An atypical residue in the pore of Varroa destructor GABA-activated RDL receptors affects picrotoxin block and thymol modulation.

GABA-activated RDL receptors are the insect equivalent of mammalian GABAA receptors, and play a vital role in neurotransmission and insecticide action. Here we clone the pore lining M2 region of the Varroa mite RDL receptor and show that it has 4 atypical residues when compared to M2 regions of most other insects, including bees, which are the major host of Varroa mites. We create mutant Drosophila RDL receptors containing these substitutions and characterise their effects on function. Using two electrode voltage clamp electrophysiology we show that one substitution (T6'M) ablates picrotoxin inhibition and increases the potency of GABA. This mutation also alters the effect of thymol, which enhances both insect and mammalian GABA responses, and is widely used as a miticide. Thymol decreases the GABA EC50 of WT receptors, enhancing responses, but in T6'M-containing receptors it is inhibitory. The other 3 atypical residues have no major effects on either the GABA EC50, the picrotoxin potency or the effect of thymol. In conclusion we show that the RDL 6' residue is important for channel block, activation and modulation, and understanding its function also has the potential to prove useful in the design of Varroa-specific insecticidal agents.

Evaluation of anticonvulsant actions of dibromophenyl enaminones using in vitro and in vivo seizure models.

Epilepsy and other seizure disorders are not adequately managed with currently available drugs. We recently synthesized a series of dibromophenyl enaminones and demonstrated that AK6 and E249 were equipotent to previous analogs but more efficacious in suppressing neuronal excitation. Here we examined the actions of these lead compounds on in vitro and in vivo seizure models. In vitro seizures were induced in the hippocampal slice chemically (zero Mg2+ buffer and picrotoxin) and electrically using patterned high frequency stimulation (HFS) of afferents. In vivo seizures were induced in rats using the 6 Hz and the maximal electroshock models. AK6 (10 µM) and E249 (10 µM) depressed the amplitude of population spikes recorded in area CA1 of the hippocampus by -50.5±4.3% and -40.1±3.1% respectively, with partial recovery after washout. In the zero Mg2+ model, AK6 (10 µM) depressed multiple population spiking (mPS) by -59.3±6.9% and spontaneous bursts (SBs) by -65.9±7.2% and in the picrotoxin-model by -43.3±7.2% and -50.0±8.3%, respectively. Likewise, E249 (10 µM) depressed the zero-Mg2+-induced mPS by -48.8±9.5% and SBs by -55.8±15.5%, and in the picrotoxin model by -37.1±5.5% and -56.5±11.4%, respectively. They both suppressed post-HFS induced afterdischarges and SBs. AK6 and E249 dose-dependently protected rats in maximal electroshock and 6 Hz models of in vivo seizures after 30 min pretreatment. Their level of protection in both models was similar to that obtained with phenytoin Finally, while AK6 had no effect on locomotion in rats, phenytoin significantly decreased locomotion. AK6 and E249, suppressed in vitro and in vivo seizures to a similar extent. Their in vivo activities are comparable with but not superior to phenytoin. The most efficacious, AK6 produced no locomotor suppression while phenytoin did. Thus, AK6 and E249 may be excellent candidates for further investigation as potential agents for the treatment of epilepsy syndromes with possibly less CNS side effects.

Infralimbic cortex controls core body temperature in a histamine dependent manner.

An increase in body temperature accelerates biochemical reactions and behavioral and physiological responses. A mechanism to actively increase body temperature would be beneficial during motivated behaviors. The prefrontal cortex is implicated in organizing motivated behavior; the infralimbic cortex, a subregion of the medial prefrontal cortex, has the necessary connectivity to serve the role of initiating such thermogenic mechanism at the beginning of the appetitive phase of motivated behavior; further, this cortex is active during motivated behavior and its disinhibition produces a marked behavioral and vegetative arousal increase, together with increases in histamine levels. We wanted to explore if this arousal was related to histaminergic activation after pharmacological infralimbic disinhibition and during the appetitive phase of motivated behavior. We measured core temperature and motor activity in response to picrotoxin injection in the infralimbic cortex, as well as during food-related appetitive behavior, evoked by enticing hungry rats with food. Pretreatment with the H1 receptor antagonist pyrilamine decreased thermal response to picrotoxin and enticement and completely blunted motor response to enticement. Motor and temperature responses to enticement were also completely abolished by infralimbic cortex inhibition with muscimol. To assess if this histamine dependent temperature increase was produced by an active sympathetic mediated thermogenic mechanism or was just a consequence of increased locomotor activity, we injected propranolol (i.p.), a ß adrenergic receptor blocker, before picrotoxin injection into the infralimbic cortex. Propranolol reduced the temperature increase without affecting locomotor activity. Altogether, these results suggest that infralimbic activation is necessary for appetitive behavior by inducing a motor and a vegetative arousal increase mediated by central histamine.

