Resilience of network activity in preconditioned neurons exposed to 'stroke-in-a-dish' insults.

Exposing cultured cortical neurons to stimulatory agents - the K+ channel blocker 4-aminopyridine (4-ap), and the GABAA receptor antagonist bicuculline (bic) - for 48 h induces down-regulated synaptic scaling, and preconditions neurons to withstand subsequent otherwise lethal 'stroke-in-a-dish' insults; however, the degree to which usual neuronal function remains is unknown. As a result, multi-electrode array and patch-clamp electrophysiological techniques were employed to characterize hallmarks of spontaneous synaptic activity over a 12-day preconditioning/insult experiment. Spiking frequency increased 8-fold immediately upon 4-ap/bic treatment but declined within the 48 h treatment window to sub-baseline levels that persisted long after washout. Preconditioning resulted in key markers of network activity - spiking frequency, bursting and avalanches - being impervious to an insult. Surprisingly, preconditioning resulted in higher peak NMDA mEPSC amplitudes, resulting in a decrease in the ratio of AMPA:NMDA mEPSC currents, suggesting a relative increase in synaptic NMDA receptors. An investigation of a broad mRNA panel of excitatory and inhibitory signaling mediators indicated preconditioning rapidly up-regulated GABA synthesis (GAD67) and BDNF, followed by up-regulation of neuronal activity-regulated pentraxin and down-regulation of presynaptic glutamate release (VGLUT1). Preconditioning also enhanced surface expression of GLT-1, which persisted following an insult. Overall, preconditioning resulted in a reduced spiking frequency which was impervious to subsequent exposure to 'stroke-in-a-dish' insults, a phenotype initiated predominantly by up-regulation of inhibitory neurotransmission, a lower neuronal postsynaptic AMPA: NMDA receptor ratio, and trafficking of GLT-1 to astrocyte plasma membranes.

Comparison of the toxicokinetics of the convulsants picrotoxinin and tetramethylenedisulfotetramine (TETS) in mice.

Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 µM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2ß3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.

Neurochemical effects of motor cortex stimulation in the periaqueductal gray during neuropathic pain.

OBJECTIVE: Motor cortex stimulation (MCS) is a neurosurgical technique used to treat patients with refractory neuropathic pain syndromes. MCS activates the periaqueductal gray (PAG) matter, which is one of the major centers of the descending pain inhibitory system. However, the neurochemical mechanisms in the PAG that underlie the analgesic effect of MCS have not yet been described. The main goal of this study was to investigate the neurochemical mechanisms involved in the analgesic effect induced by MCS in neuropathic pain. Specifically, we investigated the release of γ-aminobutyric acid (GABA), glycine, and glutamate in the PAG and performed pharmacological antagonism experiments to validate of our findings. METHODS: Male Wistar rats with surgically induced chronic constriction of the sciatic nerve, along with sham-operated rats and naive rats, were implanted with both unilateral transdural electrodes in the motor cortex and a microdialysis guide cannula in the PAG and subjected to MCS. The MCS was delivered in single 15-minute sessions. Neurotransmitter release was evaluated in the PAG before, during, and after MCS. Quantification of the neurotransmitters GABA, glycine, and glutamate was performed using a high-performance liquid chromatography system. The mechanical nociceptive threshold was evaluated initially, on the 14th day following the surgery, and during the MCS. In another group of neuropathic rats, once the analgesic effect after MCS was confirmed by the mechanical nociceptive test, rats were microinjected with saline or a glycine antagonist (strychnine), a GABA antagonist (bicuculline), or a combination of glycine and GABA antagonists (strychnine+bicuculline) and reevaluated for the mechanical nociceptive threshold during MCS. RESULTS: MCS reversed the hyperalgesia induced by peripheral neuropathy in the rats with chronic sciatic nerve constriction and induced a significant increase in the glycine and GABA levels in the PAG in comparison with the naive and sham-treated rats. The glutamate levels remained stable under all conditions. The antagonism of glycine, GABA, and the combination of glycine and GABA reversed the MCS-induced analgesia. CONCLUSIONS: These results suggest that the neurotransmitters glycine and GABA released in the PAG may be involved in the analgesia induced by cortical stimulation in animals with neuropathic pain. Further investigation of the mechanisms involved in MCS-induced analgesia may contribute to clinical improvements for the treatment of persistent neuropathic pain syndromes.

