A novel allosteric modulator of the cannabinoid CB1 receptor ameliorates hyperdopaminergia endophenotypes in rodent models.

The endocannabinoid system (eCBs) encompasses the endocannabinoids, their synthetic and degradative enzymes, and cannabinoid (CB) receptors. The eCBs mediates inhibition of neurotransmitter release and acts as a major homeostatic system. Many aspects of the eCBs are altered in a number of psychiatric disorders including schizophrenia, which is characterized by dysregulation of dopaminergic signaling. The GluN1-Knockdown (GluN1KD) and Dopamine Transporter Knockout (DATKO) mice are models of hyperdopaminergia, which display abnormal psychosis-related behaviors, including hyperlocomotion and changes in pre-pulse inhibition (PPI). Here, we investigate the ability of a novel CB1 receptor (CB1R) allosteric modulator, ABM300, to ameliorate these dysregulated behaviors. ABM300 was characterized in vitro (receptor binding, ß-arrestin2 recruitment, ERK1/2 phosphorylation, cAMP inhibition) and in vivo (anxiety-like behaviors, cannabimimetic effects, novel environment exploratory behavior, pre-pulse inhibition, conditioned avoidance response) to assess the effects of the compound in dysregulated behaviors within the transgenic models. In vitro, ABM300 increased CB1R agonist binding but acted as an inhibitor of CB1R agonist induced signaling, including ß-arrestin2 translocation, ERK phosphorylation and cAMP inhibition. In vivo, ABM300 did not elicit anxiogenic-like or cannabimimetic effects, but it decreased novelty-induced hyperactivity, exaggerated stereotypy, and vertical exploration in both transgenic models of hyperdopaminergia, as well as normalizing PPI in DATKO mice. The data demonstrate for the first time that a CB1R allosteric modulator ameliorates the behavioral deficits in two models of increased dopamine, warranting further investigation as a potential therapeutic target in psychiatry.

Development of propagated discharge and behavioral arrest in hippocampal and amygdala-kindled animals.

Patients with focal temporal lobe seizures often experience transient episodes of impaired awareness with behavioral arrest, but the precise mechanism remains unknown. The Network Inhibition Hypothesis attributes these deficits to a loss of cholinergic input to the cortex. This is presumed to result from increased activation of inhibitory regions that suppress subcortical arousal, giving rise to cortical delta wave activity. Recently, this hypothesis has been tested in animal experiments, where triggering dorsal hippocampal seizures is associated with behavioral arrest. To further test this hypothesis in animals - and, more specifically, to characterize the relationship between propagated discharge, cortical delta waves and behavioral arrest - we performed partial kindling studies in three different limbic sites in rats. We found that seizure discharge took longer to spread from the amygdala than the hippocampus, and took more stimulations to elicit behavioral arrest. In addition, the onset of propagated discharge in subcortical and cortical sites did not always match with the onset of behavioral arrest. Importantly, the activity seen in the cortex did not resemble the slow waves seen in deep sleep. Together, these findings suggest that limbic discharge triggers epileptic discharge in downstream pacemakers, including the cortex, and that these secondarily cause behavioral arrest.

Dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats.

PURPOSE: Linoleic and alpha-linolenic polyunsaturated fatty acids, derived from plant oils, have been reported to reduce neuronal excitability ex vivo and in cell culture. The evidence derived from animal seizure models, however, has been contradictory. The goal of the present study was to assess the dose-dependent anticonvulsant effects of a fatty acid mixture containing linoleic and alpha-linolenic acids in a 4 to 1 ratio (the "SR-3" compound). METHODS: The maximal pentylenetetrazol seizure model and Long-Evans hooded rats were used. RESULTS: Daily intraperitoneal injection of SR-3 for 21 consecutive days raised omega-3 polyunsaturated fatty acid (n-3 PUFA) composition in the unesterified fatty acid fraction of brain lipids (p < 0.05), and increased latency to seizure onset when administered at 200 mg/kg (p < 0.05), but not at 40 mg/kg (p > 0.05). There were no significant effects of SR-3 on seizure occurrence or on seizure severity (p > 0.05). A toxic effect of the SR-3 compound on peristalsis was observed at a dose of 400 mg/kg and above. CONCLUSION: Linoleic and alpha-linolenic polyunsaturated fatty acids in a 4 to 1 ratio raises n-3 PUFA composition of unesterified fatty acids in the brain and increases resistance to pentylenetetrazol-induced seizures.

