Glutamate dynamics in the dorsolateral striatum of rats with goal-directed and habitual cocaine-seeking behavior.

The shift from drug abuse to addiction is considered to arise from the transition between goal-directed and habitual control over drug behavior. Habitual responding for appetitive and skill-based behaviors is mediated by potentiated glutamate signaling in the dorsolateral striatum (DLS), but the state of the DLS glutamate system in the context of habitual drug-behavior remains undefined. Evidence from the nucleus accumbens of cocaine-experienced rats suggests that decreased transporter-mediated glutamate clearance and enhanced synaptic glutamate release contribute to the potentiated glutamate signaling that underlies the enduring vulnerability to relapse. Preliminary evidence from the dorsal striatum of cocaine-experienced rats suggests that this region exhibits similar alterations to glutamate clearance and release, but it is not known whether these glutamate dynamics are associated with goal-directed or habitual control over cocaine-seeking behavior. Therefore, we trained rats to self-administer cocaine in a chained cocaine-seeking and -taking paradigm, which yielded goal-directed, intermediate, and habitual cocaine-seeking rats. We then assessed glutamate clearance and release dynamics in the DLS of these rats using two different methods: synaptic transporter current (STC) recordings of patch-clamped astrocytes and the intensity-based glutamate sensing fluorescent reporter (iGluSnFr). While we observed a decreased rate of glutamate clearance in STCs evoked with single-pulse stimulation in cocaine-experienced rats, we did not observe any cocaine-induced differences in glutamate clearance rates from STCs evoked with high frequency stimulation (HFS) or iGluSnFr responses evoked with either double-pulse stimulation or HFS. Furthermore, GLT-1 protein expression in the DLS was unchanged in cocaine-experienced rats, regardless of their mode of control over cocaine-seeking behavior. Lastly, there were no differences in metrics of glutamate release between cocaine-experienced rats and yoked-saline controls in either assay. Together, these results suggest that glutamate clearance and release dynamics in the DLS are largely unaltered by a history of cocaine self-administration on this established cocaine seeking-taking paradigm, regardless of whether the control over the cocaine seeking behavior was habitual or goal directed.

Ascorbic Acid Reduces Neurotransmission, Synaptic Plasticity, and Spontaneous Hippocampal Rhythms in In Vitro Slices.

Ascorbic acid (AA; a.k.a. vitamin C) is well known for its cellular protection in environments of high oxidative stress. Even though physiological concentrations of AA in the brain are significant (0.2-10 mM), surprisingly little is known concerning the role of AA in synaptic neurotransmission under normal, non-disease state conditions. Here, we examined AA effects on neurotransmission, plasticity and spontaneous network activity (i.e., sharp waves and high frequency oscillations; SPW-HFOs), at the synapse between area 3 and 1 of the hippocampal cornu ammonis region (CA3 and CA1) using an extracellular multi-electrode array in in vitro mouse hippocampal slices. We found that AA decreased evoked field potentials (fEPSPs, IC50 = 0.64 mM) without affecting V50s or paired pulse facilitation indicating normal neurotransmitter release mechanisms. AA decreased presynaptic fiber volleys but did not change fiber volley-to-fEPSP coupling, suggesting reduced fEPSPs resulted from decreased fiber volleys. Inhibitory effects were also observed in CA1 stratum pyramidale where greater fEPSPs were required for population spikes in the presence of AA suggesting an impact on the intrinsic excitability of neurons. Other forms of synaptic plasticity and correlates of memory (i.e., short- and long-term potentiation) were also significantly reduced by AA as was the incidence of spontaneous SPW-HFOs. AA decreased SPW amplitude with a similar IC50 as fEPSPs (0.65 mM). Overall, these results indicate that under normal conditions AA significantly regulates neurotransmission, plasticity, and network activity by limiting excitability. Thus, AA may participate in refinement of signal processing and memory formation, as well as protecting against pathologic excitability.

Effects of methamphetamine-induced neurotoxicity on striatal long-term potentiation.

