Ketogenic diet attenuates neuronal injury via autophagy and mitochondrial pathways in pentylenetetrazol-kindled seizures.

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.

Descifrando la fisiopatología de la epilepsia en un modelo animal: el pentilentetrazol induce la activación pero no la muerte de las neuronas de la amígdala extendida medial / Understanding the pathophysiology of epilepsy in an animal model: pentylenetetrazole induces activation but not death of neurons of the medial extended amygdala

Introducción: Desde mitad del siglo XX se ha apuntado a la importancia de la amígdala en la epilepsia, aunque los mecanismos básicos de esta participación en su mayoría son aún desconocidos. Esta ignorancia es aún mayor cuando se tienen en cuenta las distintas subdivisiones de la amígdala, especialmente sus partes mediales. En este trabajo evaluamos la participación de la amígdala extendida medial en un modelo animal de epilepsia, así como las consecuencias que tiene el epileptógeno en esta estructura cerebral. Material y métodos: Se utilizaron ratas adultas Wistar machos (n = 48); 24 animales recibieron inyecciones intraperitoneales de pentilentetrazol y 24, de salina. Luego de 2, 6, 12 y 24 h de sobrevida, los animales se fijaron, y sus cerebros se cortaron seriadamente y se procesaron para fos (inmunoquímica) y muerte neuronal con la técnica A-Cu-Ag. Los datos se analizaron con un ANOVA de 2 vías seguido de un test post-hoc (LSD de Fisher). Resultados: Muy poca activación fos se halla en animales controles. En animales experimentales, fos fue rápidamente inducida en la amígdala extendida medial a las 2 h. Esta activación fue sostenida hasta las 12 h y retornó a valores basales a las 24 h. Sin embargo, el estado epiléptico no produjo muerte neuronal. Conclusiones: Se demuestra así una participación de la amígdala extendida medial en mecanismos epilépticos en los cuales subyace un componente inhibitorio. Sin embargo, el estado epiléptico inducido no produce muerte neuronal en esta estructura (AU)

Introduction: Since middle of the 20th century the importance of amygdala in epilepsy it has suggested, although the basic mechanisms of this participation are still unknown. This ignorance increases when the different subdivisions of amygdala are considered, especially the medial amygdala. In this work we assess the involvement of the medial extended amygdala in an animal model of epilepsy and the consequences of its application in this brain structure. Material and methods: Forty eight adult Wistar male rats were used, of which 24 of them received i.p. injections of pentylenetetrazole, and 24 (controls) were injected with saline. After 2, 6, 12 and 24 h survival, animals were fixed; the brains were sectioned serially and stained for fos (immunochemistry) and for neuronal death with the A-Cu-Ag technique. Data were analysed using two-way ANOVA followed by the Fisher post hoc test. Results: Very few or no fos-immunoreactive neurons were seen in control animals. In experimental animals, fos was rapidly induced in structures of medial extended amygdala with peak levels at 2 h. Marked fos immunoreactivity persisted up to 12 h followed by a gradual return to baseline at 24 h. However, status epilepticus did not induced neuronal death. Conclusions: These results show involvement of medial extended amygdala in epileptic mechanisms with an inhibitory component. However, neuronal death is not a consequence of status epilepticus-induced by pentylentetrazole (AU)

Design and synthesis of 6-amino-1,4-oxazepane-3,5-dione derivatives as novel broad spectrum anticonvulsants.

Based on the structural estimations of the typical anticonvulsant drugs, a series of 6-amino-1,4-oxazepane-3,5-dione derivatives, novel structures of 7-membered heterocyclic imides, which were hybridized with pharmacophores such as cyclic imide and N-CO-C-N group in their molecule were designed and synthesized. Their anticonvulsant activities were evaluated by the maximal electroshock induced seizure (MES) and subcutaneous pentylenetetrazole (PTZ) tests. Almost all the designed compounds except 1c and 1f showed comparable anticonvulsant activities in at least one of the anticonvulsant tests. Moreover, some of the tested compounds exhibited moderate anticonvulsant activities in both MES and PTZ tests. From these results, 6-amino-1,4-oxazepane-3,5-dione derivatives could be recommended as novel structures of broad spectrum anticonvulsants.

