Seizures induced by pentylenetetrazole in the adult zebrafish: a detailed behavioral characterization.

Pentylenetetrazole (PTZ) is a common convulsant agent used in animal models to investigate the mechanisms of seizures. Although adult zebrafish have been recently used to study epileptic seizures, a thorough characterization of the PTZ-induced seizures in this animal model is missing. The goal of this study was to perform a detailed temporal behavior profile characterization of PTZ-induced seizure in adult zebrafish. The behavioral profile during 20 min of PTZ immersion (5, 7.5, 10, and 15 mM) was characterized by stages defined as scores: (0) short swim, (1) increased swimming activity and high frequency of opercular movement, (2) erratic movements, (3) circular movements, (4) clonic seizure-like behavior, (5) fall to the bottom of the tank and tonic seizure-like behavior, (6) death. Animals exposed to distinct PTZ concentrations presented different seizure profiles, intensities and latencies to reach all scores. Only animals immersed into 15 mM PTZ showed an increased time to return to the normal behavior (score 0), after exposure. Total mortality rate at 10 and 15 mM were 33% and 50%, respectively. Considering all behavioral parameters, 5, 7.5, 10, and 15 mM PTZ, induced seizures with low, intermediate, and high severity, respectively. Pretreatment with diazepam (DZP) significantly attenuated seizure severity. Finally, the brain PTZ levels in adult zebrafish immersed into the chemoconvulsant solution at 5 and 10 mM were comparable to those described for the rodent model, with a peak after a 20-min of exposure. The PTZ brain levels observed after 2.5-min PTZ exposure and after 60-min removal from exposure were similar. Altogether, our results showed a detailed temporal behavioral characterization of a PTZ epileptic seizure model in adult zebrafish. These behavioral analyses and the simple method for PTZ quantification could be considered as important tools for future investigations and translational research.

Determination of stimulants using gas chromatography/high-resolution time-of-flight mass spectrometry and a soft ionization source.

RATIONALE: The aim of this study was to investigate the mass spectral fragmentation of a small set of stimulants in a high-resolution time-of-flight mass spectrometer equipped with a soft ionization source using vacuum ultraviolet (VUV) photons emitted from different plasma gases. It was postulated that the use of a plasma gas such as Xe, which emits photons at a lower energy than Kr or Ar, would lead to softer ionization of the test compounds, and thus to less fragmentation. METHODS: A set of nine stimulants: cocaine, codeine, nicotine, methadone, phenmetrazine, pentylenetetrazole, niketamide, fencamfamine, and caffeine, was analyzed by gas chromatography/time-of-flight mass spectrometry (GC/TOFMS) in positive ion mode with this soft ionization source, using either Xe, Kr, or Ar as plasma gases. Working solutions of the test compounds at 0.1 to 100 ng/µL were used to establish instrument sensitivity and linearity. RESULTS: All test compounds, except methadone and pentylenetetrazole, exhibited strong molecular ions and no fragmentation with Xe-microplasma photoionization (MPPI). Methadone exhibited significant fragmentation not only with Xe, but also with Kr and Ar, and pentylenetetrazole could not be ionized with Xe, probably because its ionization energy is above 8.44 eV. The Kr- and Ar-MPPI mass spectra of the test compounds showed that the relative intensity of the molecular ion decreased as the photon energy increased. CONCLUSIONS: When coupled to a TOF mass spectrometer this soft ionization source has demonstrated signal-to-noise (S/N) ratios from 7 to 730 at 100 pg per injection (depending on the compound), and a dynamic range of three orders of magnitude (100 pg to 100 ng) for some of the test compounds.

Effect of age on pentylenetetrazol-kindling and kindling-induced impairments of learning performance.

Epileptogenesis during ontogeny may not be linearly related to time. It is known that the behavioral manifestations of seizures are age-dependent, but more research is needed to clarify ontogenetic aspects of epilepsies and related alterations, including cognitive deficits. Kindling is an accepted animal model for the study of the convulsive component of epilepsy and its consequences on behavior. Recently, we demonstrated an impairment in acquisition of a conditioned reaction in young adult kindled rats, using pentylenetetrazol (PTZ) as the kindling stimulus. The present study was undertaken to investigate the dependence on age of alterations in the induction of PTZ kindling in rats. We started the kindling protocol in 4-, 6-, and 8-week- and 6-, 12-, 18-, and 24-month-old rats. The PTZ kindling showed an age-dependent decrease in expression of convulsions. The diminished kindling capacity was already seen in 6-month-old rats. In contrast, kindling-related impairment effects on cognitive functions increased with age. Thus, the correlation between learning impairment and occurrence of tonic-clonic seizures that we had demonstrated in 8-week-old rats was abolished in older rats. On the other hand, when the kindling procedure was started in 6-week-old rats, no impairment was found in fully kindled rats.

[Interaction of NAD with the GABA-ergic system of the rat brain]. / Vzaimodeistvie NAD s GAMK-ergicheskoi sistemi golovnogo mozga krys.

After administration of corazole content of gamma-aminobutyric acid (GABA) was increased in the rat brain within 2 min and 10 min by 28% and 80%, respectively. Content of the GABA was distinctly decreased in the prespastic phase. During this period specific binding of 3H-muscimol to the GABA receptors was decreased. NAD at concentrations 10(-6) M and 10(-7) M activated the GABA receptors and inhibited binding of 14C-GABA to the synaptosomes of both intact rats and the animals treated with the convulsant agent. NAD appears to cause an effect as an inhibitory neurotransmitter at the postsynaptic level.

High-performance liquid chromatographic procedure for quantitative determination of pentylenetetrazol in serum and discrete areas of rat brain.

A precise and reproducible high-performance liquid chromatographic method for the determination of pentylenetetrazol in serum and brain tissue is described. The procedure employs reversed-phase chromatography, monitoring the eluant at 202 nm. Quantification is based on peak-height ratio of the drug to the internal standard (p-methylphenobarbital). A linear response is obtained to 100 micrograms/ml (serum) or micrograms/g (brain tissue). Within-day and between-day precision are smaller than 5%, and analytical recovery is greater than 95%. Numerous drugs tested do not interfere with the assay. The method has been used to investigate the kinetics of pentylenetetrazol distribution in serum and in discrete areas of rat brain.

Continuous measurement of pentylenetetrazol concentration by a liquid ion exchanger microelectrode.

A double-barrelled microelectrode is described, which permits the continuous measurement of the concentration of the epileptogenic agent pentylenetetrazol (PTZ). The electrode is based on the liquid potassium exchanger (Corning No. 477 317) and enables measurements of PTZ concentration in physiological salines down to 1 mM. The electromotive behaviour of the liquid membrane against PTZ cannot directly be described by the Nicolsky-Eisenman formalism. It is suggested that specific interactions of the PTZ molecule with the Corning ligand are involved in the potential generating mechanisms.

Quantitative GLC determination of pentylenetetrazol in biological fluids.

A quantitative, sensitive, and specific GLC method was developed for the determination of pentylenetetrazol in water, plasma, and urine. The assay involves a single extraction of the sample into chloroform followed by centrifugation, evaporation, and chromatography. The method for pentylenetetrazol is reproducible, and the sensitivity limit of the assay is 0.5 mug of pentylenetetrazol/ml of biological fluid using a 2-ml sample. This method has a sensitivty sufficient to detect human plasma levels after therapeutic clinical doses and was successfully applied to monitor complete plasma level profiles of this drug in dogs. The data indicate that this drug is very rapidly absorbed following an oral dose, and the half-life of the drug in plasma is approximately 1 hr.