Antiepileptic potential and behavioral profile of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog.

Thyrotropin-releasing hormone (TRH) and its analogs have a number of neurobiological functions and therapeutic uses in disorders of the central nervous system. In this study, the newly synthesized TRH analogs were evaluated for central nervous system activity in pentobarbital-induced sleeping in mice. The most potent TRH analog (L-pGlu-(2-propyl)-L-His-L-ProNH(2) coded as NP-647) was evaluated for its antiepileptic potential in various seizure models in mice in comparison with TRH. Intravenous pretreatment with NP-647 (10 and 20 micromol/kg body wt) significantly delayed the onset and reduced the frequency of convulsions in the pentylenetetrazole model, but not in the maximum electroshock seizure model. Also, it was found to be protective against picrotoxin- and kainic acid-induced seizures. However, NP-647 did not significantly affect theophylline-induced seizures. Further study of the effect of NP-647 on locomotor activity and a functional observational battery revealed that it did not significantly exhibit any undesirable effects as compared with vehicle and TRH. NP-647 did not significantly affect cerebral blood flow, whereas the native peptide TRH markedly increased cerebral blood flow. Furthermore, NP-647 exerted antiepileptic activity without significantly altering plasma thyroid-stimulating hormone levels and mean arterial blood pressure. This suggests that NP-647 is more selective for central nervous system activity and devoid of hormonal and cerebrovascular system effects. In contrast, TRH exhibited cardiac and endocrine effects as marked by significant elevation in mean arterial blood pressure and plasma thyroid-stimulating hormone levels. This study demonstrates that NP-647 has potential antiepileptic activity devoid of undesirable effects and, thus, can be exploited for the prevention and treatment of epilepsy.

Zimelidine decreases seizure susceptibility in stressed mice.

To further evaluate whether selective serotonin reuptake inhibitors (SSRIs) have pro- or anticonvulsant properties and whether these properties will be modified by stress, we studied the effect of zimelidine on the convulsions produced by picrotoxin, a GABA(A) receptor antagonist, in unstressed and swim stressed mice. Zimelidine potentiated the ability of swim stress to enhance the threshold doses of intravenously administered picrotoxin producing convulsant signs and death, without having an effect in unstressed mice. The anticonvulsant effect of zimelidine was counteracted with mianserin, the antagonist of 5-HT(2A/2C), and diminished with WAY-100635, a selective antagonist of 5-HT(1A) receptors. In stressed mice, WAY-100635 prevented the anticonvulsant effect of 8-OH-DPAT, a 5-HT(1A) receptor agonist. SB-269970 and ketanserin, the antagonists of 5-HT(7) and 5-HT(2A) receptors, respectively, failed to reduce the effect of zimelidine. The results suggest the involvement of 5-HT(2C) and 5-HT(1A) receptors in the anticonvulsant effects of zimelidine and possibly other SSRIs in stress.

Stimulation of 5-HT 1A receptors increases the seizure threshold for picrotoxin in mice.

