Beneficial effects of desacyl-ghrelin, hexarelin and EP-80317 in models of status epilepticus

It has been reported that ghrelin exerts anticonvulsive effects in models of epilepsy. In this study we aimed to characterize the anticonvulsive activity of ghrelin and other growth hormone secretagogue receptor 1a (GHSR_1a) ligands in rats exposed to status epilepticus induced by pilocarpine or kainate. Firstly, in three independent experiments, before receiving pilocarpine (380 mg/kg, i.p.), rats were pretreated with one among ghrelin (1.5 mg/kg), desacyl-ghrelin (1.5 mg/kg), hexarelin (330 μg/kg), EP-80317 (330 μg/kg), JMV-1843 (330 μg/kg), JMV-2959 (330 μg/kg) or saline. Secondly, in the fourth experiment, rats were pretreated with i.p. ghrelin, desacyl-ghrelin, hexarelin, EP-80317 or saline, followed by kainate (15 mg/kg, i.p.). We evaluated: induction of generalized seizures, latency to generalized seizures, status epilepticus, latency to status epilepticus (the time lag between the first tonic–clonic convulsion and the switch to continuous seizures) and mortality. In the pilocarpine model, 60% of rats pretreated with EP-80317 (P < 0.05) showed no seizure. Hexarelin and EP-80317 were both able to prevent progression to status epilepticus in pilocarpine-treated rats (P < 0.05). When status epilepticus was induced by kainate, seizures developed with few exceptions. However, latency to status epilepticus was significantly (P < 0.01) longer in rats pretreated with desacyl-ghrelin, whereas hexarelin and EP-80317 did not display any effect. Almost all GHSR_1a ligands prevented pilocarpine-induced mortality, which was observed only in rats pretreated with saline or JMV-2959. After kainate administration, all rats survived to status epilepticus. These findings demonstrate that desacyl-ghrelin, hexarelin and EP-80317 but not other GHSR_1a ligands display relevant anticonvulsive properties in models of limbic seizures.     

Modulation of the locomotor responses induced by D1-like and D2-like dopamine receptor agonists and D-amphetamine by NMDA and non-NMDA glutamate receptor agonists and antagonists in the core of the rat nucleus accumbens

Dopamine and glutamate interactions in the nucleus accumbens (NAcc) play a crucial role in both the development of a motor response suitable for the environment and in the mechanisms underlying the motor-activating properties of psychostimulant drugs such as amphetamine. We investigated the effects of the infusion in the NAcc of NMDA and non-NMDA receptor agonists and antagonists on the locomotor responses induced by the selective D(1)-like receptor agonist SKF 38393, the selective D(2)-like receptor agonist quinpirole, alone or in combination, and D-amphetamine. Infusion of either the NMDA receptor agonist NMDA, the NMDA receptor antagonist D-AP5, the non-NMDA receptor antagonist CNQX, or the non-NMDA receptor agonist AMPA resulted in an increase in basal motor activity. Conversely, all of these ionotropic glutamate (iGlu) receptor ligands reduced the increase in locomotor activity induced by focal infusion of D-amphetamine. Interactions with dopamine receptor activation were not so clear: (i). infusion of NMDA and D-AP5 respectively enhanced and reduced the increase in locomotor activity induced by the infusion of the D(1)-like receptor agonist of SKF 38393, while AMPA or CNQX decreased it; (ii). infusion of NMDA, D-AP5, and CNQX reduced the increase in locomotor activity induced by co-injection of SKF 38393+quinpirole--a pharmacological condition thought to activate both D(1)-like and D(2)-like presynaptic and postsynaptic receptors, while infusion of AMPA potentiated it; (iii). infusion of either NMDA, D-AP5 or CNQX, but not of AMPA, potentiated the decrease in motor activity induced by the D(2)-like receptor agonist quinpirole, a compound believed to act only at presynaptic D(2)-like receptors when injected by itself. Our results show that NMDA receptors have an agonist action with D(1)-like receptors and an antagonist action with D(2)-like receptors, while non-NMDA receptors have the opposite action. This is discussed from a anatamo-functional point of view.     