Assessing the link between omega-3 fatty acids, cardiac arrest, and sudden unexpected death in epilepsy.

People with epilepsy may have abnormal cardiac function. This has been linked to a greater incidence of sudden unexpected death in epilepsy (SUDEP). In the present review, we assess the evidence linking cardiac failure to SUDEP, and propose the use of the maximal pentylenetetrazol seizure test to model SUDEP in animals to identify causal links between cardiac failure and SUDEP. We also discuss recent claims on the use of omega-3 polyunsaturated fatty acids to reduce the incidence of SUDEP because of their cardioprotective and anticonvulsant effects.

G protein-coupled receptors disrupted in human genetic disease.

Genetic variation in G protein-coupled receptors (GPCRs) results in the disruption of GPCR function in a wide variety of human genetic diseases. In vitro strategies have been used to elucidate the molecular pathologies that underlie naturally occurring GPCR mutations. Various degrees of inactive, overactive, or constitutively active receptors have been identified. These mutations often alter ligand binding, G protein coupling, receptor desensitization, and receptor recycling. The role of inactivating and activating calcium-sensing receptor (CASR) mutations is discussed with respect to familial hypocalciuric hypercalemia (FHH) and autosomal dominant hypocalemia (ADH). Among ADH mutations, those associated with tonic-clonic seizures are discussed. Other receptors discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone, luteinizing hormone, gonadotropin-releasing hormone (GnRHR), adrenocorticotropic hormone, vasopressin, endothelin-beta, purinergic, and the G protein associated with asthma (GPRA). Diseases caused by mutations that disrupt GPCR function are significant because they might be selectively targeted by drugs that rescue altered receptors. Examples of drug development based on targeting GPCRs mutated in disease include the calcimimetics used to compensate for some CASR mutations, obesity therapeutics targeting melanocortin receptors, interventions that alter GnRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor in a rare bleeding disorder. The discovery of GPRA suggests that drug screens against variant GPCRs may identify novel drugs. This review of the variety of GPCRs that are disrupted in monogenic disease provides the basis for examining the significance of common pharmacogenetic variants.

A functional G300S variant of the cysteinyl leukotriene 1 receptor is associated with atopy in a Tristan da Cunha isolate.

Atopy is a well-defined immune phenotype that is reported to be a risk factor for asthma. Among the many loci that contribute to a genetic predisposition to asthma, the cysteinyl leukotriene receptor genes and their variants have been important subjects of study because they are functionally and pharmacologically implicated in the atopy phenotype affecting many asthma subjects. Moreover, the product of cysteinyl-leukotriene 1 receptor gene (CysLT1), located at Xq13.2, is targeted by LT receptor antagonists. In our earlier association study, the M201V variant of the cysteinyl-leukotriene 2 receptor gene (CysLT2), located at 13q14, was implicated in atopic asthma. Here we report the screening of the coding region of the CysLT1, gene in the highly asthmatic Tristan da Cunha population. In this population, we discovered a CysLT1 G300S variant that is carried with a significantly higher frequency in atopics and asthmatics from the Tristan da Cunha population. Furthermore, we report the asthma independent association of the CysLT1 G300S variant with atopy. Subsequently, we compared the changes conferred by each SNP on CysLT function. The CysLT1 300S receptor interacts with LTD4 with significantly greater potency. For the 300S variant, a statistically significant decrease in the effector concentration for half-maximum response (EC50) for intracellular Ca flux and total InsP generation is observed. Other aspects of the receptor function and activity, such as desensitization, pharmacologic profile in response to montelukast, and cellular localization, are unchanged. These in vitro analyses provide evidence that the 300S CysLT1 variant, found more commonly in atopics in the Tristan da Cunha population, encodes a functionally more sensitive variant.

The anticonvulsant effects of progesterone and its metabolites on amygdala-kindled seizures in male rats.

