Effects of Stevia rebaudiana Bertoni extracts in the rat model of epilepsy induced by pentylenetetrazol: Sirt-1, at the crossroads between inflammation and apoptosis.

The current study investigated the effects of stevia extracts on a PTZ-induced epileptic rat model and its potential mechanism. Thirty male Sprague-Dawley rats were equally subdivided into 3 groups; (1) normal control (NC) group, (2) PTZ-group: received PTZ (50 mg/kg, i.p. every other day) for 2 weeks, and (3) PTZ+ Stevia group: received PTZ and stevia (200 mg/kg orally daily) for 4 weeks (2 weeks before the start of PTZ treatment and 2 weeks with PTZ administration). The first jerk latency and the seizure score were assessed in rats. Also, brain tissue samples were collected by the end of the experiment, and oxidative stress markers (catalase, MDA, and total antioxidant capacity (TAC)) were measured by biochemical analysis in hippocampal brain homogenates. Also, in the hippocampus, the expression of IL6 and Bcl-2 at the mRNA level and expression of Sirt-1, P53, caspase-3, GFAP, and NF-kB in CA3 hippocampal region by immunohistochemistry was investigated. PTZ substantially increased the seizure score and decreased the seizure latency. Also, PTZ significantly increased MDA, GFAP, IL-6, NF-kB, caspase-3, and p53 and significantly reduced Sirt-1, TAC, and Bcl-2 in hippocampal tissues compared to the control group (p < 0.01). However, Stevia Rebaudiana Bertoni (Stevia R.) significantly attenuated the PTZ-induced seizures, improved oxidative stress markers, downregulated GFAP, IL-6, NF-kB, caspase-3, and p53, and upregulated Sirt-1 and Bcl-2 in the CA3 hippocampal region (p < 0.01). In conclusion, Stevia R. exhibits neuroprotective and antiepileptic actions in PTZ-induced epilepsy due to its antioxidant, anti-apoptotic, and anti-inflammatory effects. Additionally, the Sirt-1 pathway might be involved in the antiepileptic and neuroprotective effects of stevia in PTZ-kindled epileptic rat model.

Comparative Antiseizure Analysis of Diverse Natural Coumarin Derivatives in Zebrafish.

Coumarins are a well-known group of plant secondary metabolites with various pharmacological activities, including antiseizure activity. In the search for new antiseizure drugs (ASDs) to treat epilepsy, it is yet unclear which types of coumarins are particularly interesting as a systematic analysis has not been reported. The current study performed behavioral antiseizure activity screening of 18 different coumarin derivatives in the larval zebrafish pentylenetetrazole (PTZ) model using locomotor measurements. Activity was confirmed for seven compounds, which lowered seizure-like behavior as follows: oxypeucedanin 38%, oxypeucedanin hydrate 74%, notopterol 54%, nodakenetin 29%, hyuganin C 35%, daphnoretin 65%, and pimpinellin 60%. These coumarins, together with nodakenin, underwent further antiepileptiform analysis by local field potential recordings from the zebrafish opticum tectum (midbrain). All of them, except for nodakenetin, showed pronounced antiepileptiform activity, decreasing PTZ-induced elevation in power spectral density (PSD) by 83-89% for oxypeucedanin, oxypeucedanin hydrate, and notopterol, 77% for nodakenin, 26% for nodakenetin, 65% for hyuganin C, 88% for daphnoretin, and 81% for pimpinellin. These data demonstrate the potential of diverse coumarin scaffolds for ASD discovery. Finally, the structural differences between active and inactive coumarins were investigated in silico for oxypeucedanin hydrate and byacangelicin for their interaction with GABA-transaminase, a hypothetical target.

Early α-linolenic acid exposure to embryo reduces pentylenetetrazol-induced seizures in zebrafish larva.

