors protect against partial seizures induced by 6-Hz electrical stimulation in mice.

Low-frequency (6 Hz), long-duration (3 s) electrical stimulation in mice produces seizures characterized by immobility, focal clonus, and automatic behaviors reminiscent of human limbic epilepsy. Renewed interest has been expressed in this seizure model with the recognition that it is sensitive to a broad spectrum of anticonvulsants (AEDs) and may have distinct pharmacologic responsiveness from other in vivo tests of AED efficacy. Here we sought to determine whether the progesterone-derived neuroactive steroid allopregnanolone (5alpha,3alpha-P) and several structural analogues with varying degrees of activity as positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors are protective in the 6-Hz seizure model. Mice were pretreated with neuroactive steroids (15 min before) or clonazepam (CZP; 30 min before) to 6-Hz corneal stimulation (32 mA). Animals that failed to exhibit immobility were considered protected. The neuroactive steroids prevented 6-Hz seizures with rank order of potencies (ED50 values): ganaxolone (6.3 mg/kg) > 5alpha,3alpha-P (14.2 mg/kg) > or = 5beta,3alpha-P (14.4 mg/kg) > 5alpha,3beta-P (>100 mg/kg). CZP also was protective (ED(50) value, 0.075 mg/kg). The potencies of the neuroactive steroids and CZP are similar to their previously reported activities in the pentylenetetrazol (PTZ) seizure model. Neuroactive steroids have comparable potencies in the 6-Hz and PTZ models. Their structural specificity in both models corresponds with their activities as positive allosteric modulators of GABAA receptors, although ganaxolone is more potent than expected, probably because it has greater bioavailability. The 6-Hz model may be a valuable tool in drug development for the identification of GABAergic AEDs.

Microwave synthesis and anticonvulsant activity of 2‐benzyl‐1(2H)‐phthalazinones

AbstractMicrowave irradiation of 1(2H)‐phthalazinone with benzyl halides and potassium carbonate in dimethyl‐formamide afforded 2‐benzyl‐1(2H)phthalazinones in modest yields. These products were tested in mice and rats in maximal electroshock and pentylenetetrazole seizure models for anticonvulsant activity, and in the rotorod test for neurotoxicity. A majority of the compounds exhibited anticonvulsant protection. The p‐aminobenzyl analog 1f was the most active compound in the maximal electroshock assay; it's ED50 value was 19.46 mg/kg and the TD50 was 61.35 mg/kg. In the rat, the values were 3.71 mg/kg and &gt;125 mg/kg, respectively.

Fos in locus coeruleus neurons following audiogenic seizure in the genetically epilepsy-prone rat: comparison to electroshock and pentylenetetrazol seizure models

Seizures in genetically epilepsy-prone rats (GEPRs) may result from hypoactivity of locus coeruleus (LC) neurons during seizures. This study examined Fos-like-immunoreactivity (FLI) in the LC following audiogenic seizures in two strains of GEPRs (GEPR-9s and -3s), and following pentylenetetrazol (PTZ) or maximal electroshock seizures (MES) in normal rats. After tonic seizure, GEPR-9s showed an identical LC-FLI response to that of normal rats following tonic seizures induced by either PTZ or MES. GEPR-3s, having clonic seizures, had less FLI in the LC. Therefore, stimulus-transcription coupling in the GEPR LC is apparently normo-typic in its FLI response to seizure and thus is not likely the root cause of NE abnormalities in this seizure model.

Microwave synthesis and anticonvulsant activity of new 3‐benzyl‐1,2,3‐benzotriazin‐4(3H)‐ones

AbstractSeveral 3‐benzyl‐1,2,3‐benzotriazin‐4(3H)‐ones 1 were prepared by alkylation of 1,2,3‐benzotriazin‐4(3H)‐one with benzyl halides in dimethylformamide in a microwave oven in moderate yields. Accompanying 1 were minor amounts of products believed to be the O‐alkylated derivatives. Support for 3‐benzylation is shown by an alternate synthesis of la from o‐amino‐N‐benzylbenzamide by nitrous acid cyclization. The title compounds were evaluated in mice and rats in maximal electroshock (MES) and pentylenetetrazole (scMet) seizure models for anticonvulsant activity, and in the rotorod test for neurotoxi‐city. They were generally non‐toxic. The 3‐benzyl analog was the most active (maximal electroshock) compound; it's maximal electroshock ED50 value was 93 mg/kg (mouse).

