Suzanne L. Hansen

Anticonvulsant and antiepileptogenic effects of GABAA receptor ligands in pentylenetetrazole-kindled mice

Although animal models based on pentylenetetrazole (PTZ) are widely used, the mechanism by which PTZ elicits its action is not very well understood. At the molecular level, a generally accepted mechanism of PTZ is noncompetitive antagonism of the gamma-aminobutyric acid (GABA)(A) receptor complex. By a systematic pharmacological investigation of various GABA(A) receptor ligands, our aim was to gain a better understanding of the GABAergic mechanisms involved in different PTZ-induced seizures. We investigated anticonvulsant effects of various specific GABA(A) receptor ligands, which are believed to bind to different binding sites on the GABA(A) receptor complex, on PTZ-induced clonic seizures in drug naive and PTZ-kindled mice as well as their effects on the development of PTZ kindling. Diazepam and alphaxalone produced potent anticonvulsant effects and completely suppressed the development of kindling. In contrast, the antagonists bicuculline and dehydroepiandrosterone sulfate (DHEAS) displayed neither anticonvulsant nor antiepileptogenic effects. Flumazenil, often used as a reference antagonist at the GABA(A) receptor benzodiazepine (BZ) binding site, lacked anticonvulsant effects but surprisingly inhibited the development of PTZ-kindled seizures. The agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol (THIP) was devoid of both anticonvulsant and antiepileptogenic effects. Marked differences in drug sensitivity were observed between models based on single and chronic administration of PTZ showing that the two sets of models are fundamentally different. These results describe the pharmacology of a set of ligands believed to bind to different sites at the GABA(A) receptor complex in animal models based on PTZ and demonstrate that a drugs action in these models cannot be readily explained by agonistic or antagonistic properties at the receptor level.

First Demonstration of a Functional Role for Central Nervous System Betaine/γ-Aminobutyric Acid Transporter (mGAT2) Based on Synergistic Anticonvulsant Action among Inhibitors of mGAT1 and mGAT2

In a recent study, EF1502 [N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo [d]isoxazol-3-ol], which is an N-substituted analog of the GAT1-selective GABA uptake inhibitor exo-THPO (4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol), was found to inhibit GABA transport mediated by both GAT1 and GAT2 in human embryonic kidney (HEK) cells expressing the mouse GABA transporters GAT1 to 4 (mGAT1–4). In the present study, EF1502 was found to possess a broad-spectrum anticonvulsant profile in animal models of generalized and partial epilepsy. When EF1502 was tested in combination with the clinically effective GAT1-selective inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid] or LU-32-176B [N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol], another GAT1-selective N-substituted analog of exo-THPO, a synergistic rather than additive anticonvulsant interaction was observed in the Frings audiogenic seizure-susceptible mouse and the pentylenetetrazol seizure threshold test. In contrast, combination of the two mGAT1-selective inhibitors, tiagabine and LU-32-176B, resulted in only an additive anticonvulsant effect. Importantly, the combination of EF1502 and tiagabine did not result in a greater than additive effect in the rotarod behavioral impairment test. In subsequent in vitro studies conducted in HEK-293 cells expressing the cloned mouse GAT transporters mGAT1 and mGAT2, EF1502 was found to noncompetitively inhibit both mGAT1 and the betaine/GABA transporter mGAT2 (Ki of 4 and 5 μM, respectively). Furthermore, in a GABA release study conducted in neocortical neurons, EF1502 did not act as a substrate for the GABA carrier. Collectively, these findings support a functional role for mGAT2 in the control of neuronal excitability and suggest a possible utility for mGAT2-selective inhibitors in the treatment of epilepsy.

GAD65 is essential for synthesis of GABA destined for tonic inhibition regulating epileptiform activity

J. Neurochem. (2010) 115, 1398–1408.

Extrasynaptic GABAA receptor activation reverses recognition memory deficits in an animal model of schizophrenia