Over the past few years, there has been a tremendous increase in interest of general population toward food-based therapies for management of chronic clinical conditions due to their lesser adverse effects with prolonged use over pharmacotherapies. Foods enriched with omega-3 fatty acids have shown some promising results in case of epilepsy. The present study was envisioned to investigate the effect of early exposure of α-linolenic acid (ALA), an essential omega-3 fatty acid in developing zebrafish (Danio rerio) embryos toward pentylenetetrazol (PTZ)-induced seizure susceptibility. The healthy wild-type zebrafish embryos were incubated in system water or system water containing different ALA concentrations (1-20 µM) till 7 dpf (days post fertilization). Each larva at 7 dpf was placed in 8 mM PTZ solution and seizure event was recorded. ALA incubation at 10 µM and 20 µM concentrations showed a dose-dependent reduction in PTZ-mediated hyperactive responses in larvae indicated by a marked decrease in total distance travelled and speed, as compared to vehicle control. Furthermore, both the treated groups showed increase in the latency to PTZ-induced clonus-like seizures in larvae, as compared to vehicle control. ALA incubated larvae at 10 µM and 20 µM concentrations also showed a significant reduction in c-fos mRNA level. A marked increase in the level of ALA and docosahexaenoic acid was also observed in the larvae incubated at highest effective concentration of ALA. The present study concluded that embryonic exposure of ALA reduced PTZ-induced seizures in zebrafish larva.

High-throughput brain activity mapping and machine learning as a foundation for systems neuropharmacology.

Technologies for mapping the spatial and temporal patterns of neural activity have advanced our understanding of brain function in both health and disease. An important application of these technologies is the discovery of next-generation neurotherapeutics for neurological and psychiatric disorders. Here, we describe an in vivo drug screening strategy that combines high-throughput technology to generate large-scale brain activity maps (BAMs) with machine learning for predictive analysis. This platform enables evaluation of compounds' mechanisms of action and potential therapeutic uses based on information-rich BAMs derived from drug-treated zebrafish larvae. From a screen of clinically used drugs, we found intrinsically coherent drug clusters that are associated with known therapeutic categories. Using BAM-based clusters as a functional classifier, we identify anti-seizure-like drug leads from non-clinical compounds and validate their therapeutic effects in the pentylenetetrazole zebrafish seizure model. Collectively, this study provides a framework to advance the field of systems neuropharmacology.

Subtle improvement of seizure susceptibility by atorvastatin treatment during epileptogenesis.

BACKGROUND: The process by which a brain insult elicits epilepsy is termed epileptogenesis and it is characterized by numerous molecular and functional alterations. Statins are first-line drugs for hypercholesterolemia and related diseases, and display neuroprotective properties in clinical and experimental studies. Considering the importance in developing therapeutic strategies to prevent or modify epileptogenesis, we aimed the present study to test the hypothesis that atorvastatin modifies seizure susceptibility of mice after status epilepticus (SE). METHODS: Male and female C57BL/6 mice were submitted to the pilocarpine-induced SE and then treated with atorvastatin (10 or 100mg/kg, once daily by gavage) for 14days. At days 7 and 14 post SE we evaluated the susceptibility of mice to the convulsant effects of a low dose of PTZ (30mg/kg). Cell loss in the hilus of dentate gyrus was evaluated by Giemsa staining. RESULTS: Latencies to myoclonic jerks and to tonic-clonic seizures decreased between baseline (before SE) and days 7 and 14 after SE, confirming the development of seizure susceptibility. Atorvastatin protected against PTZ-induced tonic-clonic seizures in both sexes at day 14 post-SE. Protective effects were similar in both female and male mice, except that a high dose of atorvastatin was required for females (protection at 100mg/kg versus 10mg/kg in males). Giemsa staining did not reveal neuroprotective effects of atorvastatin. CONCLUSIONS: Atorvastatin treatment during epileptogenesis had slight beneficial effects on seizure susceptibility. These seem not related to neuroprotection. Further studies are needed to determine the disease-modifying potential of atorvastatin in epilepsy.

Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice.