Anticonvulsant profile and teratogenicity of 3,3-dimethylbutanoylurea: a potential for a second generation drug to valproic acid.

PURPOSE: The purpose of this study was to evaluate the anticonvulsant activity and teratogenic potential of branched aliphatic acylureas represented by isovaleroylurea (IVU), pivaloylurea (PVU) and 3,3-dimethylbutanoylurea (DBU), as potential second-generation drugs to valproic acid (VPA). METHODS: The anticonvulsant activity of IVU, PVU, and DBU was determined in mice and rats utilizing the maximal electroshock seizure (MES) and the pentylenetetrazole (scMet) tests. The ability of DBU to block electrical-, or chemical-induced seizures was further examined in three acute seizure models: the psychomotor 6 Hz model, the bicuculline and picrotoxin models and one model of chronic epilepsy (i.e., the hippocampal kindled rat model). The induction of neural tube defects (NTDs) by IVU, PVU, and DBU was evaluated after i.p. administration at day 8.5 of gestation to a mouse strain highly susceptible to VPA-induced teratogenicity. The pharmacokinetics of DBU was studied following i.v. administration to rats. RESULTS: DBU emerged as the most potent compound having an MES-ED(50)of 186 mg/kg (mice) and 64 mg/kg (rats) and an scMet-ED(50)of 66 mg/kg (mice) and 26 mg/kg (rats). DBU underwent further evaluation in the hippocampal kindled rat (ED(50)= 35 mg/kg), the psychomotor 6 Hz mouse model (ED(50)= 80 mg/kg at 32 mA and ED(50)= 133 mg/kg at 44 mA), the bicuculline- and picrotoxin-induced seizure mouse model (ED(50)= 205 mg/kg and 167 mg/kg, respectively). In contrast to VPA, DBU, IVU, and PVU did not induce a significant increase in NTDs as compared to control. DBU was eliminated by metabolism with a half-life of 4.5 h. CONCLUSIONS: DBU's broad spectrum and potent anticonvulsant activity, along with its high safety margin and favorable pharmacokinetic profile, make it an attractive candidate to become a new, potent, and safe AED.

Pharmacological and behavioral characteristics of interactions between vigabatrin and conventional antiepileptic drugs in pentylenetetrazole-induced seizures in mice: an isobolographic analysis.

To characterize the anticonvulsant effects and types of interactions exerted by mixtures of vigabatrin (VGB) and conventional antiepileptic drugs (valproate (VPA), ethosuximide (ESM), phenobarbital (PB), and clonazepam (CZP)) in pentylenetetrazole (PTZ)-induced seizures in mice, the isobolographic analysis for three fixed-ratio combinations of 1 : 3, 1 : 1, and 3 : 1 was used. The adverse-effect profile of the combinations tested, at the doses corresponding to their median effective doses (ED(50)) at the fixed-ratio of 1 : 1 against PTZ-induced seizures, was determined by the chimney (motor performance), step-through passive avoidance (long-term memory), pain threshold (pain sensitivity), and Y-maze (general explorative locomotor activity) tests in mice. Additionally, the observed isobolographic interactions were verified in terms of a pharmacokinetic interaction existence. VGB combined with PB or ESM exerted supra-additive (synergistic) interactions against the clonic phase of PTZ-induced seizures, which was associated with the increment of PB or ESM concentrations in the brains of examined animals. The remaining combinations tested (ie VGB+VPA and VGB+CZP) occurred additive in the PTZ test, which was associated with no significant changes in the brain concentrations of VPA and CZP. None of the examined combinations exerted motor impairment in the chimney test in mice. In the standard variant of passive avoidance task (current of 0.6 mA; 2 s of stimulus duration), the combinations of VGB+CZP and VGB+VPA significantly affected long-term memory in mice. Moreover, VGB in a dose-dependent manner lengthened the latency to the first pain reaction in the pain threshold test in mice. The modified variant of step-through passive avoidance task (current of 0.6 mA; stimulus duration based on the latency from the pain threshold test) revealed no significant changes in the long-term memory of animals for the combinations of VGB+VPA and VGB+CZP; so the observed effects in the standard variant of passive avoidance task were a result of the antinociceptive effects produced by VGB. In the Y-maze test, VGB also, in a dose-dependent manner, increased the general explorative locomotor activity of the animals tested. Similarly, the total number of arm entries in the Y-maze was significantly increased for the combinations of VGB+CZP and VGB+ESM, but not for VGB+PB and VGB+VPA. The application of VGB in combination with PB, ESM, CZP, and VPA suppressed the clonic phase of PTZ-induced seizures, having no harmful or deleterious effects on behavioral functioning of the animals tested, which might be advantageous in further clinical practice.