To evaluate the possible role of 5-HT 1A and 5-HT 2A receptors in the anticonvulsant effect of swim stress, mice were pre-treated with agonists and antagonists of these receptors prior to exposure to stress and the intravenous infusion of picrotoxin. 8-OH-DPAT ((+/-)-8-hydroxy-2-(di-n-propylamino) tetralin) and WAY-100635 (a selective agonist and antagonist of 5-HT 1A receptors), DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) and ketanserin (a 5-HT 2A/2C receptor agonist and antagonist) were used. Results demonstrated that 1 and 3 mg/kg of 8-OH-DPAT increased the doses of picrotoxin producing running/bouncing clonus, tonic hindlimb extension and death in stressed and unstressed mice, respectively. Pre-treatment with WAY (0.3 mg/kg) prevented the effect of 8-OH-DPAT (3 mg/kg). DOI (2.5 mg/kg) and ketanserin (1 mg/kg) failed to affect the seizure threshold for picrotoxin. The results show that stimulation of 5-HT 1A receptors exerts anticonvulsant actions in stressed and unstressed mice, while stimulation of 5-HT 2A/2C receptors does not interfere with the effect of stress on picrotoxin-induced convulsions.

Prevention of picrotoxin convulsions-induced learning and memory impairment by nitric oxide increasing dose of L-arginine in rats.

Learning and memory processes were tested in adult male rats using a traditional pole-climbing apparatus 30 min after the administration of L-arginine (500 and 1000 mg/kg), the precursor of nitric oxide (NO), and N-nitro-L-arginine methyl ester (L-NAME) (50 and 100 mg/kg), the inhibitor of NO synthesis. The effects of the convulsant (5.0 mg/kg) and a smaller nonconvulsant (2.5 mg/kg) dose of picrotoxin were tested on learning and memory 120 min and 24 h after their administration. The tests were carried out 30 min after L-arginine in animals treated 120 min previously with the convulsant dose of picrotoxin. A dose-dependent enhancement and an inhibition of learning and memory were observed in animals treated with L-arginine and L-NAME, respectively. The convulsant dose of picrotoxin impaired both learning and memory processes. The effect of picrotoxin was reverted following the administration of L-arginine (1000 mg/kg). An interpretation of these results indicates that convulsions induced by picrotoxin produces learning and memory impairment, and that this defect is reversible if NO synthesis is increased in the brain by the systemic administration of L-arginine.

Evidence for an involvement of nitric oxide and gamma aminobutyric acid in the anticonvulsant action of L-arginine on picrotoxin-induced convulsions in rats.

Five, 30, and 60 min pretreatment of 1000 mg/kg and not 500 mg/kg of L-arginine inhibited convulsions induced by picrotoxin. The concentrations of nitric oxide (NO) and gamma aminobutyric acid (GABA) were increased in the brain 5, 30, and 60 min after administration of 1000 mg/kg and not 500 mg/kg of L-arginine. A much higher dose of L-arginine (2000 mg/kg), 30 min after administration, produced a lesser anticonvulsant and NO and GABA increasing actions as compared to that produced by 1000 mg/kg of L-arginine. The same dose of L-arginine, 60 min after administration, decreased the concentrations of both NO and GABA and increased the convulsion frequency of picrotoxin. An NO decreasing dose of nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine methyl ester (L-NAME) decreased brain GABA concentration and increased the convulsant action of picrotoxin. Further, L-NAME pretreatment prevented L-arginine (1000 mg/kg) from producing anticonvulsant and NO and GABA increasing effects. An interpretation of these results suggests that NO synthesized from systemically administered L-arginine inhibits convulsions by increasing the concentration of GABA in the brain. However, the effects of L-arginine are reversible, if it is administered at a higher dose (2000 mg/kg) 60 min prior to the test. It is concluded that L-arginine produces anticonvulsant or proconvulsant action depending upon the dose and time of its administration-related changes in the concentrations of NO and GABA in the brain.

Anticonvulsive activity of Butea monosperma flowers in laboratory animals.