The use of acupuncture in the treatment of central nervous system (CNS) disorders is an age-old practice. Although only a few studies have proved its efficacy, evidence has indicated the use of acupuncture to treat different types of seizures. Therefore, the present study aimed to evaluate the effect of manual acupuncture (MAC) using the chemical kindling model. The role of MAC in oxidative stress and inflammation after pentylenetetrazole (PTZ)-induced kindling was investigated by measuring reactive oxygen species (ROS) production, superoxide dismutase (SOD), and catalase (CAT) activities, nitrite content, and deoxyribonucleic acid (DNA) damage in cerebral cortex. Mice received PTZ (60mg/kgs.c.) once every three days for 16days, totaling six treatments. MAC was applied at acupoint GV20 daily during the entire experimental protocol. Diazepam (DZP) (2mg/kg) was used as positive control. Also, we evaluated the MAC effect associated with DZP (MAC/DZP) at a low dose (0.15mg/kg). The results demonstrated that MAC or MAC/DZP were not able to reduce significantly seizure occurrence or to increase the latency to the first seizure during treatment. MAC/DZP promoted a difference in the first latency to seizure only on the third day. PTZ-induced kindling caused significant neuronal injury, oxidative stress, increased DNA damage, nitric oxide production, and expression of the pro-inflammatory Tumor Necrosis Factor-α (TNF-α). These effects were reversed by treatment with MAC or MAC/DZP. These results indicated that the stimulation of acupoint GV20 by MAC showed no potential antiepileptogenic effect in the model used, although it greatly promoted neuronal protection, which may result from antioxidant and anti-inflammatory effects observed here.

Gastrodin Attenuates Pentylenetetrazole-Induced Seizures by Modulating the Mitogen-Activated Protein Kinase-Associated Inflammatory Responses in Mice.

Gastrodin, the major component isolated from the rhizome of the Chinese traditional medicinal herb Gastrodia elata ("Tianma"), has a long history in the treatment of epilepsy and other neurological disorders. However, the molecular mechanisms are not clear. Here, we found that gastrodin ameliorated pentylenetetrazole (PTZ)-induced epileptic seizures with improvement of the electroencephalographic pattern in mice. Further studies demonstrated that gastrodin decreased the levels of the pro-inflammatory cytokines interleukin-1ß and tumor necrosis factor-α while increasing interleukin-10, an anti-inflammatory cytokine in the brain. Furthermore, gastrodin attenuated the PTZ-induced microglial activation along with inhibition of mitogen-activated protein kinases, cAMP response element binding protein, and NF-κB. Our data suggest that gastrodin attenuates seizures by modulating the mitogen-activated protein kinase-associated inflammatory responses.

Omega 3 polyunsaturated fatty acids enhance the protective effect of levetiracetam against seizures, cognitive impairment and hippocampal oxidative DNA damage in young kindled rats.

Levetiracetam (LEV) is a unique, effective, relatively safe antiepileptic drug that preferentially interacts with synaptic vesicle protein 2A (SV2A). This study aimed to explore the effect of combined treatment of LEV with omega 3 (OM3) on cognitive impairment and hippocampal oxidative stress and DNA damage induced by seizures in the PTZ-kindled young rat model. Cognitive functions, biomarkers of oxidative stress, and DNA damage were assessed in PTZ-kindled young rats pretreated with single and combined treatment of LEV (30mg/kg, i.p.) and OM3 (200mg/kg, p.o.). Pretreatment with LEV and OM3 at the tested doses significantly attenuated PTZ-induced seizures and decreased cognitive impairment in both passive avoidance and elevated plus maze tests in the PTZ-kindled rats. Moreover, the increase in hippocampal glutamate, malondialdehyde and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels, as well as the decrease in reduced glutathione (GSH) levels and GSH-peroxidase and superoxide dismutase activities induced by PTZ kindling, significantly decreased. These effects were higher with combined treatment of LEV with OM3 and significantly more than the observed effects of single LEV or OM3. In conclusion, the combined treatment of LEV with OM3 is more effective in seizure control and alleviating the cognitive impairment induced by PTZ kindling in the young rat model, the effects that result from the decrease in hippocampal oxidative stress and DNA damage which can be attributed to the antioxidant properties of both LEV and OM3. These results may be promising for the use of LEV and OM3 combination in the treatment of epileptic children.