Validation of a modified mirrored chamber sensitive to anxiolytics and anxiogenics in mice.

RATIONALE: Anxiety is a common disorder in humans that exists in many forms, and animal models of human anxiety are typically employed for the discovery of anxiolytic drugs with human therapeutic potential. OBJECTIVES: Ideally, animal models of anxiety are validated for the detection of both anxiogenic and anxiolytic effects, but most animal models can effectively only measure anxiolytic-like effects. As control animals typically spend small amounts of time in the aversive portion of an apparatus, decreases in time spent in this portion are difficult to detect. METHODS: We have modified an existing test of murine anxiety, the mirrored chamber, and have validated this test using several anxiolytic and anxiogenic drugs. In addition, nine mouse strains were compared on the elevated plus maze and modified mirrored chamber. RESULTS: Increasing doses of ethanol, diazepam, and pentobarbital produced an anxiolytic-like profile while pentylenetetrazol (PTZ), D-amphetamine, and methyl-6, 7-dimethoxyl-4-ethyl-beta-carboline-3-carboxylate (DMCM) appeared anxiogenic. This modified test also dissociated drug effects on anxiety from those on activity for d-amphetamine and diazepam. The inbred mouse strains tested produced a similar range of scores for time spent on the open arms of the elevated plus maze and voluntary reentry time in the mirrored chamber, with an overall genetic correlation of 0.68. CONCLUSIONS: Since control animals reliably reentered the more aversive portion of the apparatus for 25% of the total time available, the modified mirrored chamber may be able to detect anxiogenic states produced by various stressors and drug withdrawal. Further, the strain differences detected suggest that the modified mirrored chamber will be a valuable tool in the discovery of the genetic bases of anxiety states and disorders.

Design and synthesis of semicarbazones and their bio-isosteric analogues as potent anticonvulsants: the role of hydrogen bonding.

A series of p-nitrophenyl substituted semicarbazones (4a-c) and phenoxy/p-bromophenoxy acetyl hydrazones (8a-q) were synthesized and their anticonvulsant activity was screened against maximal electroshock seizure (MES), subcutaneous metrazole (ScMet) and subcutaneous strychnine (ScSty) tests. Compounds 4a-c with -NHCO- were found to be the most active in all these tests. These compounds were also active in the MES test after oral administration in rats. On the other hand, compounds 8a-q with -OCH2- were devoid of anticonvulsant activity. The studies revealed that the hydrogen bonding domain in semicarbazones, adjacent to the lipophilic aryl ring, is essential for the anticonvulsant activity.