The bioassay-guided fractionation of dried flowers of Butea monosperma (BM) was carried out to isolate the active principle responsible for its anticonvulsant activity. The petroleum ether extract was fractionated by column chromatography using solvents of varying polarity such as n-hexane, n-hexane:ethyl acetate, ethyl acetate, and methanol. The anticonvulsive principle of B. monosperma was found to be a triterpene (TBM) present in the n-hexane:ethyl acetate (1:1) fraction of the petroleum ether extract. TBM exhibited anticonvulsant activity against seizures induced by maximum electroshock (MES) and its PD(50) was found to be 34.2+/-18.1 mg/kg. TBM also inhibited seizures induced by pentylenetetrazol (PTZ), electrical kindling, and the combination of lithium sulfate and pilocarpine nitrate (Li-Pilo). However, TBM was not effective against seizures induced by strychnine and picrotoxin. TBM exhibited depressant effect on the central nervous system. After repeated use for 7 days, the PD(50) (MES) of TBM increased to 51.5+/-12.1 mg/kg. Similarly, after repeated use of TBM, the duration of sleep induced by pentobarbital was not reduced significantly. Further studies are required to investigate its usefulness in the treatment of epilepsy.

Antagonism of picrotoxin-induced changes in dopamine and serotonin metabolism by allopregnanolone and midazolam.

The effects of allopregnanolone and midazolam, given intracerebroventricularly, on the behavioral and biochemical effects of picrotoxin, were examined in a model of neurotoxin-induced seizures, in mice. After acute injections, midazolam (ED(50)=39.8 nmol) and allopregnanolone (ED(50)=11.0 nmol) produced similar and dose-dependent protection against picrotoxin-induced seizures. Picrotoxin given intraperitoneally at the ED(85) dose decreased significantly the concentration of serotonin (5-HT), dopamine (DA), homovanilic acid (HVA) and 3,4-dihydroxyindolacetic acid (DOPAC), in the mouse striatum and the frontal cortex, in the period of time immediately preceding the onset of seizures. A single injection of allopregnanolone more potently, in comparison to midazolam, antagonized the biochemical action of picrotoxin, abolishing its effects on DA, HVA and 5-HT concentration, in the mouse striatum and the frontal cortex. These results for the first time provide a direct argument for an involvement of central dopaminergic and serotonergic systems in the seizure development. The present data add also to the accumulating evidence suggesting a favorable pharmacological profile for some neurosteroids currently considered to have a future role in the management of epilepsy.

Roles of group II metabotropic glutamate receptors in modulation of seizure activity.

Evidence suggests that metabotropic glutamate receptors (mGluR) are involved in mediating seizures and epileptogenesis. In the present experiments, the selective, group II mGluR agonist (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid (LY354740, 0.1-1.0 microM) inhibited spontaneous epileptiform discharges which developed in rat cortical slices in Mg2+-free medium. LY354740 (4-16 mg/kg) administered prior to an injection of pentylenetetrazol (80 mg/kg) or picrotoxin (3.2 mg/kg) produced a dose-dependent decrease in the number of mice exhibiting clonic convulsions, but had no effect on N-methyl-D-aspartate (NMDA, 150 mg/kg)-induced convulsions. LY354740 (4-16 mg/kg) did not affect lethality induced in mice by pentylenetetrazol, picrotoxin or NMDA. LY354740 potentiated the anticonvulsant activity of the conventional antiepileptic drug diazepam, significantly decreasing the ED50 for that drug's effect on pentylenetetrazol-induced convulsions by 30%, but had no influence on anticonvulsant activity of ethosuximide and valproic acid. A pharmacokinetic interaction between LY354740 and diazepam, leading to the lowering of the plasma level of free diazepam, was also demonstrated. Our data suggest that the group II mGluR agonist LY354740 possesses anti-seizure activity and may modify the effects of some conventional antiepileptic drugs.

Anxiolytic and anticonvulsant properties of doramectin in rats: behavioral and neurochemistric evaluations.

Doramecin is an antiparasitic drug that may interfere with gamma-aminobutyric acid (GABA) neurotransmission. Some behavioral manifestations are related with GABAergic neurotransmissions as anxiety and seizures. The objective of the present study was to examine the possible central nervous system (CNS) effects of doramectin (100, 300 and 1000 microg/kg, SC) in rats, using anxiety behavioral models, susceptibility to seizures and central neurotransmitter evaluations. The open-field results showed (i) few alterations in locomotion frequency; (ii) a biphasic effect on rearing frequency that may be the consequence of least habituation in open-field; (iii) the reduction of grooming durations might be attributed to a possible anxiolytic effect of doramectin since GABAergic agonists reduced this parameter in apparatus. Our data in the hole board showed no effects in locomotion and rearing frequencies but increased head dipping frequency of rats administered doramectin similarly to anxiolytic drugs. In plus-maze test, doramectin administration increased the number of entries and time into open arms, indicating also an anxiolytic effect. Doramectin protected animals from convulsant effects of picrotoxin, indicative of an anxiolytic pharmacological profile of a drug with GABAergic properties. The alterations observed in central dopaminergic, noradrenergic and serotoninergic neurotransmissions might be the consequence of reinforcement in central GABAergic neurotransmission induced by doramectin. The present results suggest that doramectin has the pharmacological profile of an anxiolytic/anticonvulsant drug with GABAergic properties.