Treatment with lacosamide impedes generalized seizures in a rodent model of cortical dysplasia.

OBJECTIVE: Epilepsy is a common neurologic disorder resulting in spontaneous, recurrent seizures. About 30-40% of patients are not responsive to pharmacologic therapies. This may be due to the differences between individual patients such as etiology, underlying pathophysiology, and seizure focus, and it highlights the importance of new drug discovery and testing in this field. Our goal was to determine the efficacy of lacosamide (LCM), a drug approved for the treatment of focal seizures, in a model of generalized epilepsy with cortical dysplasia (CD). We sought to compare LCM to levetiracetam (LEV), a drug that is currently used for the treatment of both partial and generalized epilepsy and to test its proficiency. METHODS: Pregnant rats were irradiated to produce pups with malformed cortices in a model of CD, which will be referred to as the "first hit." Adult animals, developed normally (NL) and irradiated (XRT), were surgically implanted with electroencephalography (EEG) electrodes. Baseline EEG was recorded on all rats prior to pretreatments with either LCM, LEV, or placebo (PBO). After 30 min, all rats were injected with a subconvulsive dose of pentylenetetrazole (PTZ), a γ-aminobutyric acid receptor A (GABAA ) antagonist used to provoke generalized seizures as a "second hit." RESULTS: LCM and LEV were both effective against seizures induced by PTZ. XRT rats had a higher seizure incidence with longer and more severe seizures than NL rats. Seizure duration was decreased with both LCM and LEV in all animals. In XRT rats, there was a significant reduction in acute seizure incidence and severity with both LCM and LEV after PTZ injection. SIGNIFICANCE: Our results suggest that LCM could be used as a potential treatment option for generalized epilepsy with CD as the underlying pathology.

Mechanisms of Action, Resistance and Toxicity of Insecticides Targeting GABA Receptors.

BACKGROUND: γ-Aminobutyric acid (GABA) receptors play a central role in fast inhibitory neurotransmission in insects. Several classes of insecticides targeting insect GABA-gated chloride channels have been developed. The important resistant to dieldrin GABA receptor subunit (RDL) has been used to investigate insecticide sites of action using radioligands, electrophysiology and site-directed mutagenesis. Although this important subunit readily forms robust functional homomeric receptors when expressed, alternative splicing and RNA A-to-I editing can generate diverse forms of the receptor. METHODS: We have reviewed studies on native and recombinant insect GABA-gated chloride channels, their interactions with ligands acting at orthosteric and allosteric sites and their interactions with insecticides. Since some GABA receptor modulators act on L-glutamate-gated chloride channels, some comparisons are included. RESULTS: The actions on GABA-gated chloride channels of polychlorocycloalkanes, cyclodienes, macrocyclic lactones, phenylpyrazoles, isoxazolines, and metadiamides are described and the mechanisms of action of members of these insecticide classes are addressed. Mutations that lead to resistance are discussed as they can be important in developing field diagnostic tests. Toxicity issues relating to insecticides targeting GABA-gated chloride channels are also addressed. An overview of all major insecticide classes targeting insect GABA-gated chloride channels has enhanced our understanding of these important receptors and their insecticide binding sites. However, the subunit composition of native GABA receptors remains unknown and studies to clarify this are needed. Also, the precise sites of action of the recently introduced isoxazolines and meta-diamides will be of interest to pursue.

Chronic GABAergic blockade in the spinal cord in vivo induces motor alterations and neurodegeneration.