Effects of D-penicillamine on pentylenetetrazole-induced seizures in mice: involvement of nitric oxide/NMDA pathways.

Besides the clinical applications of penicillamine, some reports show that use of D-penicillamine (D-pen) has been associated with adverse effects such as seizures. So, the purpose of this study was to evaluate the effects of D-pen on pentylenetetrazole (PTZ)-induced seizures in male NMRI mice. It also examined whether N-methyl-D-aspartate (NMDA) receptor/nitrergic system blockage was able to alter the probable effects of D-pen. Different doses of D-pen (0.1, 0.5, 1, 10, 100, 150, and 250 mg/kg) were administered intraperitoneally (i.p.) 90 min prior to induction of seizures. D-Penicillamine at a low dose (0.5 mg/kg, i.p.) had anticonvulsant effects, whereas at a high dose (250 mg/kg, i.p.), it was proconvulsant. Both anti- and proconvulsant effects of D-pen were blocked by a single dose of a nonspecific inhibitor of nitric oxide synthase (NOS), L-NAME (10 mg/kg, i.p.), and a single dose of a specific inhibitor of neuronal nitric oxide synthase (nNOS), 7-nitroindazole (30 mg/kg, i.p.). A selective inhibitor of iNOS, aminoguanidine (100 mg/kg, i.p.), had no effect on these activities. An NMDA receptor antagonist, MK-801 (0.05 mg/kg, i.p.), alters the anti- and proconvulsant effects of D-pen. The results of the present study showed that the nitric oxide system and NMDA receptors may contribute to the biphasic effects of D-pen, which remain to be clarified further.

Curcumin supplementation improves mitochondrial and behavioral deficits in experimental model of chronic epilepsy.

The present study was aimed to investigate the potential beneficial effect of curcumin, a polyphenol with pleiotropic properties, on mitochondrial dysfunctions, oxidative stress and cognitive deficits in a kindled model of epilepsy. Kindled epilepsy was induced in rats by administering a sub-convulsive dose of pentylenetetrazole (PTZ, 40 mg/kg body weight) every alternate day for 30 days. PTZ administered rats exhibited marked cognitive deficits assessed using active and passive avoidance tasks. This was accompanied by a significant decrease in NADH:cytochrome-c reductase (complex I) and cytochrome-c oxidase (complex IV) activities along with an increase in ROS, lipid peroxidation and protein carbonyls. The levels of glutathione also decreased in the cortex and hippocampus. Electron micrographs revealed disruption of mitochondrial membrane integrity with distorted cristae in PTZ treated animals. Histopathological examination showed pyknotic nuclei and cell loss in the hippocampus as well as in the cortex of PTZ treated animals. Curcumin administration at a dose of 100 mg/kg, p.o. throughout the treatment paradigm was able to ameliorate cognitive deficits with no significant effect on seizure score. Curcumin was able to restore the activity of mitochondrial complexes. In addition, significant reduction in ROS generation, lipid peroxidation and protein carbonyls was observed in PTZ animals supplemented with curcumin. Moreover, glutathione levels were also restored in PTZ treated rats supplemented with curcumin. Curcumin protected mitochondria from seizure induced structural alterations. Further, the curcumin supplemented PTZ rats had normal cell morphology and reduced cell loss. These results suggest that curcumin supplementation has potential to prevent mitochondrial dysfunctions and oxidative stress with improved cognitive functions in a chronic model of epilepsy.

The anti-seizure drugs vinpocetine and carbamazepine, but not valproic acid, reduce inflammatory IL-1ß and TNF-α expression in rat hippocampus.