RATIONALE: Methamphetamine (METH) exposure is associated with damage to central monoamine systems, particularly dopamine signaling. Rodent models of such damage have revealed a decrease in the amplitude of phasic dopamine signals and significant striatal dysfunction, including changes in the molecular, system, and behavioral functions of the striatum. Dopamine signaling through D1 receptors promotes corticostriatal long-term potentiation (LTP), a critical substrate of these striatal functions. OBJECTIVES: Therefore, the purpose of this study was to determine if METH-induced dopamine neurotoxicity would impair D1 receptor-dependent striatal LTP in mice. METHODS: Mice were treated with a METH binge regimen (4 × 10 mg/kg d,l-methamphetamine, s.c.) that recapitulates all of the known METH-induced neurotoxic effects observed in humans, including dopamine toxicity. Three weeks later, acute brain slices containing either the dorsomedial striatum (DMS) or dorsolateral striatum (DLS) were prepared, and plasticity was assessed using white matter, high-frequency stimulation (HFS), and striatal extracellular electrophysiology. RESULTS: Under these conditions, LTP was induced in brain slices containing the DMS from saline-pretreated mice, but not mice with METH-induced neurotoxicity. Furthermore, the LTP observed in DMS slices from saline-pretreated mice was blocked by the dopamine D1 receptor antagonist SCH23390, indicating that this LTP is dopamine D1 receptor-dependent. Finally, acute in vivo treatment of METH-pretreated mice with bupropion (50 mg/kg, i.p.) promoted LTP in DMS slices. CONCLUSIONS: Together, these studies demonstrate that METH-induced neurotoxicity impairs dopamine D1 receptor-dependent LTP within the DMS and that the FDA-approved drug bupropion restores induction of striatal LTP in mice with METH-induced dopamine neurotoxicity.

Spontaneous recurrent seizures in an intra-amygdala kainate microinjection model of temporal lobe epilepsy are differentially sensitive to antiseizure drugs.

The discovery and development of novel antiseizure drugs (ASDs) that are effective in controlling pharmacoresistant spontaneous recurrent seizures (SRSs) continues to represent a significant unmet clinical need. The Epilepsy Therapy Screening Program (ETSP) has undertaken efforts to address this need by adopting animal models that represent the salient features of human pharmacoresistant epilepsy and employing these models for preclinical testing of investigational ASDs. One such model that has garnered increased interest in recent years is the mouse variant of the Intra-Amygdala Kainate (IAK) microinjection model of mesial temporal lobe epilepsy (MTLE). In establishing a version of this model, several methodological variables were evaluated for their effect(s) on pertinent quantitative endpoints. Although administration of a benzodiazepine 40 min after kainate (KA) induced status epilepticus (SE) is commonly used to improve survival, data presented here demonstrates similar outcomes (mortality, hippocampal damage, latency periods, and 90-day SRS natural history) between mice given midazolam and those that were not. Using a version of this model that did not interrupt SE with a benzodiazepine, a 90-day natural history study was performed and survival, latency periods, SRS frequencies and durations, and SRS clustering data were quantified. Finally, an important step towards model adoption is to assess the sensitivities or resistances of SRSs to a panel of approved and clinically used ASDs. Accordingly, the following ASDs were evaluated for their effects on SRSs in these mice: phenytoin (20 mg/kg, b.i.d.), carbamazepine (30 mg/kg, t.i.d.), valproate (240 mg/kg, t.i.d.), diazepam (4 mg/kg, b.i.d.), and phenobarbital (25 and 50 mg/kg, b.i.d.). Valproate, diazepam, and phenobarbital significantly attenuated SRS frequency relative to vehicle controls at doses devoid of observable adverse behavioral effects. Only diazepam significantly increased seizure freedom. Neither phenytoin nor carbamazepine significantly altered SRS frequency or freedom under these experimental conditions. These data demonstrate that SRSs in this IAK model of MTLE are pharmacoresistant to two representative sodium channel-inhibiting ASDs (phenytoin and carbamazepine) and partially sensitive to GABA receptor modulating ASDs (diazepam and phenobarbital) or a mixed-mechanism ASD (valproate). Accordingly, this model is being incorporated into the NINDS-funded ETSP testing platform for treatment resistant epilepsy.

Screening of prototype antiseizure and anti-inflammatory compounds in the Theiler's murine encephalomyelitis virus model of epilepsy.