Inhibitory GABAergic and glycinergic neurotransmission in the spinal cord play a central role in the regulation of neuronal excitability, by maintaining a balance with the glutamate-mediated excitatory transmission. Glutamatergic agonists infusion in the spinal cord induce motor neuron death by excitotoxicity, leading to motor deficits and paralysis, but little is known on the effect of the blockade of inhibitory transmission. In this work we studied the effects of GABAergic and glycinergic blockade, by means of microdialysis perfusion (acute administration) and osmotic minipumps infusion (chronic administration) of GABA and glycine receptors antagonists directly in the lumbar spinal cord. We show that acute glycinergic blockade with strychnine or GABAergic blockade with bicuculline had no significant effects on motor activity and on motor neuron survival. However, chronic bicuculline infusion, but not strychnine, induced ipsilateral gait alterations, phalange flaccidity and significant motor neuron loss, and these effects were prevented by AMPA receptor blockade with CNQX but not by NMDA receptor blockade with MK801. In addition, we demonstrate that the chronic infusion of bicuculline enhanced the excitotoxic effect of AMPA, causing faster bilateral paralysis and increasing motor neuron loss. These findings indicate a relevant role of GABAergic inhibitory circuits in the regulation of motor neuron excitability and suggest that their alterations may be involved in the neurodegeneration processes characteristic of motor neuron diseases such as amyotrophic lateral sclerosis.

Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels.

Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 µM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 µM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to 10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1ß2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs.

Combined diazepam and MK-801 therapy provides synergistic protection from tetramethylenedisulfotetramine-induced tonic-clonic seizures and lethality in mice.

The synthetic rodenticide, tetramethylenedisulfotetramine (TMDT), is a persistent and highly lethal GABA-gated Cl(-) channel blocker. TMDT is clandestinely produced, remains popular in mainland China, and causes numerous unintentional and deliberate poisonings worldwide. TMDT is odorless, tasteless, and easy to manufacture, features that make it a potential weapon of terrorism. There is no effective treatment. We previously characterized the effects of TMDT in C57BL/6 mice and surveyed efficacies of GABAergic and glutamatergic anticonvulsant treatments. At 0.4 mg/kg i.p., TMDT produced neurotoxic symptomatology consisting of twitches, clonic and tonic-clonic seizures, often progressing to status epilepticus and death. If administered immediately after the occurrence of the first clonic seizure, the benzodiazepine diazepam (DZP) effectively prevented all subsequent seizure symptoms, whereas the NMDA receptor antagonist dizocilpine (MK-801) primarily prevented tonic-clonic seizures. The latter agent, however, appeared to be more effective at preventing delayed death. The present study further explored these phenomena, and characterized the therapeutic actions of DZP and MK-801 as combinations. Joint treatment with both DZP and MK-801 displayed synergistic protection against tonic-clonic seizures and 24 h lethality as determined by isobolographic analysis. Clonic seizures, however, remained poorly controlled. A modification of the treatment regimen, where DZP was followed 10 min later by MK-801, yielded a reduction in both types of seizures and improved overall outcome. Simultaneous monitoring of subjects via EEG and videography confirmed effectiveness of this sequential regimen. We conclude that TMDT blockage at GABAA receptors involves early activation of NMDA receptors, which contribute to persistent ictogenic activity. Our data predict that a sequential combination treatment with DZP followed by MK-801 will be superior to either individual therapy with, or simultaneous administration of, these two agents in treating TMDT poisoning.

Distinct Neuropsychological Mechanisms May Explain Delayed- Versus Rapid-Onset Antidepressant Efficacy.

The biochemical targets for antidepressants are relatively well established, but we lack a clear understanding of how actions at these proteins translate to clinical benefits. This study used a novel rodent assay to investigate how different antidepressant drugs act to modify affective biases that have been implicated in depression. In this bowl-digging task, rats encounter two equal value learning experiences on separate days (one during an affective manipulation and the other during control conditions). This induces an affective bias that is quantified using a preference test in which both digging substrates are presented together and the individual rats' choices recorded. The assay can be used to measure affective biases associated with learning (when the treatment is given at the time of the experience) or examine the modification of previously acquired biases (when the treatment is administered before the preference test). The rapid-onset antidepressant ketamine, but not the delayed-onset antidepressant, venlafaxine, attenuated the previously acquired FG7142-induced negative bias following systemic administration. Venlafaxine but not ketamine induced a positive bias when administered before learning. We then used local drug infusions and excitotoxic lesions to localize the effects of ketamine to the medial prefrontal cortex and venlafaxine to the amygdala. Using a modified protocol we also showed that positive and negative biases amplified further when the numbers of substrate-reinforcer associations are increased. We propose that this pattern of results could explain the delayed onset of action of venlafaxine and the rapid onset of action but lack of long-term efficacy seen with ketamine.