In the present study, the effects of the two classical anti-epileptic drugs, carbamazepine and valproic acid, and the non-classical anti-seizure drug vinpocetine were investigated on the expression of the pro-inflammatory cytokines IL-1ß and TNF-α in the hippocampus of rats by PCR or western blot after the administration of one or seven doses. Next, the effects of the anti-seizure drugs were investigated on the rise in cytokine expression induced by lipopolysaccharides (LPS) inoculation in vivo. To validate our methods, the changes induced by the pro-convulsive agents 4-aminopyridine, pentylenetetrazole and pilocarpine were also tested. Finally, the effect of the anti-seizure drugs on seizures and on the concomitant rise in pro-inflammatory cytokine expression induced by 4-aminopyridine was explored. Results show that vinpocetine and carbamazepine reduced the expression of IL-1ß and TNF-α from basal conditions, and the increase in both pro-inflammatory cytokines induced by LPS. In contrast, valproic acid failed to reduce both the expression of the cytokines from basal conditions and the rise in IL-1ß and TNF-α expression induced by LPS. Tonic-clonic seizures induced either by 4-aminopyridine, pentylenetetrazole or pilocarpine increased the expression of IL-1ß and TNF-α markedly. 4-aminopyridine-induced changes were reduced by all the tested anti-seizure drugs, although valproic acid was less effective. We conclude that the anti-seizure drugs, vinpocetine and carbamazepine, whose mechanisms of action involve a decrease in ion channels permeability, also reduce cerebral inflammation. The mechanism of action of anti-seizure drugs like vinpocetine and carbamazepine involves a decrease in Na(+) channels permeability. We here propose that this mechanism of action also involves a decrease in cerebral inflammation.

Anticonvulsant activity of 2-(substituted-imino)thiazolidin-4-ones.

A novel series of 2-(substituted-imino)thiazolidin-4-ones were synthesized and evaluated for anticonvulsant activity using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (Sc-PTZ) assays and their neurotoxicity was measured by the rotarod test. The results of these tests demonstrated that 2-(4-(pentyloxy)phenylimino)thiazolidin-4-one (5d) was the most potent anticonvulsant, with ED50 value of 18.5 mg/kg and 15.3 mg/kg in the MES and Sc-PTZ tests, and protective index (PI=TD50/ED50) values of 10.6 and 12.8 respectively. 5d was much safer than a reference drug Carbamazepine.

Novel anticonvulsive effects of progesterone in a mouse model of hippocampal electrical kindling.

Progesterone is a known anticonvulsant, with its inhibitory effects generally attributed to its secondary metabolite, 5α,3α-tetrahydroprogesterone (THP), and THP's enhancement of GABAA receptor activity. Accumulating evidence, however, suggests that progesterone may have non-genomic actions independent of the GABAA receptor. In this study, we explored THP/GABAA-independent anticonvulsive actions of progesterone in a mouse model of hippocampal kindling and in mouse entorhinal slices in vitro. Specifically, we examined the effects of progesterone in kindled mice with or without pretreatments with finasteride, a 5α-reductase inhibitor known to block the metabolism of progesterone to THP. In addition, we examined the effects of progesterone on entorhinal epileptiform potentials in the presence of a GABAA receptor antagonist picrotoxin and finasteride. Adult male mice were kindled via a daily stimulation protocol. Electroencephalographic (EEG) discharges were recorded from the hippocampus or cortex to assess "focal" or "generalized" seizure activity. Kindled mice were treated with intra-peritoneal injections of progesterone (10, 35, 100 and 160mg/kg) with or without finasteride pretreatment (50 or 100mg/kg), THP (1, 3.5, 10 and 30mg/kg), midazolam (2mg/kg) and carbamazepine (50mg/kg). Entorhinal cortical slices were prepared from naïve young mice, and repetitive epileptiform potentials were induced by 4-aminopyridine (100µM), picrotoxin (100µM) and finasteride (1µM). Pretreatment with finasteride did not abolish the anticonvulsant effects of progesterone. In finasteride-pretreated mice, progesterone at 100 and 160mg/kg decreased cortical but not hippocampal afterdischarges (ADs). Carbamazepine mimicked the effects of progesterone with finasteride pretreatments in decreasing cortical discharges and motor seizures, whereas midazolam produced effects similar to progesterone alone or THP in decreasing hippocampal ADs and motor seizures. In brain slices, progesterone at 1µM inhibited entorhinal epileptiform potentials in the presence of picrotoxin and finasteride. We suggest that progesterone may have THP/GABAA-dependent and independent anticonvulsive actions in the hippocampal-kindled mouse model.