Additivities of compounds that increase the numbers of high affinity [3H]muscimol binding sites by different amounts define more than 9 GABA(A) receptor complexes in rat forebrain: implications for schizophrenia and clozapine research.

BACKGROUND: The numbers of [3H]MUS binding sites were reported to be elevated in layers II and III, but not V or VI, in cingulate cortex of schizophrenic brains post mortem. These increases in [3H]MUS binding sites are probably due to compensatory up-regulation of GABA(A) receptors on pyramidal cells as a consequence of a selective loss of GABAergic interneurons in layer II of cingulate cortex. The number of [3H]flunitrazepam binding sites was reported to be reduced in schizophrenic cingulate cortex, and this may directly reflect the loss of GABAergic interneurons. Chronic administration of clozapine to rats was reported to significantly reduce the numbers of [3H]MUS binding sites in temporal cortex and hippocampus which may be due to selective blockade of GABA(A) receptors on GABAergic interneurons that make synaptic contact with pyramidal cells. Basket cells are GABAergic interneurons that make synaptic contact with pyramidal cells as well as other interneurons. Basket cells can also generate both theta and gamma oscillations. Clozapine increases the power of theta and gamma EEG. Schizophrenic patients show reduced EEG power at 40 Hz (gamma frequency) but not at lower frequencies during auditory stimulation. The GABA(A) receptor blocker bicuculline at 10 nM, but not 10 microM, was reported to increase the amplitude of slow oscillations (< or =1 Hz) in rat hippocampal slices. It therefore seems possible that clozapine, by selectively blocking another GABA(A) receptor, could increase the amplitude of gamma oscillations. FINDINGS: Twenty-six compounds that inhibit [35S]TBPS binding in ways that are reversible by 10 nM R-5135 were found to increase [3H]MUS binding to membranes prepared from rat whole forebrain. In almost all cases the increases in binding were due to increases in the number of binding sites with little effect on affinity (Kd) for [3H]MUS. Concentration-response curves for the compounds revealed maximum increases in [3H]MUS (Esat) binding ranging from 140% (for meclizine) to 313% of control for honokiol. Additivity experiments showed that propofol (44% above control) and diflunisal (50% above control) were almost entirely additive, but there was also a small, but significant overlap, suggesting the existence of three groups of [3H]MUS binding sites defined by propofol and diflunisal. Meclizine was entirely additive with both propofol and diflunisal, indicating the existence of a fourth [3H]MUS binding site. Alphaxalone is also completely additive with meclizine, and has an Esat value significantly larger than that for propofol + diflunisal suggesting a fifth [3H]MUS binding site. The Esat for mefenamate is significantly greater than the Esat for alphaxalone, and mefenamate is also completely additive with meclizine, suggesting the existence of a sixth [3H]MUS binding site. The Esat for magnolol is significantly greater then the Esat, for mefenamate, and the Esat for honokiol is greater than that for magnolol, suggesting, but not proving, the existence of a seventh and an eighth group of [3H]MUS binding sites. The binding of [3H]MUS alone, without enhancers may represent a ninth group of binding sites which is probably heterogeneous as indicated by the very low pseudo Hill coefficients for bicuculline and strychnine in displacing [3H]MUS without enhancer. Altogether, our results suggest the existence of more than 9 different [3H]MUS binding sites. Clozapine was a very weak overall displacer of [3H]MUS (IC50 = 280 microM). However, 5 microM clozapine reduced [3H]MUS binding 6% (P < 0.0001, n = 10) and significantly reduced [3H]MUS binding enhanced by propofol (approximately 14%) or clotrimazole (approximately 17%) but not 17 other compounds tested. TENTATIVE CONCLUSIONS: In the absence of enhancers [3H]MUS may bind preferentially to GABA(A) receptors on pyramidal cells and less to interneurons in cerebral cortex. Conversely, [3H]flunitrazepam may bind preferentially to GABA(A) receptors (allosterically) on interneurons and less to pyramidal cells. Clozapine appears to selectively block a small fraction (10-20%) of [3H]MUS binding sites with an IC50 value in the low micromolar range. This fraction may be preferentially located on certain GABAergic interneurons (basket cells?) that make synaptic contact with pyramidal cells. The blockade of these GABA(A) receptors by clozapine would be expected to increase the firing rate of the interneurons and the release of GABA onto pyramidal cells. Such blockade would also increase the generation of gamma oscillations by the basket cells. Some of these interneurons appear to be destroyed selectively, probably during the second trimester of gestation by a non-paralytic polio virus, in individuals who wil