Exploration of Daphnia behavioral effect profiles induced by a broad range of toxicants with different modes of action.

Behavior is increasingly reported as a sensitive and early indicator of toxicant stress in aquatic organisms. However, the systematic understanding of behavioral effects and comparisons between effect profiles is hampered because the available studies are limited to few chemicals and differ in the exposure conditions and effect parameters examined. The aims of the present study were 1) to explore behavioral responses of Daphnia magna exposed to different toxicants, 2) to compare behavioral effect profiles with regard to chemical modes of action, and 3) to determine the sensitivity and response time of behavioral parameters in a new multi-cell exposure system named Multi-DaphTrack compared with currently utilized tests. Twelve compounds covering different modes of toxic action were selected to sample a wide range of potential effect profiles. Acute standard immobilization tests and 48 h of behavioral tracking were performed in the customized Multi-DaphTrack system and a single-cell commercialized biological early warning system. Contrasting behavioral profiles were observed for average speed (i.e., intensity, time of effect onset, effect duration), but no distinct behavioral profiles could be drawn from the chemical mode of action. Most compounds tested in the Multi-DaphTrack system induced an early and significant average speed increase at concentrations near or below the 10% effective concentration (48 h) of the acute immobilization test, demonstrating that the Multi-DaphTrack system is fast and sensitive. To conclude, behavior endpoints could be used as an alternative or complement to the current acute standard test or chemical analysis for the predictive evaluation of ecotoxic effects of effluents or water bodies.

Postnatal alterations in GABAB receptor tone produce sensorimotor gating deficits and protein level differences in adulthood.

The GABA transmitter system plays a vital role in modulating synaptic formation and activity during development. The GABAB receptor subtype in particular has been implicated in cell migration, promotion of neuronal differentiation, neurite outgrowth, and synapse formation but it's role in development is not well characterized. In order to investigate the effects of brief alterations in GABAB signaling in development, we administered to rats the GABAB agonist baclofen (2.0mg/kg) or antagonist phaclofen (0.3mg/kg) on postnatal days 7, 9, and 12, and evaluated sensorimotor gating in adulthood. We also examined tissue for changes in multiple proteins associated with GABAB receptor function and proteins associated with synapse formation. Our data indicate that early postnatal alterations to GABAB receptor-mediated signaling produced sex differences in sensorimotor gating in adulthood. Additionally, we found differences in GABAB receptor subunits and kalirin protein levels in the brain versus saline treated controls. Our data demonstrate that a subtle alteration in GABAB receptor function in early postnatal life induces changes that persist into adulthood.

Parvalbumin immunoreactivity and expression of GABAA receptor subunits in the thalamus after experimental TBI.