RationaleSchizophrenia is a complex psychiatric disorder comprised of three main classes of symptoms: positive, negative and cognitive symptoms. Currently, no approved treatment exists for the cognitive symptoms. There is thus a great need for research aiming at identifying novel targets for treatment of this indication. Several neurotransmitter systems are affected in schizophrenia patients, including the γ-amino butyric acid (GABAergic) system, demonstrated by reduced parvalbumin-containing interneurons, glutamate decarboxylase (GAD) and the GABA transporter GAT-1. Furthermore, gene expression of several GABAA receptor sub-units, such as α1, α4 and δ is reduced in the dorsolateral prefrontal cortex of schizophrenia patients.ObjectivesThe psychotomimetic NMDA receptor antagonist phencyclidine (PCP) is frequently employed to model schizophrenia in animal disease models. Sub-chronic PCP treatment of female hooded Lister rats has repeatedly been shown to induce impairments in object recognition memory, and this model was therefore chosen for the examination of the potential of positive modulation of extrasynaptic GABAA receptors in alleviating the PCP-induced deficit.ResultsRats treated sub-chronically with PCP showed significant impairments in recognition memory. This deficit was reversed by positive modulation of extrasynaptic GABAA receptors.ConclusionThe present study shows that extrasynaptic GABAA receptors may present a novel target for the development of therapeutics aimed at improving cognitive deficits in schizophrenia.

Positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reverses sub-chronic PCP-induced deficits in the novel object recognition task in rats.

Cognitive deficits are a major clinical unmet need in schizophrenia. The psychotomimetic drug phencyclidine (PCP) is widely applied in rodents to mimic symptoms of schizophrenia, including cognitive deficits. Previous studies have shown that sub-chronic PCP induces an enduring episodic memory deficit in female Lister Hooded rats in the novel object recognition (NOR) task. Here we show that positive modulation of AMPA receptor (AMPAR) mediated glutamate transmission alleviates cognitive deficits induced by sub-chronic PCP treatment. Female Lister hooded rats were treated sub-chronically with either vehicle (0.9% saline) or PCP (2mg/kg two doses per day for 7 days), followed by a 7 days washout period. 30 min prior to the acquisition trial of the NOR task animals were dosed with either vehicle, CX546 (10, 40 or 80 mg/kg) or CX516 (0.5, 2.5, 10, 40 or 80 mg/kg). Our results show that sub-chronic PCP treatment induced a significant decrease in the discrimination index (DI) and both ampakines CX546 and CX516 were able to reverse this disruption of object memory in rats in the novel object recognition task. These data suggest that positive AMPAR modulation may represent a mechanism for treatment of cognitive deficits in schizophrenia.

Ketogenic diet is antiepileptogenic in pentylenetetrazole kindled mice and decrease levels of N-acylethanolamines in hippocampus

The ketogenic diet (KD) is used for the treatment of refractory epilepsy in children, however, the mechanism(s) remains largely unknown. Also, the antiepileptogenic potential in animal models of epilepsy has been poorly addressed. Activation of cannabinoid type-1 receptor (CB(1)-R) upon seizure activity may mediate neuroprotection as may several N-acylethanolamines. It is unknown how the KD interfere with the endocannabinoid system. We investigated the antiepileptogenic potential of the KD in the pentylenetetrazole kindling model in young mice and measured the hippocampal levels of CB(1)-R by Western blot and of endocannabinoids and N-acylethanolamines by mass spectrometry. The KD significantly decreased incidence and severity of seizures, and significantly increased the latency to clonic convulsions. There were no changes in levels of endocannabinoids or CB(1)-R expression by either seizure activity or type of diet. The level of oleoylethanolamide as well as the sum of N-acylethanolamines were significantly decreased by the KD, but were unaffected by seizure activity. The study shows that the KD had clear antiepileptogenic properties in the pentylenetetrazole kindling model and does not support a role of endocannabinoids in this model. The significance of the decreased hippocampal level of oleoylethanolamide awaits further studies.

Pharmacological screening of Malian medicinal plants used against epilepsy and convulsions.

ETHNOPHARMACOLOGICAL RELEVANCE Several medicinal plants are used in Mali to treat epilepsy and convulsions. So far, no studies have investigated the pharmacological effect of these plants. AIMS The aim of this study was to investigate the in vitro and in vivo antiepileptic potential of the ethanolic extracts of 11 Malian medicinal plants. MATERIALS AND METHODS The extracts were screened for antiepileptic properties in the mouse cortical wedge preparation and in the [3H]-flumazenil binding assay. Two of the plant extracts were investigated for anticonvulsive properties in the pentylenetetrazol (PTZ) kindling model in mice. Possible side effects on motor impairment were evaluated using the rota-rod test. RESULTS Extracts of 10 of the 11 medicinal plants showed affinity to the benzodiazepine binding site on the GABAA receptor. Seven of the 11 extracts inhibited the spontaneous discharges (SEDs) in the mouse cortical wedge preparation, with the extracts of Flueggea virosa and Psorospermum senegalense being the most potent. However, when tested for in vivo anticonvulsive properties these two extracts failed to show any effect on PTZ-induced seizures in mice. CONCLUSIONS The pharmacological screening of the ethanolic extracts of 11 Malian medicinal plants in vitro lead to the identification of several extracts with potential anticonvulsant properties.