Activación del sistema de los opiodes endógenos por dosis subconvulsivamantes de metrazol / Activation of the endogenous opioid system with metrazol subconvulsivant dosis

Se investigaron las alteraciones en el sistema de los opioides endógenos en el cerebro de la rata, inducidas por la administración de una dósis subconvulsivante de metrazol (PTZ) (30 mg/kg i.p.). Por medio de experimentos de microdiálisis, encontramos durante los primeros 60 min después del tratamiento, una liberación importante de opiodes endógenos en el hipocampo y la amígdala cerebral. Posteriormente, los valores regresaron a los niveles basales. Por autorradiografía se observó un decremento en los niveles de los receptores mu en varias estructuras cerebrales. Mediante el análisis de la unión a receptores las membranas cerebrales, se confirmó un decremento en el número de estos receptores, sin cambios en su afinidad. En la aplicación de la prueba de Randall-Sellito, se encontró un aumento en el umbral de respuesta a estímulos dolorosos, durante los primeros 30 min. después del PTZ. Finalmente, experimentos de hibridación in situ revelaron un incremento en los niveles de la proencefalina a las 24 hrs después del tratamiento. Nuestros resultados indican que la administración de dosis subconvulsivante de PTZ activan de manera importante al sistema de los opiodes endógenos. Estos cambios resultan relevantes para entender el proceso del epileptogénesis y los mecanismos involucrados en el mismo

Critical volume of rat cortex and extracellular threshold concentration for a pentylenetetrazol-induced epileptic focus.

The initiation of focal interictal epileptiform activity (FIEA) has been shown to depend on the activation of a sufficiently large volume of brain tissue. We estimated the size of this 'critical volume' for the convulsant pentylenetetrazol (PTZ) by analyzing the diffusion following its microinjection into rat motor cortex. PTZ concentration was monitored 100-200 microm away from the injection site with a PTZ-sensitive microelectrode. Diffusion analysis in 0.3% agar yielded the free diffusion coefficient D (8.50 +/- 0.15 X 10(-6) cm2 x s(-1) at 37 degrees C, median +/- S.E.M.). In brain tissue, diffusion was modified by extracellular volume fraction (alpha), tortuosity (lambda = (D/ADC)1/2; ADC = apparent diffusion coefficient) and non-specific uptake (k'). Using a value of 0.2 for alpha from previous studies, we found values of lambda = 1.61 +/- 0.01, k' = 3.37 +/- 0.15 X 10(-3) s(-1) and an injected volume U of 5.16 +/- 0.45 X 10(-10) l for pulses without FIEA, and lambda = 1.95 +/- 0.06, k' = 6.24 +/- 1.73 X 10(-3) s(-1) and U = 7.40 +/- 0.66 X 10(-10) l for pulses with FIEA. From the calculated concentration distribution of PTZ during FIEA we estimated a threshold concentration of about 1.77 mM PTZ and a volume with a radius of about 219 microm in which this concentration had to be exceeded. Since this critical volume was comparable in size to foci elicited by penicillin or electric stimuli in previous studies, it is concluded that it is determined by intrinsic tissue properties rather than by the convulsive agent being used.

Effects of pentylenetetrazol on GABA receptors expressed in oocytes of Xenopus laevis: extra- and intracellular sites of action.

The convulsive agent pentylenetetrazol (PTZ) antagonized gamma-aminobutyric acid (GABA)-induced membrane currents on RNA-injected Xenopus oocytes with both extra- and intracellular applications. With extracellular administration PTZ enters the cell within a few minutes and reaches concentrations in the millimolar range. The permeability of the plasma membrane makes it possible for systemically applied PTZ to elicit its effect on the GABA-induced currents via extra- as well as intracellular sites of action.

NG-nitro-L-arginine, a nitric oxide synthase inhibitor, and seizure susceptibility in four seizure models in mice.

Nitric oxide may be involved in seizure phenomena even though data often seem to be contradictory. This prompted us to study the influence of nitric oxide upon electrically and chemically induced seizures. The effects of nitric oxide synthase inhibitor, NG-nitro-L-arginine (NNA), on pentylenetetrazol-, aminooxyacetic acid-, aminophylline-induced seizures or electroconvulsive shock were evaluated. NNA was applied at 1, 10 and 40 mg/ kg 0.5 and 2.0 h before chemical seizures and at 1 and 40 mg/kg 0.5 and 2.0 h prior to electroconvulsions. The nitric oxide synthase inhibitor (up to 40 mg/ kg) did not affect the susceptibility of mice to pentylenetetrazol, amino-oxyacetic acid or electroconvulsions. However, NNA significantly enhanced the convulsive properties of aminophylline when applied at 40 mg/kg, 0.5 h before the test. The CD50 value for aminophylline-induced clonus and tonus/ mortality was decreased from 233 to 191 and from 242 to 212 mg/kg, respectively. However, this pretreatment also led to a significant increase in the plasma levels of theophylline. Our results suggest that differential effects of NNA on chemically-induced convulsions might in some cases be associated with a pharmacokinetic interaction.