Traumatic brain injury (TBI) causes 10-20% of acquired epilepsy in humans, resulting in an ictogenic region that is often located in the cerebral cortex. The thalamus provides heavy projections to the cortex and the activity of thalamocortical pathways is controlled by GABAergic afferents from the reticular nucleus of the thalamus (RT). As rats with TBI induced by lateral fluid-percussion injury (FPI) undergo epileptogenesis, we hypothesized that damage to the parvalbumin (PARV)-immunoreactive (ir) neurons in the RT is associated with seizure susceptibility after lateral FPI. To address this hypothesis, adult Sprague-Dawley rats (n=13) were injured with lateral FPI. At 6months post-TBI, each animal underwent a pentylenetetrazol (PTZ) seizure susceptibility test and 2weeks of continuous video-electroencephalography (EEG) monitoring for detection of the occurrence of spontaneous seizures. Thereafter, the brain was processed for PARV immunohistochemistry. We (a) estimated the total number of PARV-ir neurons in the RT using unbiased stereology, (b) measured the volume of the ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei of the thalamus, which receive PARV-ir inputs from the RT and project to the perilesional cortex, (c) quantified the density of PARV-ir terminals in the VPM-VPL, and (d) studied the expression of GABAA receptor subunits in a separate group of rats using laser-dissection of the thalamus followed by Real-Time polymerase chain reaction (RT-PCR) array studies. At 6months post-TBI, only 64% of PARV-ir neurons were remaining in the RT ipsilaterally (p<0.001 as compared to controls) and 84% contralaterally (p<0.05). Accordingly, the volume of the ipsilateral RT was 58% of that in controls ipsilaterally (p<0.001) and 90% contralaterally (p>0.05). Also, the volume of the VPM-VPL was only 51% of that in controls ipsilaterally (p<0.001) and 91% contralaterally (p<0.05). The density of PARV-ir axonal labeling was remarkably increased in the lateral aspects of the VPM and VPL (both p<0.001). Expression of the ε- and θ-subunits of the GABAA receptor was down-regulated (0.152, p<0.01 and 0.302, p<0.05, respectively), which could relate to the inclusion of the hypothalamus into the tissue analyzed with RT-PCR arrays. In controls, the lower the number of PARV-ir neurons in the RT, the higher the seizure susceptibility in the PTZ test. Rats with TBI showed seizure susceptibility comparable to that in controls with the lowest number of PARV-ir neurons in the RT. Our data show that the RT and VPM-VPL undergo remarkable degeneration after lateral-FPI which results in reorganization of PARV-ir terminals in the VPM-VPL. The contribution of RT damage to seizure susceptibility and post-traumatic epileptogenesis deserves further studies.

Insect γ-aminobutyric acid receptors and isoxazoline insecticides: toxicological profiles relative to the binding sites of [³H]fluralaner, [³H]-4'-ethynyl-4-n-propylbicycloorthobenzoate, and [³H]avermectin.

Isoxazoline insecticides, such as fluralaner (formerly A1443), are noncompetitive γ-aminobutyric acid (GABA) receptor (GABA-R) antagonists with selective toxicity for insects versus mammals. The isoxazoline target in house fly ( Musca domestica ) brain has subnanomolar affinity for [³H]fluralaner and a unique pattern of sensitivity to isoxazolines and avermectin B(1a) (AVE) but not to fipronil and α-endosulfan. Inhibitor specificity profiles for 15 isoxazolines examined with Musca GABA-R and [³H]fluralaner, [³H]-4'-ethynyl-4-n-propylbicycloorthobenzoate ([³H]EBOB), and [³H]AVE binding follow the same structure-activity trends although without high correlation. The 3 most potent of the 15 isoxazolines tested in Musca [³H]fluralaner, [³H]EBOB, and [³H]AVE binding assays and in honeybee (Apis mellifera) brain [³H]fluralaner assays are generally those most toxic to Musca and four agricultural pests. Fluralaner does not inhibit [³H]EBOB binding to the human GABA-R recombinant ß3 homopentamer, which is highly sensitive to all of the commercial GABAergic insecticides. The unique isoxazoline binding site may resurrect the GABA-R as a major insecticide target.

FG7142, yohimbine, and ßCCE produce anxiogenic-like effects in the elevated plus-maze but do not affect brainstem activated hippocampal theta.