OBJECTIVE: Infection with Theiler's murine encephalomyelitis virus (TMEV) in C57Bl/6J mice results in handling-induced seizures and is useful for evaluating compounds effective against infection-induced seizures. However, to date only a few compounds have been evaluated in this model, and a comprehensive study of antiseizure medications (ASMs) has not yet been performed. Furthermore, as the TMEV infection produces marked neuroinflammation, an evaluation of prototype anti-inflammatory compounds is needed as well. METHODS: Male C57Bl/6J mice were inoculated with TMEV (day 0) followed by daily administrations of test compounds (day 3-7) and subsequent handling sessions (day 3-7). Doses of ASMs, comprising several mechanistic classes, were selected based on previously published data demonstrating the effect of these compounds in reducing seizures in the 6 Hz model of pharmacoresistant seizures. Doses of anti-inflammatory compounds, comprising several mechanistic classes, were selected based on published evidence of reduction of inflammation or inflammation-related endpoints. RESULTS: Several prototype ASMs reduced acute seizures following TMEV infection: lacosamide, phenytoin, ezogabine, phenobarbital, tiagabine, gabapentin, levetiracetam, topiramate, and sodium valproate. Of these, phenobarbital and sodium valproate had the greatest effect (>95% seizure burden reduction). Prototype anti-inflammatory drugs celecoxib, dexamethasone, and prednisone also moderately reduced seizure burden. SIGNIFICANCE: The TMEV model is utilized by the Epilepsy Therapy Screening Program (ETSP) as a tool for evaluation of novel compounds. Compounds reducing seizures in the TMEV comprise distinct mechanistic classes, some with mechanisms of action that extend beyond traditional ASMs.

Development of an antiseizure drug screening platform for Dravet syndrome at the NINDS contract site for the Epilepsy Therapy Screening Program.

OBJECTIVE: Dravet syndrome (DS) is a rare but catastrophic genetic epilepsy, with 80% of patients carrying a mutation in the SCN1A gene. Currently, no antiseizure drug (ASD) exists that adequately controls seizures. In the clinic, individuals with DS often present first with a febrile seizure and, subsequently, generalized tonic-clonic seizures that can continue throughout life. To facilitate the development of ASDs for DS, the contract site of the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) has evaluated a mouse model of DS using the conditional knock-in Scn1aA1783V/WT mouse. METHODS: Survival rates and temperature thresholds for Scn1aA1783V/WT were determined. Prototype ASDs were administered via intraperitoneal injections at the time-to-peak effect, which was previously determined, prior to the induction of hyperthermia-induced seizures. ASDs were considered effective if they significantly increased the temperature at which Scn1aA1783V/WT mice had seizures. RESULTS: Approximately 50% of Scn1aA1783V/WT survive to adulthood and all have hyperthermia-induced seizures. The results suggest that hyperthermia-induced seizures in this model of DS are highly refractory to a battery of ASDs. Exceptions were clobazam, tiagabine, levetiracetam, and the combination of clobazam and valproic acid with add-on stiripentol, which elevated seizure thresholds. SIGNIFICANCE: Overall, the data demonstrate that the proposed model for DS is suitable for screening novel compounds for the ability to block hyperthermia-induced seizures and that heterozygous mice can be evaluated repeatedly over the course of several weeks, allowing for higher throughput screening.

Evaluation of subchronic administration of antiseizure drugs in spontaneously seizing rats.

OBJECTIVE: Approximately 30% of patients with epilepsy do not experience full seizure control on their antiseizure drug (ASD) regimen. Historically, screening for novel ASDs has relied on evaluating efficacy following a single administration of a test compound in either acute electrical or chemical seizure induction. However, the use of animal models of spontaneous seizures and repeated administration of test compounds may better differentiate novel compounds. Therefore, this approach has been instituted as part of the National Institute of Neurological Disorders and Stroke Epilepsy Therapy Screening Program screening paradigm for pharmacoresistant epilepsy. METHODS: Rats were treated with intraperitoneal kainic acid to induce status epilepticus and subsequent spontaneous recurrent seizures. After 12 weeks, rats were enrolled in drug screening studies. Using a 2-week crossover design, selected ASDs were evaluated for their ability to protect against spontaneous seizures, using a video-electroencephalographic monitoring system and automated seizure detection. Sixteen clinically available compounds were administered at maximally tolerated doses in this model. Dose intervals (1-3 treatments/d) were selected based on known half-lives for each compound. RESULTS: Carbamazepine (90 mg/kg/d), phenobarbital (30 mg/kg/d), and ezogabine (15 mg/kg/d) significantly reduced seizure burden at the doses evaluated. In addition, a dose-response study of topiramate (20-600 mg/kg/d) demonstrated that this compound reduced seizure burden at both therapeutic and supratherapeutic doses. However, none of the 16 ASDs conferred complete seizure freedom during the testing period at the doses tested. SIGNIFICANCE: Despite reductions in seizure burden, the lack of full seizure freedom for any ASD tested suggests that this screening paradigm may be useful for testing novel compounds with potential utility in pharmacoresistant epilepsy.