Pharmacodynamics of phenobarbital anesthesia and pentylenetetrazol-induced maximal seizures in a rat model of neoplastic spinal cord compression.

The purpose of this investigation was to determine whether paraplegia induced by neoplastic cord compression affects the pharmacodynamics of phenobarbital general anesthesia or of pentylenetetrazol (PTZ)-induced convulsions. Paraplegic rats harboring a thoracolumbar epidural tumor, or an identical hindlimb tumor mass, received an i.v. infusion of phenobarbital until the onset of anesthesia. At that point, the phenobarbital concentrations in the CSF and serum were measured. Similarly, PTZ was infused until the onset of maximal seizures. It was found that changes related to systemic tumor growth and newly developed paraplegia due to neoplastic spinal cord compression did not attenuate the pharmacodynamics of phenobarbital. However, sustained paraplegia of 4 days' duration reduced CNS sensitivity to the hypnotic action of the barbiturate as evidenced by the higher cerebrospinal fluid phenobarbital concentration required to induce anesthesia (170 +/- 31 vs 125 +/- 20 mg/L; P < 0.05). On the other hand, sustained paraplegia did not affect brain threshold concentration for PTZ-induced seizures.

Neural sensitivity to pentylenetetrazol convulsions in inbred and selectively bred mice.

In these experiments, sensitivity to the convulsant drug pentylenetetrazol (PTZ) was measured in 10 inbred mouse strains, and in 4 mouse lines selectively bred for severe (WSP1, WSP2) or minimal (WSR1, WSR2) ethanol withdrawal convulsions. Using a timed infusion procedure, sensitivity to convulsions was assessed by measures of latency to convulsion, effective dose (ED) infused at time of convulsion, and brain concentration (BC) of PTZ at time of convulsion. In addition, ED and latency to convulsion were measured in WSP and WSR mice at 5 different concentrations of PTZ. Higher concentrations, which increased rate of drug infusion, reduced latency but had little effect on ED. WSP1 mice were slightly more sensitive to PTZ than WSR1 mice, but WSP2 mice were equally or less sensitive than WSR2 mice. Among the inbred strains, latency, ED and brain PTZ concentration were found to be highly correlated, suggesting that pharmacokinetic factors do not significantly influence access of PTZ to sites of action in the central nervous system. The C57BL/6J strain was least sensitive by all measures, while DBA/2J mice were highly sensitive. The BALB/cJ strain was the most sensitive strain as assessed by ED and latency, but BC indicated relatively average sensitivity. Apparently, pharmacokinetic factors in this strain result in a relatively rapid accumulation of drug in brain, making it appear to be more sensitive. Thus, although ED provides a reliable estimate of neural sensitivity in general, genetic factors exist which, in some strains, modify access of PTZ and possibly other drugs to brain, potentially affecting determination of sensitivity in the absence of a measure of brain drug concentration.

Central nervous system uptake kinetics of pentylenetetrazol in the developing rat.

The present investigation was undertaken to examine the brain uptake kinetics of the central nervous system (CNS) stimulant pentylenetetrazol (PTZ) during postnatal development. This study represents part of an ongoing effort to develop an appropriate seizure model for investigations of anticonvulsant action in the developing rat. The systemic pharmacokinetics of PTZ were examined in male and female adult animals following both intravenous (IV) and subcutaneous (SC) injection. PTZ administered SC was absorbed rapidly and was completely bioavailable in both genders. No statistically significant gender-dependent differences in the disposition of PTZ were identified. To examine the CNS uptake kinetics of PTZ, animals of four different age groups (5, 10, 20, and 60 days postnatal) received timed SC infusions of PTZ. Significant age-related differences in the rate of uptake of PTZ into the CNS were observed. These differences paralleled the previously reported age-dependent changes in the dose of PTZ required to elicit seizure activity. The results of this investigation indicate that the apparent age-related change in sensitivity to the convulsant effects of PTZ is due in part to age-dependent uptake of the convulsant into brain tissue.