Rat retinal ganglion cell loss caused by kainate,NMDA and ischemia correlates with a reduction in mRNA and protein of Thy-1 and neurofilament light

Quantification of retinal ganglion cell (RGC) loss/survival following a defined insult to the retina is a prerequisite in order to allow a comparison to be made between the effectiveness of potential neuroprotective drugs. The purpose of the present study was to extend the characterisation of our previously published semiquantitative RT-PCR assay to assess RGC loss/survival. Comparisons were made between the total mRNA levels of the ganglion cell-specific markers Thy-1 and neurofilament light (NF-L) in the retina at specific times after an intravitreal injection of N-methyl-D-aspartate (NMDA) or kainate or after 45 min of ischemia/reperfusion and also between the levels of NF-L mRNA and protein at various times after NMDA injection. Changes in Thy-1 and NF-L immunoreactivities were also observed. NMDA, kainate and ischemia/reperfusion all caused a reduction in the retinal content of Thy-1 and NF-L mRNAs and immunoreactivities. An excellent correlation was observed between the levels of the two mRNAs after these treatments. After NMDA, loss of NF-L mRNA was shown to precede loss of NF-L protein but total loss of each marker was similar after 7 days. The results of the study demonstrate that injury and subsequent death of RGCs, which occurs after ischemia/reperfusion and after intraocular injection of NMDA or kainate, can be followed by measurement of total retinal levels of Thy-1 and NF-L mRNAs and NF-L protein. The assays provides accurate, practical and complementary methods for assessing the potential benefits of neuroprotective drugs on RGCs which have been injured by a variety of experimental modalities.     

Zinc released from metallothionein-iii may contribute to hippocampal CA1 and thalamic neuronal death following acute brain injury

Vesicular zinc was initially considered the sole source of toxic intraneuronal zinc accumulation in response to acute brain injury, but recent evidence suggests that additional sources also exist. Because metallothioneins (MTs) can bind and release zinc, we examined the possibility that the brain-specific form, MT-III, is such a zinc source. After kainate-induced seizures, cytoplasmic zinc accumulation and neuronal death in the hippocampal CA1 region and the thalamus were substantially lower in Mt3-null mice than in wild-type mice. Furthermore, compared with zinc transporter 3 (Znt3)-null mice, Znt3/Mt3 double-null mice exhibited further reductions in neuronal death in CA1 following kainate-induced seizures. Similar reductions in zinc accumulation and neuronal death in hippocampal CA1 and the dentate gyrus in Mt3-null mice were observed in a sodium nitroprusside model of acute brain injury. In contrast to CA1, more neuronal death occurred after kainate-induced seizures in CA3 of Mt3-null mice. These results suggest that intracellular zinc release from MT-III may contribute substantially to zinc-mediated neuronal death in certain brain areas, including the hippocampal CA1 region and the thalamus.     

Some properties of membrane current fluctuations induced by kainate, quisqualate, and NMDA in cultured septal neurons of rat

Ionic currents induced by glutamate, kainate, quisqualate, and (NMDA) in cultured septal neurons were analyzed by fluctuation analysis. The power spectrum (PWS) of NMDA currentt fluctuations always fitted a single Lorentzian. PWSs of the other agonists fitted the sum of two Lorentzians; however, the slopes of PWSs became larger and the PWSs became closer to single Lorentzians as the number of drug application increased. This may be explained in such a way that, in multiple conductance channels activated by these agonists, the high frequency component decreases the gating activity in later recordings, whereas the low frequency component keeps its gating kinetics.     

C-fos immunoreactivity in the brain following electrical or chemical stimulation of the medial hypothalamus of freely moving rats

c-fos immunoreactivity was used to map brain areas in which neurons reacted either to electrical stimulation or to microinjection of the excitatory amino acid kainate and of the GABA_A antagonist, SR-95531, applied to the medial hypothalamus of freely moving rats. All these stimulations induced flight behavior of moderate intensity. Immunoreactive cells were found within a radius of 0.5 mm around the stimulated area. Distally, clusters of labeled cells were found ipsilaterally in the piriform and entorhinal cortices, in several amygdaloid nuclei, in the bed nucleus of the stria terminalis, in the septo-hypothalamic nucleus, in the paraventricular, anterior and dorsomedial hypothalamic nuclei, in the paraventricular thalamic nucleus, in the dorsal periaqueductal gray extending to the cuneiform nucleus, and bilaterally in the supramamillary decussation and the locus coeruleus. The specificity of the brain areas thus labeled was indicated by the unilateral pattern of activation as well as by the different pattern obtained after control microinjection of saline. Therefore, these results are likely to provide sound information about the brain structures involved in defensive/aversive behavior evoked from the medial hypothalamus.     