Picrotoxin in the medial prefrontal cortex impairs sensorimotor gating in rats: reversal by haloperidol.

RATIONALE: Neuropathological data indicate a GABAergic dysfunction in the prefrontal cortex and hippocampus of schizophrenics. On this basis, the construct validity of an animal model of schizophrenia was tested. OBJECTIVE: This study assessed prepulse inhibition (PPI) of startle in rats after injections of the GABA antagonist picrotoxin into the prefrontal cortex and the ventral hippocampus. It was also tested if reductions in PPI are reversed by the dopamine antagonist haloperidol. PPI is a measure of sensorimotor gating and is impaired in schizophrenia patients. The hypothesis underlying this study was that blockade of prefrontocortical and hippocampal GABA receptors disrupts PPI in a dopamine-dependent way. This hypothesis was based on neuropathological data from schizophrenics indicating a loss of GABAergic neurons in the prefrontal cortex and hippocampus and on the observation that PPI is reduced in schizophrenics. METHODS: Picrotoxin (0, 5, 10 ng/0.5 microl) was infused through chronically indwelling cannulae into the medial prefrontal cortex (mPFC), into the lateral prefrontal cortex and into the ventral hippocampus. The effect on PPI was measured directly after picrotoxin infusion. The neuroleptic compound haloperidol (0.1 mg/kg) was administered intraperitoneally 30 min before testing. RESULTS: Picrotoxin in the mPFC dose-dependently reduced PPI and this effect was antagonized by systemic pretreatment with the dopamine antagonist haloperidol. No significant effects on PPI were observed after picrotoxin infusions into the lateral prefrontal cortex or into the ventral hippocampus. CONCLUSIONS: These findings indicate that acute blockade of GABA receptors in the mPFC impairs sensorimotor gating in a dopamine-dependent manner. Since PPI in rats has been shown to possess face, predictive, and construct validity as an animal model for some psychotic symptoms, we discuss the potential relevance of our findings for the pathophysiology of schizophrenia.

Role of GABA-A and mitochondrial diazepam binding inhibitor receptors in the anti-stress activity of neurosteroids in mice.