Discovery of the First Vitamin K Analogue as a Potential Treatment of Pharmacoresistant Seizures.

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.

The current approach of the Epilepsy Therapy Screening Program contract site for identifying improved therapies for the treatment of pharmacoresistant seizures in epilepsy.

The Epilepsy Therapy Screening Program (ETSP), formerly known as the Anticonvulsant Screening Program (ASP), has played an important role in the preclinical evaluation of many of the antiseizure drugs (ASDs) that have been approved by the FDA and thus made available for the treatment of seizures. Recent changes to the animal models used at the contract site of the ETSP at the University of Utah have been implemented in an attempt to better model the unmet clinical needs of people with pharmacoresistant epilepsy and thus identify improved therapies. In this review, we describe the changes that have occurred over the last several years in the screening approach used at the contract site and, in particular, detail the pharmacology associated with several of the animal models and assays that are either new to the program or have been recently characterized in more depth. There is optimism that the refined approach used by the ETSP contract site, wherein etiologically relevant models that include those with spontaneous seizures are used, will identify novel, potentially disease modifying therapies for people with pharmacoresistant epilepsy and those at risk for developing epilepsy. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century - From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'.

Conditional Knock-out of mGluR5 from Astrocytes during Epilepsy Development Impairs High-Frequency Glutamate Uptake.

Astrocyte expression of metabotropic glutamate receptor 5 (mGluR5) is consistently observed in resected tissue from patients with epilepsy and is equally prevalent in animal models of epilepsy. However, little is known about the functional signaling properties or downstream consequences of astrocyte mGluR5 activation during epilepsy development. In the rodent brain, astrocyte mGluR5 expression is developmentally regulated and confined in expression/function to the first weeks of life, with similar observations made in human control tissue. Herein, we demonstrate that mGluR5 expression and function dramatically increase in a mouse model of temporal lobe epilepsy. Interestingly, in both male and female mice, mGluR5 function persists in the astrocyte throughout the process of epileptogenesis following status epilepticus. However, mGluR5 expression and function are transient in animals that do not develop epilepsy over an equivalent time period, suggesting that patterns of mGluR5 expression may signify continuing epilepsy development or its resolution. We demonstrate that, during epileptogenesis, astrocytes reacquire mGluR5-dependent calcium transients following agonist application or synaptic glutamate release, a feature of astrocyte-neuron communication absent since early development. Finally, we find that the selective and conditional knock-out of mGluR5 signaling from astrocytes during epilepsy development slows the rate of glutamate clearance through astrocyte glutamate transporters under high-frequency stimulation conditions, a feature that suggests astrocyte mGluR5 expression during epileptogenesis may recapitulate earlier developmental roles in regulating glutamate transporter function.SIGNIFICANCE STATEMENT In development, astrocyte mGluR5 signaling plays a critical role in regulating structural and functional interactions between astrocytes and neurons at the tripartite synapse. Notably, mGluR5 signaling is a positive regulator of astrocyte glutamate transporter expression and function, an essential component of excitatory signaling regulation in hippocampus. After early development, astrocyte mGluR5 expression is downregulated, but reemerges in animal models of temporal lobe epilepsy (TLE) development and patient epilepsy samples. We explored the hypothesis that astrocyte mGluR5 reemergence recapitulates earlier developmental roles during TLE acquisition. Our work demonstrates that astrocytes with mGluR5 signaling during TLE development perform faster glutamate uptake in hippocampus, revealing a previously unexplored role for astrocyte mGluR5 signaling in hypersynchronous pathology.

Recurrent epileptiform discharges in the medial entorhinal cortex of kainate-treated rats are differentially sensitive to antiseizure drugs.