Anticonvulsant activity of ethanol extracts of Vetiveria zizanioides roots in experimental mice.

CONTEXT: Vetiveria zizanioides Linn. (Gramineae), an aromatic plant commonly known as vetiver, is traditionally used for various ailments. Ethanol and aqueous extract of this plant found extensive use in Indian folklore medicine and used in treatment of a wide range of disorders including seizure. However, the anticonvulsant activity of this plant has not been studied. OBJECTIVE: To evaluate anticonvulsant activity of ethanol extract of V. zizanioides (EEVZ) in experimental mice. MATERIALS AND METHODS: Anticonvulsant activity of EEVZ was determined by maximal electroshock stimulation (MES) and pentylenetetrazole (PTZ) in mice for 8 d experimental protocol. The extract at a dose of 100, 200 and 400 mg/kg body weight was administered once by oral route. RESULTS: LD50 value of EEVZ in mice was found at a dose of 600 mg/kg body weight. EEVZ at a dose of 400 mg/kg significantly (p < 0.001) reduced flexion (l5.98 to 3.73 s), extension (13.73 to 0.96 s), clonus (14.07 to 4.93 s), stupor (6.29 to 1.22 s) in the MES model. Further, it increases onset of clonic (88.25 to 708.32 s/30 min) and tonic (139.52 to 1126.39 s/30 min) in the PTZ model. In the PTZ model, 33% normal control and 83% EEVZ (100 mg/kg) animals were alive, while 100% protection was achieved in standard drug phenobarbital (20 mg/kg), EEVZ (200 mg/kg) and EEVZ (400 mg/kg) animals. DISCUSSION AND CONCLUSION: Findings demonstrate that V. zizanioides shows significant anticonvulsant activity in mice.

The effects of zinc treatment on the blood-brain barrier permeability and brain element levels during convulsions.

We evaluated the effect of zinc treatment on the blood-brain barrier (BBB) permeability and the levels of zinc (Zn), natrium (Na), magnesium (Mg), and copper (Cu) in the brain tissue during epileptic seizures. The Wistar albino rats were divided into four groups, each as follows: (1) control group, (2) pentylenetetrazole (PTZ) group: rats treated with PTZ to induce seizures, (3) Zn group: rats treated with ZnCl(2) added to drinking water for 2 months, and (4) Zn + PTZ group. The brains were divided into left, right hemispheres, and cerebellum + brain stem regions. Evans blue was used as BBB tracer. Element concentrations were analyzed by inductively coupled plasma optical emission spectroscopy. The BBB permeability has been found to be increased in all experimental groups (p < 0.05). Zn concentrations in all brain regions in Zn-supplemented groups (p < 0.05) showed an increase. BBB permeability and Zn level in cerebellum + brain stem region were significantly high compared to cerebral hemispheres (p < 0.05). In all experimental groups, Cu concentration decreased, whereas Na concentrations showed an increase (p < 0.05). Mg content in all the brain regions decreased in the Zn group and Zn + PTZ groups compared to other groups (p < 0.001). We also found that all elements' levels showed hemispheric differences in all groups. During convulsions, Zn treatment did not show any protective effect on BBB permeability. Chronic Zn treatment decreased Mg and Cu concentration and increased Na levels in the brain tissue. Our results indicated that Zn treatment showed proconvulsant activity and increased BBB permeability, possibly changing prooxidant/antioxidant balance and neuronal excitability during seizures.

Hyperthermic seizures enhance responsiveness to pentylenetetrazole and induce cognitive dysfunction: protective effect of 3-alkynyl selenophene.