The neurobiological underpinnings of anxiety are of paramount importance to selective and efficacious pharmaceutical intervention. Hippocampal theta frequency in urethane anaesthetized rats is suppressed by all known (and some previously unknown) anti-anxiety (anxiolytic) drugs. Although these findings support the predictive validity of this assay, its construct validity (i.e., whether theta frequency actually indexes anxiety per se) has not been a subject of systematic investigation. We reasoned that if anxiolytic drugs suppress hippocampal theta frequency, then drugs that increase anxiety (i.e., anxiogenic agents) should increase theta frequency, thus providing evidence of construct validity. We used three proven anxiogenic drugs--two benzodiazepine receptor inverse agonists, N-methyl-ß-carboline-3-carboxamide (FG7142) and ß-carboline-3-carboxylate ethyl ester (ßCCE), and one α2 noradrenergic receptor antagonist, 17α-hydroxy-yohimban-16α-carboxylic acid methyl ester (yohimbine) as pharmacological probes to assess the construct validity of the theta model. Although all three anxiogenic drugs significantly increased behavioural measures of anxiety in the elevated plus-maze, none of the three increased the frequency of hippocampal theta oscillations in the neurophysiological model. As a positive control, we demonstrated that diazepam, a proven anxiolytic drug, decreased the frequency of hippocampal theta, as in all other studies using this model. Given this discrepancy between the significant effects of anxiogenic drugs in the behavioural model and the null effects of these drugs in the neurophysiological model, we conclude that the construct validity of the hippocampal theta model of anxiety is questionable.

Decreased GABABR expression and increased neuronal cell death in developing rat brain after PTZ-induced seizure.

The objective of this study was to evaluate the PTZ-induced seizures effects on GABAB receptor (R) expression and to observe its neurodegenerative effect in hippocampal part of developing rat brain. In the present study, high dose of pentylenetetrazol (PTZ 40 mg/kg) was injected in developing rats of age 5 weeks having average weight of 60-65 g for 4 days. Further, baclofen (B 3 mg/kg i.p) agonist and phaclofen (P 30 µg/rat) antagonist of GABABR were injected along with PTZ. Western blot analysis was used to elucidate expression of GABABR protein upon PTZ, baclofen and phaclofen exposure in the developing rat brain. Furthermore, PTZ-induced apoptotic neurodegeneration was also observed through the release of caspase-3 antibody and propidium iodide (PI) staining using confocal microscopy. Seizure was confirmed using electroencephalography (EEG) data obtained from the Laxtha EEG-monitoring device in the EEG recording room and EEG was monitored 5-15 min after PTZ injection. The results of the present study showed that PTZ-induced seizure significantly decreased GABABR expression and induced neuronal apoptosis in cortical and hippocampal part of brain. While, baclofen reverse the effect of PTZ by increasing the expression of GABABR as compared to the PTZ- , PTZ plus B- and PTZ plus P-treated groups. Our findings indicated that PTZ-induced seizure showed not only decrease in GABABR expression but also cause neuronal apoptosis in the developing rat brain.

GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABA(A) receptors (GABA(A)-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABA(A)-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na(+)K(+)2Cl(-) co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABA(A)-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics.

Modelling neurological diseases has proven extraordinarily difficult due to the phenotypic complexity of each disorder. The zebrafish has become a useful model system with which to study abnormal neurological and behavioural activity and holds promise as a model of human disease. While most of the disease modelling using zebrafish has made use of adults, larvae hold tremendous promise for the high-throughput screening of potential therapeutics. The further development of larval disease models will strengthen their ability to contribute to the drug screening process. Here we have used zebrafish larvae to model the symptoms of bipolar disorder by treating larvae with sub-convulsive concentrations of the GABA antagonist pentylenetetrazol (PTZ). A number of therapeutics that act on different targets, in addition to those that have been used to treat bipolar disorder, were tested against this model to assess its predictive value. Carbamazepine, valproic acid, baclofen and honokiol, were found to oppose various aspects of the PTZ-induced changes in activity. Lidocaine and haloperidol exacerbated the PTZ-induced activity changes and sulpiride had no effect. By comparing the degree of phenotypic rescue with the mechanism of action of each therapeutic we have shown that the low-concentration PTZ model can produce a number of intermediate phenotypes that model symptoms of bipolar disorder, may be useful in modelling other disease states, and will help predict the efficacy of novel therapeutics.

Voluntary exercise offers anxiolytic potential and amplifies galanin gene expression in the locus coeruleus of the rat.