OBJECTIVE: Approximately 30% of patients with epilepsy are refractory to existing antiseizure drugs (ASDs). Given that the properties of the central nervous systems of these patients are likely to be altered due to their epilepsy, tissues from rodents that have undergone epileptogenesis might provide a therapeutically relevant disease substrate for identifying compounds capable of attenuating pharmacoresistant seizures. To facilitate the development of such a model, this study describes the effects of classical glutamate receptor antagonists and 20 ASDs on recurrent epileptiform discharges (REDs) in brain slices derived from the kainate-induced status epilepticus model of temporal lobe epilepsy (KA-rats). METHODS: Horizontal brain slices containing the medial entorhinal cortex (mEC) were prepared from KA-rats, and REDs were recorded from the superficial layers. 6-cyano-7-nitroquinoxaline-2,3-dione, (2R)-amino-5-phosphonovaleric acid, tetrodotoxin, or ASDs were bath applied for 20 minutes. Concentration-dependent effects and half maximal effective concentration values were determined for RED duration, frequency, and amplitude. RESULTS: ASDs targeting sodium and potassium channels (carbamazepine, eslicarbazepine, ezogabine, lamotrigine, lacosamide, phenytoin, and rufinamide) attenuated REDs at concentrations near their average therapeutic plasma concentrations. γ-aminobutyric acid (GABA)ergic synaptic transmission-modulating ASDs (clobazam, midazolam, phenobarbital, stiripentol, tiagabine, and vigabatrin) attenuated REDs only at higher concentrations and, in some cases, prolonged RED durations. ASDs with other/mixed mechanisms of action (bumetanide, ethosuximide, felbamate, gabapentin, levetiracetam, topiramate, and valproate) and glutamate receptor antagonists weakly or incompletely inhibited RED frequency, increased RED duration, or had no significant effects. SIGNIFICANCE: Taken together, these data suggest that epileptiform activity recorded from the superficial layers of the mEC in slices obtained from KA-rats is differentially sensitive to existing ASDs. The different sensitivities of REDs to these ASDs may reflect persistent molecular, cellular, and/or network-level changes resulting from disease. These data are expected to serve as a foundation upon which future therapeutics may be differentiated and assessed for potentially translatable efficacy in patients with refractory epilepsy.

Heterophilic Type II Cadherins Are Required for High-Magnitude Synaptic Potentiation in the Hippocampus.

Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show that SO synapses normally have significantly more mushroom spines and higher-magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered that these differences require the Type II classic cadherins, cadherins-6, -9, and -10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and -10 to regulate mushroom spine density and high-magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower-magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, -9, and -10 are gatekeepers for high-magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.

Corneal kindled C57BL/6 mice exhibit saturated dentate gyrus long-term potentiation and associated memory deficits in the absence of overt neuron loss.

Memory deficits have a significant impact on the quality of life of patients with epilepsy and currently no effective treatments exist to mitigate this comorbidity. While these cognitive comorbidities can be associated with varying degrees of hippocampal cell death and hippocampal sclerosis, more subtle changes in hippocampal physiology independent of cell loss may underlie memory dysfunction in many epilepsy patients. Accordingly, animal models of epilepsy or epileptic processes exhibiting memory deficits in the absence of cell loss could facilitate novel therapy discovery. Mouse corneal kindling is a cost-effective and non-invasive model of focal to bilateral tonic-clonic seizures that may exhibit memory deficits in the absence of cell loss. Therefore, we tested the hypothesis that corneal kindled C57BL/6 mice exhibit spatial pattern processing and memory deficits in a task reliant on DG function and that these impairments would be concurrent with physiological remodeling of the DG as opposed to overt neuron loss. Following corneal kindling, C57BL/6 mice exhibited deficits in a DG-associated spatial memory test - the metric task. Compatible with this finding, we also discovered saturated, and subsequently impaired, LTP of excitatory synaptic transmission at the perforant path to DGC synapse. This saturation of LTP was consistent with evidence suggesting that perforant path to DGC synapses in kindled mice had previously experienced LTP-like changes to their synaptic weights: increased postsynaptic depolarizations in response to equivalent presynaptic input and significantly larger amplitude AMPA receptor mediated spontaneous EPSCs. Additionally, there was evidence for kindling-induced changes in the intrinsic excitability of DGCs: reduced threshold to population spikes under extracellular recording conditions and significantly increased membrane resistances observed in DGCs. Importantly, quantitative immunohistochemical analysis revealed hippocampal astrogliosis in the absence of overt neuron loss. These changes in spatial pattern processing and memory deficits in corneal kindled mice represent a novel model of seizure-induced cognitive dysfunction associated with pathophysiological remodeling of excitatory synaptic transmission and granule cell excitability in the absence of overt cell loss.