Neuroactive steroidal modulation of immobilization-stress and possible involvement of GABA-A and mitochondrial diazepam binding inhibitor (DBI) receptors (MDR) has been investigated in mice. Immobilization of mice for 2 h induced intense antinociception, anxiety state, and associated with a fall in adrenal ascorbic acid levels. Pretreatment with high dose of progesterone (10 mg/kg), a precursor of neurosteroids, significantly decreased the stress-induced antinociception, anxiety and fall in adrenal ascorbic acid, while low doses (1 and 5 mg/kg) or hydrocortisone (10 and 100 mg/kg) were ineffective. In contrast, progesterone (1 mg/kg, for 9 days) produced a significant antistress effect, which was blocked by GABA-A antagonists picrotoxin (1 mg/kg) and bicuculline (1 mg/kg), but not by flumazenil (2 mg/kg), a specific benzodiazepine (BZD) antagonist. 4'-chlordiazepam (0.1 and 0.25 mg/kg), a specific high affinity MDR agonist, produced significant anti-stress effect in a flumazenil-insensitive manner, but was blocked by pretreatment with PK11195 (1.5 mg/kg), a selective partial agonist of MDR, and with bicuculline (1 mg/kg), a potent GABA-A receptor antagonist. At higher doses, progesterone and 4'-chlordiazepam which are effective in immobilization stress also reduced locomotion. However, lower doses of progesterone (6.5 mg/kg) neither affected locomotion, nor produced any motor toxicity on rota-rod test. At the lower doses, the MDR ligand 4'-chlordiazepam (50 micrograms/kg) decreased locomotor activity, without altering motor toxicity on rota-rod test. Further, the per se effects of these treatments on unstressed mice were not significantly different from those of untreated controls, except for plus-maze test. The antistress profile of progesterone may be attributed to the in vivo production of neurosteroid allopregnanolone, thus resembled that of BZDs. Furthermore, the antistress actions are flumazenil-resistant, reaffirming that there may be an increase in the levels of pregnane neurosteroids in vivo, which may act on a specific allosteric site on GABA-A receptors distinct from BZD site. Because 4'-chlordiazepam binds to MDRs and stimulate mitochondrial neurosteroidogenesis, the anti-stress effects of 4'-chlordiazepam may be imputed to its MDR-induced neurosteroids, which then act on GABA-A receptors. These data suggest a pivotal role for GABA-A and mitochondrial DBI receptors in the antistress actions of neurosteroids and reinforces their ameliorative effect in physiological stress.

Propofol inhibits epileptiform activity in rat hippocampal slices.

Propofol (2,6 di-isopropylphenol) is an intravenous general anesthetic used widely in neuroanesthesia, as a sedative in intensive care units, and has successfully aborted refractory status epilepticus. We investigated the effects of propofol on epileptiform activity in rat hippocampal slices. Interictal epileptiform activity was produced by bath applying one of the following: picrotoxin (PTX; 10 and 50 microM), bicucculine methiodide (BMI; 10 and 50 microM), 4-aminopyridine (4-AP; 50 microM), 8.5 mM [K+]o or 0 [Mg2+]o artificial cerebrospinal fluid. Propofol was then added in increasing concentrations and the effect on the rate of extracellular field epileptiform discharges was measured. Ictal-like discharges (> 2 Hz for > 2 s) were produced by 7.5 mM [K+]o and pilocarpine (10 microM). Propofol (30 micrograms/ml, 168 microM) completely abolished discharges induced by 8.5 mM [K+]o and at 60 micrograms/ml (337 mM) completely suppressed discharges induced by 4-AP and 0 [Mg2+]o. Propofol was less effective in reducing discharges produced by GABAA/Cl- receptor complex antagonists. Propofol at a concentration of 300 micrograms/ml (1.7 mM) was needed to reduce BMI-induced (50 microM) discharges by 77% and only reduced PTX-induced (50 microM) discharges by 20%. Ictal-like discharges produced by pilocarpine were disrupted by low concentrations of propofol (3-10 micrograms/ml, 16.9-56.2 microM) and the duration of the ictal-like discharge period was significantly reduced. We found that propofol has significant in vitro antiepileptic effects. Additionally, propofol was less effective against GABAA antagonists suggesting that the GABAA receptor complex is the site of its action.

Piracetam and aniracetam antagonism of centrally active drug-induced antinociception.