Progesterone is a neurosteroid that modulates neuronal excitability. The anticonvulsant effects of progesterone are largely mediated by the actions of its metabolites. The purpose of this study was to measure the anticonvulsant effects of progesterone, 5alpha-dihydroprogesterone, and allopregnanolone against amygdala-kindled seizures in male rats. The amygdala kindling model is a model of human complex partial seizures with secondary generalization. A bipolar electrode was chronically implanted in the right amygdala of male Wistar rats. All subjects were kindled to 30 stage 5 seizures and stability tested. Multiple doses of progesterone, 5alpha-dihydroprogesterone, or allopregnanolone were administered in separate dose-response studies. The antiseizure effects of each compound were determined. A progesterone time-response study was also conducted. At 30 min after injection, progesterone had an ED50 of 65.3 mg/kg against the secondarily generalized seizure and an ED50 of 114 mg/kg against the focal seizure. 5alpha-dihydroprogesterone had a low ED50 of 6.2 mg/kg against both the generalized component of the amygdala-kindled seizure and the focal seizure. Allopregnanolone had an ED50 of 15.2 mg/kg against the secondarily generalized seizure and was not effective against the focal seizure. Progesterone is an effective anticonvulsant against the secondarily generalized component of amygdala-kindled seizures in male rats. Progesterone is only effective against the focal seizure at high ataxic doses. 5alpha-dihydroprogesterone is a potent anticonvulsant against both the kindled amygdala focal discharge and the secondarily generalized seizure. Allopregnanolone is an effective anticonvulsant against the secondarily generalized component of the seizure, but not against the amygdala focal discharge.

Might astrocytes play a role in maintaining the seizure-prone state?

The amygdala-kindling model is used to study complex partial epilepsy with secondary generalization. The present study was designed to (A) quantify astrocytic changes in the piriform cortex of amygdala-kindled subjects over time and (B) investigate the role that astrocytes might play in maintaining the seizure-prone state. In Study A, once the experimental subjects reached five stage 5 seizures, stimulation was stopped, and both kindled and control rats were allowed to survive for the interval appropriate to their group (7, 18, 30, or 90 days). Following each interval, the kindled and control animals were given 10 intraperitoneal injections of bromodeoxyuridine (BrdU) and sacrificed 24 h following the last injection. Significantly higher numbers of dividing astrocytes (identified by co-labeling for BrdU and to one of the astrocytic intermediate filament proteins glial fibrillary acidic protein or vimentin) were found in the kindled brains. All kindled groups had significantly higher numbers of double-labeled cells on the side contralateral to the stimulation site, except for those in the 90 day survival group. In Study B, rats were implanted with chemotrodes, were kindled as in Study A, and were subsequently infused with either saline or with L alpha-AA (to lesion astrocytes) during a further 25 stimulations (1/day). L alpha-AA infused rats had significantly diminished levels of behavioral seizures, higher after discharge thresholds, lower after discharge durations, and decreased numbers of double-labeled astrocytes in piriform cortex than did saline infused rats. Together, the data indicate that astrocytes may play a role in maintaining the seizure-prone state.

Effect of the ketogenic diet on the activity level of Wistar rats.

Children, adolescents, and adults with epilepsy often also show symptoms associated with attention-deficit/hyperactivity disorder (ADHD). The ketogenic diet, which is administered to children with epilepsy refractory to drug therapy, seems to improve behavior in individuals with symptoms of ADHD. The basis for this improvement is unknown, although it seems to be unrelated to seizure control. The present research was designed to investigate the effect of two ketogenic diets on the behavior of normal adult male rats. Two experiments were conducted. In experiment 1, 36 subjects were placed on one of three diets: a control diet, a 6.3:1 ketogenic diet, and a 4:1 ketogenic diet. In experiment 2, 20 subjects were placed either on a control diet or on a 4:1 ketogenic diet. The activity level of each subject was measured using an open field test. Time spent immobile, grooming, and in exploratory behavior was measured for 600 s. Subjects were tested once before initiation of the diets and once while on the diets. No significant group differences were found in activity level before initiation of the diets. After initiation of the diets, subjects in both ketogenic groups showed a significantly lower activity level than the rats on the control diet. The ketogenic diet decreases activity level in an animal model. This behavioral change may relate to the improved behavior seen when children with symptoms of ADHD are placed on the diet.

The effect of the 'classic' ketogenic diet on animal seizure models.