Hippocampal TNFα Signaling Contributes to Seizure Generation in an Infection-Induced Mouse Model of Limbic Epilepsy.

Central nervous system infection can induce epilepsy that is often refractory to established antiseizure drugs. Previous studies in the Theiler's murine encephalomyelitis virus (TMEV)-induced mouse model of limbic epilepsy have demonstrated the importance of inflammation, especially that mediated by tumor necrosis factor-α (TNFα), in the development of acute seizures. TNFα modulates glutamate receptor trafficking via TNF receptor 1 (TNFR1) to cause increased excitatory synaptic transmission. Therefore, we hypothesized that an increase in TNFα signaling after TMEV infection might contribute to acute seizures. We found a significant increase in both mRNA and protein levels of TNFα and the protein expression ratio of TNF receptors (TNFR1:TNFR2) in the hippocampus, a brain region most likely involved in seizure initiation, after TMEV infection, which suggests that TNFα signaling, predominantly through TNFR1, may contribute to limbic hyperexcitability. An increase in hippocampal cell-surface glutamate receptor expression was also observed during acute seizures. Although pharmacological inhibition of TNFR1-mediated signaling had no effect on acute seizures, several lines of genetically modified animals deficient in either TNFα or TNFRs had robust changes in seizure incidence and severity after TMEV infection. TNFR2-/- mice were highly susceptible to developing acute seizures, suggesting that TNFR2-mediated signaling may provide beneficial effects during the acute seizure period. Taken together, the present results suggest that inflammation in the hippocampus, caused predominantly by TNFα signaling, contributes to hyperexcitability and acute seizures after TMEV infection. Pharmacotherapies designed to suppress TNFR1-mediated or augment TNFR2-mediated effects of TNFα may provide antiseizure and disease-modifying effects after central nervous system infection.

Development and pharmacologic characterization of the rat 6 Hz model of partial seizures.

OBJECTIVE: The mouse 6 Hz model of psychomotor seizures is a well-established and commonly used preclinical model for antiseizure drug (ASD) discovery. Despite its widespread use both in the identification and differentiation of novel ASDs in mice, a corresponding assay in rats has not been developed. We established a method for 6 Hz seizure induction in rats, with seizure behaviors similar to those observed in mice including head nod, jaw clonus, and forelimb clonus. METHODS: A convulsive current that elicits these seizure behaviors in 97% of rats (CC97 ) was determined using a Probit analysis. Numerous prototype ASDs were evaluated in this model using stimulus intensities of 1.5× and 2× the CC97 , which is comparable to the approach used in the mouse 6 Hz seizure model (e.g., 32 and 44 mA stimulus intensities). The ASDs evaluated include carbamazepine, clobazam, clonazepam, eslicarbazepine, ethosuximide, ezogabine, gabapentin, lacosamide, lamotrigine, levetiracetam, phenobarbital, phenytoin, rufinamide, tiagabine, topiramate, and sodium valproate. Median effective dose (ED50 ) and median toxic (motor impairment) dose (TD50 ) values were obtained for each compound. RESULTS: Compounds that were effective at the 1.5 × CC97 stimulus intensity at protective index (PI) values >1 included clobazam, ethosuximide, ezogabine, levetiracetam, phenobarbital, and sodium valproate. Compounds that were effective at the 2 × CC97 stimulus intensity at PI values >1 included ezogabine, phenobarbital, and sodium valproate. SIGNIFICANCE: In a manner similar to the use of the mouse 6 Hz model, development of a rat 6 Hz test will aid in the differentiation of ASDs, as well as in study design and dose selection for chronic rat models of pharmacoresistant epilepsy. The limited number of established ASDs with demonstrable efficacy at the higher stimulus intensity suggests that, like the mouse 6 Hz 44 mA model, the rat 6 Hz seizure model may be a useful screening tool for pharmacoresistant seizures.