Effect of temperature on kainic acid-induced seizures

The effects of body temperature on kainic acid-induced seizures and seizure-related brain damage were examined in rats. In rats with status epilepticus induced by intraperitoneal injection of 12 mg/kg of kainic acid (KA), ictal discharges were decreased by 50% when body temperature was lowered to 28°C and nearly abolished when body temperature was lowered to 23°C. In rats with mild hypothermia (28°C), the duration of ictal discharges following KA injection was significantly lower than in rats with normal body temperature. No detectable hippocampal cell loss was observed in rats with hypothermia to 28°C whereas gross cell loss in the hippocampus was observed in all rats with KA injection at normal body temperature. In contract to hypothermia, hyperthermia markedly aggravated the seizures and hippocampal damage induced by KA. Following elevation of body temperature to 42°C KA (12 mg/kg) resulted in severe seizures and all rats died of tonic seizures within 2 h. Furthermore, 6 mg/kg of KA administered to rats with a body temperature of 41–42°C, resulted in up to 4 h of continuous ictal discharges whereas no continuous ictal discharges were observed after the same injections in rats with normal body temperature. Histological examination in rats receiving 6 mg/kg of KA revealed severe cell loss in the hippocampus in rats with hyperthermia but not in rats with normal temperature. These results demonstrate that body temperature plays an important role in the control of epileptic seizures and seizure-related brain damage. These data suggest that hypothermia may be useful in reducing seizures and associated brain damage and that hyperthermia should be avoided in status epilepticus.     

Enhancement of kainate-gated currents in retinal horizontal cells by cyclic AMP-dependent protein kinase

Dopamine, acting via cyclic adenosine 3′:5′-monophosphate (cAMP), has been shown to enhance a kainate-gated ionic conductance in white perch retinal horizontal cells in vitro. To determine whether this effect involves stimulation of a protein kinase, kainate-gated currents were observed in cultured horizontal cells that were dialyzed with the catalytic subunit of cAMP-dependent protein kinase. Intracellular application of catalytic subunit or cAMP, but not heat-inactivated catalytic subunit, caused significant enhancement of the kainate-evoked currents. These results suggest that kainate-gated channels in horizontal cells may be modified by a phosphorylation event.     

Excitatory amino acid-evoked release of [H]GABA from hippocampal neurons in primary culture

The novel glutamate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited glutamate stimulated [³H]GABA release from cortical neurons in vitro. Kainate-induced release was blocked in a competitive fashion butN-methyl-d-aspartate (NMDA)-induced release was blocked non-competitively by CNQX. 7-Chlorokynurenate (7-CK) also inhibited NMDA evoked [³H]GABA release non-competitively, but had no effect on kainate induced release. The effects of both CNQX and 7-CK on NMDA-induced release were reversed by addition of exogenous glycine but the effects of CNQX on kainate-induced release were not altered by glycine. This suggests that both CNQX and 7-CK may interact with the glycine regulatory site of the NMDA receptor.     

The neuroprotective actions of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) in a rat focal ischaemia model

The neuroprotective effects of NBQX, a selective antagonist for the AMPA/kainate subtype of excitatory amino acid receptors, were investigated in a rat focal ischaemia model, involving permanent occlusion of the left middle cerebral artery (MCA). NBQX (3, 10 or 30 mg/kg) was administered i.v. immediately after MCA occlusion and again 1 h later. The highest dose of NBQX (2 x 30 mg/kg) gave significant protection against hemispheric (24%) and cortical (27%) ischaemic damage. The lower doses of NBQX (2 x 3 or 2 x 10 mg/kg) were ineffective. No protection was seen against caudate damage for any of the doses of NBQX tested. NBQX has a t1/2 of 30 min, therefore, a second experiment was done in which a dose of 30 mg/kg was given as an i.v. bolus followed immediately by an infusion of 10 mg/kg/h for 4 h, dosing was started immediately after MCA occlusion. This dosing regimen resulted in a mean plasma level over the 4 h of 17 micrograms/ml, and significant protection against the volume of hemispheric (29%) and cortical (35%) ischaemic damage, which was slightly better than that achieved with two bolus doses of 30 mg/kg. Once again no protection was seen against caudate damage. We conclude that NBQX, an AMPA/kainate antagonist was neuroprotective in a focal ischaemia model in the rat.