Bough et al. have recently demonstrated anticonvulsant effects of the 'classic' ketogenic diet (KD) in the pentylenetetrazol infusion model in rats. Proconvulsant effects were seen, however, when the 'classic' diet was tested against maximal electroshock (MES) seizures. These differing results may reflect the fact that the two models involve different kinds of epileptogenic stimulus, or, as Bough et al. note that the two tests involve different stimulation paradigms. The pentylenetetrazol infusion paradigm is a threshold test, whereas the MES test employs a stimulus which is well above threshold. The present experiments were designed to test the effects of the 'classic' KD against seizures triggered in rats by both threshold and suprathreshold levels of electricity and pentylenetetrazol. The threshold tests employed were the pentylenetetrazol infusion test, and the threshold electroconvulsive shock (ECS) test. The subcutaneous pentylenetetrazol (scMET) test was also included, since it is sometimes considered to be a 'threshold' test. The suprathreshold tests employed were the maximal pentylenetetrazol test (MMT) and the maximal electroshock test (MES). The KD failed to suppress seizures in either of the tests involving suprathreshold stimulation (MMT and MES), although there was a significant increase in latency in the MMT test. Small but significant threshold elevations (15-20%) were seen, however, in both the pentylenetetrazol infusion test and the ECS threshold test. No seizure suppression was seen in the scMET test, which actually employs a suprasthreshold stimulus. These data indicate that the KD has significant anticonvulsant effects against both chemically and electrically triggered seizures, but that they consist of small elevations in threshold which will be seen only when threshold measures are used.

Perforant pathway kindling transiently induces the mRNA expression of GABA-B receptor subtypes R1A and R2 in the adult rat hippocampus.

We examined the gene expression responses of GABA-B R1A, R1B and R2 receptor subtypes in the hippocampus of perforant pathway-kindled rats at 24 h and 28 days after 15 consecutive daily stimulations. We found R1A expression, but not R1B expression, to be significantly induced in the dentate gyrus at 24 h. No change in the expression of R1A or R1B was observed at 28 days. R2 expression was induced throughout the hippocampus at 24 h, but also returned to control levels by 28 days. Thus, our results show that kindling induces a transient increase in GABA-B receptor mRNA in the hippocampus.

Diazepam-potentiated [3H]phenytoin binding is associated with peripheral-type benzodiazepine receptors and not with voltage-dependent sodium channels.

In the presence of diazepam, [3H]phenytoin binds with high affinity to brain membranes. The present experiments examined whether this high affinity [3H]phenytoin-binding site co-localized with the standard [3H]phenytoin-binding site on the voltage-dependent sodium channel (VDSC). Veratridine, a pharmacological activator of the voltage-dependent sodium channel, that inhibits standard [3H]phenytoin binding, failed to affect the high affinity diazepam-potentiated [3H]phenytoin binding in brain membranes, suggesting that the potentiated binding interaction resides at a site distinct from the voltage-dependent sodium channel. This possibility was confirmed by anion exchange chromatography of digitonin-solubilized rat brain membranes, as diazepam-potentiated high affinity [3H]phenytoin binding eluted in column fractions that were distinct from [3H]saxitoxin-defined voltage-dependent sodium channels. To examine whether diazepam-potentiated [3H]phenytoin binding might be associated with other 'classic' benzodiazepine receptor sites, we tested whether specific ligands for benzodiazepine receptors would either produce or block potentiated [3H]phenytoin binding. Neither agonists, nor antagonists, of the high affinity central-type benzodiazepine receptor affected potentiated [3H]phenytoin binding, suggesting that the high affinity potentiated binding site is not likely associated with central benzodiazepine receptors. Peripheral-type benzodiazepine receptor agonists, however, did potentiate [3H]phenytoin binding, and a specific receptor antagonist (PK11195) attenuated the potentiation seen with diazepam. Overall, these data illustrate that [3H]phenytoin interacts with a novel site in brain membranes that is distinct from the voltage-dependent sodium channel and is allosterically revealed by peripheral-type, but not central-type, benzodiazepine receptor agonists.

Mossy fiber sprouting induced by repeated electroconvulsive shock seizures.

The elicitation of repeated focal seizures (kindling) induces mossy fiber sprouting in the hippocampus of the rat. The present study investigated whether repeated generalized seizures also induce mossy fiber sprouting. Human psychiatric patients receive repeated generalized seizures during electroconvulsive therapy (ECT). Male Long-Evans rats received a course of eight electroconvulsive shock (ECS) seizures administered on a 48-h schedule over a course of 2 1/2 weeks. Control subjects received matched handling, but no stimulation. Fourteen days after the last ECS trial, all subjects were sacrificed and their brains subjected to Timm staining. Cell counts and area measures were also taken in the hilus. Significant sprouting, but not significant cell loss, was seen in the fascia dentata of the subjects that had received ECS.