Isobolographic assessment of the convulsant interaction between theophylline and caffeine or pentylenetetrazol in rats.

To characterize the convulsant interaction between theophylline and caffeine, male Sprague-Dawley rats received an iv infusion of one of seven different combinations of these drugs and of each drug individually until the onset of maximal seizures (which occurred within 30 to 40 min after the start of the infusion). The total infused doses of the two drugs and their respective concentrations in the serum, brain, and cerebrospinal fluid (CSF) were used for isobolographic analysis. The results are consistent with classical dose- and concentration-addition and do not suggest either antagonism or synergism. The potency ratio based on the doses or serum concentrations was appreciably different from that based on brain or CSF concentrations. The brain:serum and CSF:serum concentration ratios of caffeine were appreciably higher than those of theophylline. Similar experiments were performed with seven combinations of theophylline and pentylenetetrazol (PTZ) and with each of these drugs individually. This second set of experiments also yielded essentially linear isobolographs indicative of dose- and concentration-addition. The potency ratio based on CSF concentrations was appreciably different from ratio values based on doses, and from those based on brain or serum concentrations. These results illustrate a useful strategy for the characterization of pharmacodynamic drug interactions and highlight the importance of the choice of sampling site for determinations of the potency of drug.

Kinetics of drug action in disease states. XXXIV. Effect of experimental thyroid disorders on the pharmacodynamics of phenobarbital, ethanol and pentylenetetrazol.

To determine the effect of thyroid disorders on the concentration-activity relationship of certain drugs acting on the central nervous system, rats were made hyperthyroid by administration of L-thyroxine and hypothyroid by administration of propylthiouracil. They then received a slow i.v. infusion of phenobarbital or ethanol until they lost their righting reflex, or of pentylenetetrazol until the onset of maximal seizures. Drug concentrations in serum, brain and cerebrospinal fluid (CSF) were determined at these pharmacologic endpoints. Hyperthyroidism was associated with a statistically significant increase in the infused hypnotic dose and brain concentration of phenobarbital but had no apparent effect on concentrations of the drug in serum and CSF. The hypnotic dose of ethanol was increased significantly in hyperthyroid rats and decreased in hypothyroid animals; ethanol concentrations in serum, brain and CSF at onset of effect were generally not affected by thyroid dysfunction except for a small but statistically significant increase of serum ethanol concentrations in the hyperthyroid rats. The convulsant dose of pentylenetetrazol was reduced significantly in hypothyroid animals and unaltered in hyperthyroid rats; the concentrations of the convulsant in serum, brain and CSF were not apparently changed by the thyroid dysfunctions. In general, experimental thyroid disorders had no pronounced effect on the pharmacodynamics (concentration-effect relationship) of phenobarbital, ethanol and pentylenetetrazol in rats.

Diazepam reverses the effects of pentylenetetrazole in rat pups by acting at type 2 benzodiazepine receptors.

Pentylenetetrazole (75 mg/kg) induced a characteristic coarse body tremor (accompanied by limb extension) and hyperactivity in 4-day-old rat pups. These effects were reversed by diazepam (0.5 and 2 mg/kg) but not by CL 218,872 (10 and 20 mg/kg) which is selective for type 1 benzodiazepine receptors. Diazepam did not affect the brain concentrations of pentylenetrazole, indicating that the reversal was not based on a pharmacokinetic interaction. Neither diazepam nor CL 218,872 had significant effects on the behavior of the rat pups, although diazepam (2 mg/kg) tended to increase locomotor activity. The results suggest that diazepam displays an anticonvulsant effect in the neonatal rat which is mediated by type 2 receptors.