Brain levels of N-acylethanolamine phospholipids in mice during pentylenetetrazol-induced seizure.

The N-acylethanolamine phospholipids (NAPE) are precursors for N-acylethanolamines (NAE), including anandamide (20∶4-NAE), which is a ligand for the cannabinoid receptors. Previously, NAPE were believed to be found only in injured tissue, e.g., after neurodegenerative insults. Neuronal injury may occur in response to seizure activity. Therefore, we investigated the effect of pentylenetetrazol (PTZ)-induced seizures in PTZ-kindled mice on the level of NAPE in the brain. Male NMRI mice were kindled with PTZ injections 3 times/wk, thereby developing clonic seizures in response to PTZ. Mice were killed within 30 min after the clonic seizure on the test day (12th injection) and the brains were collected. Eight species of NAPE were analyzed as the glycerophospho-N-acylethanolamines by high-performance liquid chromatography-coupled electrospray ionization mass spectrometry. No effect of the PTZ kindling on the NAPE levels in murine brains was observed. Total NAPE in control mice cortex (n=4) was 16.4±3.0 μmol/g wet weight of which 20∶4-NAPE accounted for 3.6 mol%, and the major species was 16∶0-NAPE, accounting for 52.1 mol%. Determination of the activity of NAPE-hydrolyzing phospholipase D and of N-acyltransferase in brain membrane preparations from adult and 3-d-old mice revealed an enzyme pattern in the adult mice that was favorable for NAE accumulation as opposed to NAPE accumulation. Thus, there was no difference in NAPE levels; at present, however, this does not exclude that NAE may accumulate during seizure.

Effects of GABAA receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions

Based on an unexpected high maximum response to piperidine‐4‐sulphonic acid (P4S) at human α1α6β2γ2 GABAA receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABAA receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine‐4‐sulphonic acid (P4S) as partial agonists, respectively, at α1β2γ2, α6β2γ2 and α1α6β2γ2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole‐cell patch‐clamp recordings were used to investigate the pharmacological profile of the partial GABAA receptor agonists 4,5,6,7‐tetrahydroisoxazolo‐(5,4‐c)pyridin‐3‐ol (THIP), P4S, 5‐(4‐piperidyl)isoxazol‐3‐ol (4‐PIOL), and 3‐(4‐piperidyl)isoxazol‐5‐ol (iso‐4‐PIOL), and the competitive GABAA receptor antagonists Bicuculline Methbromide (BMB) and 2‐(3‐carboxypropyl)‐3‐amino‐6‐methoxyphenyl‐pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly α1, α2, α3, and α5 containing receptors and α1, α6 and α1α6 containing receptors, respectively. 4‐PIOL and iso‐4‐PIOL, which at GABAA receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4‐PIOL was slightly more potent at cortical neurones than at granule neurones and iso‐4‐PIOL was more potent in inhibiting isoguvacine‐evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4‐PIOL corresponded to that of a partial agonist, whereas that of iso‐4‐PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.

GABAA receptor subunit interactions important for benzodiazepine and zinc modulation: a patch‐clamp and single cell RT‐PCR study

The expression of mRNAs for the GABAA receptor subunits α1, α6, β2, β3, γ2 and δ in single mouse cerebellar granule cells and cortical interneurons were analysed by RT‐PCR and correlated to their midazolam and zinc modulation of agonist‐induced receptor currents. The registration of molecular and electrophysiological data from each cell allowed us to estimate the significance of individual subunits and their two‐factor interaction for modulation. The presence of α6 decreased midazolam modulation, but statistical analysis also suggested interactions of α6 with β3 and γ2 with respect to midazolam modulation. Zinc modulation was decreased by the presence of γ2, and analysis points to an β3 effect as well as an interaction between γ2 and δ in zinc modulation. Thus, our model confirmed, in single native cells, the known effects of α6 in midazolam and γ2 in zinc modulation, and additionally pointed to significant subunit interactions that need to be further tested in recombinant receptors. The present study offers a method to identify subunit interactions in heteromeric receptor complexes.