Although exercise improves anxiety in humans, it is controversial whether exercise is anxiolytic in rodents. We tested the hypothesis that stress influences the effect of exercise on anxiety-like and defensive behaviors. To explore the neurobiological mechanisms of exercise, we also examined whether exercise alters gene expression for the stress-related peptide galanin. Rats were housed in the presence or absence of a running wheel for 21 d. A subset of these rats were (1) not injected or received a single high, dose of the ß-carboline FG7142 (inverse agonist at the benzodiazepine receptor site) immediately prior to testing or (2) were injected repeatedly with vehicle or FG7142 during the last 10d of exercise. On day 22, anxiety-like and defensive behaviors were measured in the elevated plus maze, shock probe defensive burying, and defensive withdrawal tests. Locus coeruleus prepro-galanin mRNA was measured by in situ hybridization. Exercise and sedentary rats that were not injected exhibited similar behavior in all tests, whereas FG7142 injected immediately prior to the test battery produced intense avoidance and immobility consistent with an anxiety-like response. However, exercise produced anxiolytic-like and active defensive behaviors in the test battery relative to the sedentary condition in rats injected repeatedly with vehicle or FG7142. Exercise also increased prepro-galanin mRNA in the locus coeruleus relative to sedentary controls. These data suggest that the emergence of enhanced adaptive behavior after chronic voluntary exercise is influenced by stress. Our data support a role for galanin in the beneficial consequences of wheel running.

A single amino acid determines the toxicity of Ginkgo biloba extracts.

Ginkgo biloba extracts are currently used for a wide range of health-related conditions. Some of the medical benefits of these extracts are controversial, but their lack of toxicity in humans is not in doubt. These extracts are, however, highly toxic to insects. Their active components (ginkgolides and bilobalide) have structures similar to the convulsant picrotoxin, a GABA(A) receptor antagonist, so their lack of toxicity in mammals is puzzling. Here, we show that the different compositions of insect and vertebrate GABA receptor pores are responsible for the differing toxicities. Insect GABA receptors contain Ala at their 2' position in the pore. Substitution with Val, which is the equivalent residue in vertebrate GABA(A) receptor α-subunits, decreases ginkgolide potency by up to 10,000-fold. The reverse mutation in vertebrate GABA(A) α1 subunits increased the sensitivity of α1ß2 and α1ß2γ2 receptors to ginkgolides. Mutant cycle analysis demonstrates a strong interaction between the ginkgolides and the 2' residue, a result supported by in silico docking of compounds into a model of the pore. We conclude that the insecticidal activity of G. biloba extracts can be attributed to their effects at insect GABA receptors, and the presence of a Val at the 2' position in vertebrate GABA(A) receptors explains why these compounds are not similarly toxic to humans.

Differential modulatory actions of GABAA agonists on susceptibility to GABAA antagonists-induced seizures in morphine dependent rats: possible mechanisms in seizure propensity.

In order to clarify the mechanisms involved in the susceptibility to GABA(A) antagonists-induced seizures in morphine dependent rats, we investigated how GABA(A) agonists modulate this vulnerability. Seizures were induced to animals by infusion of GABA(A) antagonists: pentylenetetrazole (PTZ), picrotoxin (PIC) and bicuculline (BIC). GABA(A) agonists, muscimol (MUS) and 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol (THIP), were administered intravenous (i.v.) before antagonists. Morphine-dependence significantly decreased the PTZ threshold dose (19.16±1.89 versus 25.74±1.25mg/kg) while, it had no effect on PIC induced seizures. BIC doses for both threshold and tonic-clonic seizures induction were significantly lower in morphine dependent rats (0.10±0.01 and 0.12±0.02 versus 0.25±0.02 and 0.39±0.07mg/kg respectively). In morphine-dependence, although pre-treatment with MUS significantly increased the required dose of PTZ for seizures threshold, THIP significantly decreased the required dose of PTZ for tonic-clonic convulsion. Moreover, MUS pretreatment completely recovered the effect of morphine dependency on BIC seizure activity. The results suggest that the capability of GABA(A) agonists on modulation of propensity to seizures induced by different antagonists in morphine-dependence is dissimilar. Therefore, it seems that long-term morphine alters some properties of GABA system so that the responsive rate of GABA(A) receptors not only to its antagonists, but also to its agonists will change differently.