The effects of the nootropic drugs piracetam and aniracetam on antinociception induced by baclofen, bicuculline, and picrotoxin and on baclofen-induced muscle relaxation were studied in mice. Antinociception was investigated using both the hot plate (thermal stimulus) and abdominal constriction (chemical stimulus) tests. Both behaviour inhibition and muscle relaxation were observed by using the rota-rod test. Piracetam (30 mg/kg, IP) and aniracetam (10 mg/kg, PO) reduced baclofen, bicuculline, and picrotoxin antinociception without modifying analgesia induced by non-GABAergic drugs such as morphine, physostigmine, clomipramine, and diphenhydramine. In this concentration range, piracetam, and aniracetam were also able to reduce the inhibition of rota-rod performance. At higher doses piracetam (100 mg/kg, IP) and aniracetam (100 mg/kg, PO) were able to completely prevent baclofen antinociception. However, when prevention of GABAergic antinociception was complete, piracetam and aniracetam were able to block non-GABAergic antinociception also. comparing the effects of piracetam and aniracetam with those exerted by the GABAB antagonist CGP 35348, a reduction of non-GABAergic analgesia was also observed using higher doses of CGP 35348 (2.5 micrograms per mouse ICV). The present results indicate that piracetam and aniracetam, by preventing both of the investigated effects of baclofen, have some selectivity against GABAB-mediated inhibition. The well-known activity of piracetam and aniracetam on learning and memory might, therefore, depend, at least in part, on the removal of inhibitory GABAB mechanisms that impair attention and cognitive functions.

Inhibitory effect of NPY on the picrotoxin-induced activity in the hippocampus: a behavioural and electrophysiological study.

The effect of neuropeptide Y (NPY) on the picrotoxin-induced activity was studied in rat brain hippocampal slices in vitro and after intrahippocampal injection in vivo. In the hippocampal slices, NPY (0.1-0.5 microM) inhibited the picrotoxin-induced epileptiform activity recorded extracellularly in CA1 and CA3 hippocampal pyramidal cells. Similar inhibition was induced by the Y2 receptor agonist NPY13-36, which indicates that the effect of NPY was due to activation of Y2 receptors. In behavioural studies, rats with chronically implanted cannulae were injected unilaterally into the CA1 hippocampal region with a 1 ml volume of the studied substances. Picrotoxin in a dose of 1 mg (1.6 nmol) induced behavioural excitation, shakes and weak signs of epileptic behaviour. NPY in a dose of 2 mg (470 pmol), but not 1 mg, inhibited some excitatory effects of picrotoxin, but did not change the epileptic symptoms. The obtained results suggest that NPY has an inhibitory action in the hippocampus, which can be observed in vitro and also in a behavioural study.

Neuroactive steroids modulate GABA inhibition of hypothalamic somatostatin release.

The reduced steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-di-hydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC) are potent ligands of GABAA receptors. This study examined the possible modulatory effect of these metabolites on GABA inhibition of somatostatin release in cultured hypothalamic neurones. Allopregnanolone potentiates GABA inhibition, and reversed picrotoxin and bicuculline-induced augmentation of somatostatin release in a dose-dependent manner. AllotetrahydroDOC also inhibits the stimulated release induced by the antagonists, but did not modify that induced by depolarizing concentrations of K+. Pregnenolone sulphate had no effect on picrotoxin-induced somatostatin release. These findings clearly establish that 3 alpha-hydroxysteroids modulate GABA inhibition of hypothalamic somatostatin release.

[Involvement of GABAergic and NMDA systems in drug-induced convulsions in mice].

Intravenous (iv) pretreatment of muscimol, a GABAA receptor agonist, inhibited the convulsive effect of bicuculline but not that of picrotoxin, whereas phenobarbital, a Cl ion channel blocker, exhibited both effects. These results suggest that the bicuculline-induced convulsion may be due to Cl ion channel blockade secondary to the direct inhibition of GABAA receptors. CPP and MK-801, competitive and noncompetitive NMDA antagonists, respectively, inhibited both the bicuculline- and picrotoxin-induced convulsion, suggesting that these convulsive effects may also involve activation of the NMDA-Ca ion channel complex, which might link to the GABA-Cl ion channel system. Anticonvulsants phenytoin, carbamazepine and diazepam as well as phenobarbital inhibited the convulsive responses of bicuculline and picrotoxin. Therefore, the anticonvulsive effects of these drugs may involve an activation of the GABA-Cl ion channel function. On the other hand, neither the GABA agonist nor anticonvulsants affected NMDA-induced convulsion. These results also provide evidence that the convulsive effects of bicuculline and picrotoxin may be mediated by indirect activation of NMDA systems through their Cl ion channel-blocking action, whereas the convulsive effects of NMDA may involve the activation of NMDA-Ca ion channel function without the GABA-Cl ion channel activities.