Anticonvulsant Pharmacodynamics and Disposition of Triazolam in Rats

Triazolam (TZ) is a triazolobenzodiazepine used in the treatment of insomnia that possesses significant anticonvulsant properties. Despite the widespread use of this drug, detailed pharmacokinetic–pharmacodynamic information is lacking, especially with respect to inhibition of seizure activity. TZ disposition has been described previously by methods with limited specificity, and the concentration–anticonvulsant effect relationship has not been characterized. The current studies were undertaken to examine TZ disposition with a specific HPLC method, and to evaluate the relationship between anticonvulsant effect and concentration in Sprague-Dawley rats. TZ pharmacokinetics were characterized after bolus or infusion administration; in a separate experiment, TZ pharmacodynamics were assessed with pentylenetetrazol-induced seizures. The systemic disposition of TZ could be described with a two-compartment model; systemic clearance ranged from 2.45 to 5.30L/h/ kg, steady-state volume of distribution ranged from 2.10 to 4.02L/kg, and mean residence time ranged from 47 to 65min. The concentration–effect relationship was well described by a simple Emax model: Emax, expressed as the ratio of post-TZ to pre-TZ threshold convulsant doses of pentylenetetrazol, was 9.9±0.7, and the EC50 values were 10.0±4.6ng/mL and 34.8±9.0ng/g in serum and whole brain tissue, respectively. Under single-dose conditions, TZ is a very potent anticonvulsant in the rat pentylenetetrazol seizure model.

The early identification of anticonvulsant activity: role of the maximal electroshock and subcutaneous pentylenetetrazol seizure models.

A number of widely different animal seizure models have been employed in the search for new and novel anticonvulsant drugs useful for the treatment of human epilepsy. At present, no single laboratory test will, in itself, establish the presence or absence of anticonvulsant activity or fully predict the clinical potential of a test substance. Of the many available animal models, the maximal electroshock (MES) and subcutaneous pentylenetetrazol (scPTZ) tests still represent the most commonly employed models for the routine screening and identification of new anticonvulsant drugs. This chapter will briefly describe how these two tests are conducted, their limitations and how they have contributed in the past and to the present day anticonvulsant drug discovery process.

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. (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. (2) the traditional PTZ seizure test with s.c. injection of the CD 97 for generalized clonic seizures in mice; 3. (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. (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. (2) effects of route of PTZ administration (i.v. infusion vs. s.c. injection) on estimation of anticonvulsant potency; 3. (3) species differences in drug metabolism; 4. (4) differences in drug potencies calculated on the basis of administered doses compared to potency calculations based on ‘active’ drug concentrations in plasma; 5. (5) qualitative and quantitative species differences in drug actions; 6. (6) endpoints used for PTZ tests; 7. (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.

Intraosseous diazepam suppression of pentylenetetrazol-induced epileptogenic activity in pigs

Intravenous access for the administration of antiepileptic drugs can be both time-consuming and difficult in an actively seizing infant. We conducted a study to examine the intraosseous route as an alternate means of vascular access for the administration of diazepam in a pentylenetetrazol-seizure model in pigs. Epileptogenic activity was induced with pentylenetetrazol 100 mg/kg in 15 domestic swine that had undergone craniotomies for electrocortical recording. Diazepam (0.1 mg/kg) was administered IV (n = 5) or intraosseously (n = 5); control animals received no drug (n = 5). Epileptogenic activity was suppressed below control levels within one minute in the IV group and within two minutes in the intraosseous group. A two-way analysis of variance did not show a significant difference between the IV and intraosseous routes; however, both were significantly different when compared to the control. There also was no significant difference in plasma diazepam levels between the two groups at one, two, five, ten, 15, and 20 minutes. Our study demonstrated that the intraosseous route is a rapid and effective alternative for diazepam administration.