Validation of a Preclinical Drug Screening Platform for Pharmacoresistant Epilepsy.

The successful identification of promising investigational therapies for the treatment of epilepsy can be credited to the use of numerous animal models of seizure and epilepsy for over 80 years. In this time, the maximal electroshock test in mice and rats, the subcutaneous pentylenetetrazol test in mice and rats, and more recently the 6 Hz assay in mice, have been utilized as primary models of electrically or chemically-evoked seizures in neurologically intact rodents. In addition, rodent kindling models, in which chronic network hyperexcitability has developed, have been used to identify new agents. It is clear that this traditional screening approach has greatly expanded the number of marketed drugs available to manage the symptomatic seizures associated with epilepsy. In spite of the numerous antiseizure drugs (ASDs) on the market today, the fact remains that nearly 30% of patients are resistant to these currently available medications. To address this unmet medical need, the National Institute of Neurological Disorders and Stroke (NINDS) Epilepsy Therapy Screening Program (ETSP) revised its approach to the early evaluation of investigational agents for the treatment of epilepsy in 2015 to include a focus on preclinical approaches to model pharmacoresistant seizures. This present report highlights the in vivo and in vitro findings associated with the initial pharmacological validation of this testing approach using a number of mechanistically diverse, commercially available antiseizure drugs, as well as several probe compounds that are of potential mechanistic interest to the clinical management of epilepsy.

sec-Butylpropylacetamide (SPD), a new amide derivative of valproic acid for the treatment of neuropathic and inflammatory pain.

Chronic pain is a multifactorial disease comprised of both inflammatory and neuropathic components that affect ∼20% of the world's population. sec-Butylpropylacetamide (SPD) is a novel amide analogue of valproic acid (VPA) previously shown to possess a broad spectrum of anticonvulsant activity. In this study, we defined the pharmacokinetic parameters of SPD in rat and mouse, and then evaluated its antinociceptive potential in neuropathic and acute inflammatory pain models. In the sciatic nerve ligation (SNL) model of neuropathic pain, SPD was equipotent to gabapentin and more potent than its parent compound VPA. SPD also showed either higher or equal potency to VPA in the formalin, carrageenan, and writhing tests of inflammatory pain. SPD showed no effects on compound action potential properties in a sciatic nerve preparation, suggesting that its mechanism of action is distinct from local anesthetics and membrane stabilizing drugs. SPD's activity in both neuropathic and inflammatory pain warrants its development as a potential broad-spectrum anti-nociceptive drug.

The anticonvulsant action of the galanin receptor agonist NAX-5055 involves modulation of both excitatory- and inhibitory neurotransmission.

The endogenous neuropeptide galanin is ubiquitously expressed throughout the mammalian brain. Through the galanin receptors GalR1-3, galanin has been demonstrated to modulate both glutamatergic and GABAergic neurotransmission, and this appears to be important in epilepsy and seizure activity. Accordingly, galanin analogues are likely to provide a new approach to seizure management. However, since peptides are generally poor candidates for therapeutic agents due to their poor metabolic stability and low brain bioavailability, a search for alternative strategies for the development of galanin-based anti-convulsant drugs was prompted. Based on this, a rationally designed GalR1 preferring galanin analogue, NAX-5055, was synthesized. This compound demonstrates anti-convulsant actions in several animal models of epilepsy. However, the alterations at the cellular level leading to this anti-convulsant action of NAX-5055 are not known. Here we investigate the action of NAX-5055 at the cellular level by determining its effects on excitatory and inhibitory neurotransmission, i.e. vesicular release of glutamate and GABA, respectively, in cerebellar, neocortical and hippocampal preparations. In addition, its effects on cell viability and neurotransmitter transporter capacity were examined to evaluate potential cell toxicity mediated by NAX-5055. It was found that vesicular release of glutamate was reduced concentration-dependently by NAX-5055 in the range from 0.1 to 1000 nM. Moreover, exposure to 1 µM NAX-5055 led to a reduction in the extracellular level of glutamate and an elevation of the extracellular level of GABA. Altogether these findings may at least partly explain the anti-convulsant effect of NAX-5055 observed in vivo.