AIMS: In this study we investigated the effect of pre-treatment with 3-alkynyl selenophene (3-ASP) against the increase in responsiveness to pentylenetetrazole [PTZ seizure threshold] and cognitive dysfunction induced by experimental febrile seizures (FS). The effects of 3-ASP were compared to those of diazepam (DZP). MAIN METHODS: Young rats, at postnatal day 21, developed seizures after exposure to a stream of heated air to approximately 41°C. A non-spatial long-term memory and PTZ seizure threshold were determined 30 days after FS. The behavioural seizures were stereotyped followed by facial automatisms, often followed by body flexion. Young rats were pre-treated with 3-ASP (50 and 100mg/kg; per oral route), DZP (1 and 5mg/kg; intraperitoneally) or vehicle. KEY FINDINGS: 3-ASP and DZP pre-treatments were not effective in protecting against seizures induced by FS. 3-ASP pre-treatment protected against the increase in responsiveness to PTZ and cognitive dysfunction induced by FS. DZP pre-treatment was effective in protecting against the increase in responsiveness to PTZ, but not, against the impaired memory induced by FS. SIGNIFICANCE: 3-ASP pre-treatment protected against impairment of memory performance in the step-down passive avoidance task and the increase in the susceptibility to seizures caused by FS early in life of rats.

Early alterations of brain cellular energy homeostasis in Huntington disease models.

Brain energy deficit has been a suggested cause of Huntington disease (HD), but ATP depletion has not reliably been shown in preclinical models, possibly because of the immediate post-mortem changes in cellular energy metabolism. To examine a potential role of a low energy state in HD, we measured, for the first time in a neurodegenerative model, brain levels of high energy phosphates using microwave fixation, which instantaneously inactivates brain enzymatic activities and preserves in vivo levels of analytes. We studied HD transgenic R6/2 mice at ages 4, 8, and 12 weeks. We found significantly increased creatine and phosphocreatine, present as early as 4 weeks for phosphocreatine, preceding motor system deficits and decreased ATP levels in striatum, hippocampus, and frontal cortex of R6/2 mice. ATP and phosphocreatine concentrations were inversely correlated with the number of CAG repeats. Conversely, in mice injected with 3-nitroproprionic acid, an acute model of brain energy deficit, both ATP and phosphocreatine were significantly reduced. Increased creatine and phosphocreatine in R6/2 mice was associated with decreased guanidinoacetate N-methyltransferase and creatine kinase, both at the protein and RNA levels, and increased phosphorylated AMP-dependent protein kinase (pAMPK) over AMPK ratio. In addition, in 4-month-old knock-in Hdh(Q111/+) mice, the earliest metabolic alterations consisted of increased phosphocreatine in the frontal cortex and increased the pAMPK/AMPK ratio. Altogether, this study provides the first direct evidence of chronic alteration in homeostasis of high energy phosphates in HD models in the earliest stages of the disease, indicating possible reduced utilization of the brain phosphocreatine pool.

The effect of spinal cord stimulation on seizure susceptibility in rats.

OBJECTIVES: Spinal cord stimulation (SCS) activates the thalamus, which may be involved in generation of seizures. SCS may therefore influence seizure susceptibility. We investigated the effect of SCS on seizure susceptibility when performed at low frequency (4 Hz) and a frequency in the typical range of SCS treatment (54 Hz). MATERIALS AND METHODS: Rats were divided in three groups: control (N = 8), 4 Hz SCS (N = 6), and 54 Hz SCS (N = 8). Tonic-clonic seizures were induced by 10-min intravenous infusion of pentylenetetrazole (PTZ). SCS was started 5 min prior to PTZ infusion and continued for 5 min after infusion offset. Seizure susceptibility was accessed via the latency, number, and total duration of seizures. RESULTS: Four Hz SCS significantly increased seizure susceptibility. Fifty-four Hz SCS produced a nonsignificant trend toward decreased seizure susceptibility. CONCLUSIONS: Low-frequency SCS is proconvulsive in rats. Further research needs to investigate if this also applies to humans.

Upregulation of arginine vasopressin synthesis in the rat hypothalamus after kainic acid-induced seizures.

We examined the effects of kainic acid (KA)-induced seizures on arginine vasopressin (AVP) gene expression in the paraventricular (PVN) and the supraoptic nuclei (SON) of normal rats using in situ hybridization histochemistry. We also investigated the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene after KA-induced seizures in transgenic rats. AVP heteronuclear (hn) RNA levels in the PVN and the SON were significantly increased at 3h and 24h after subcutaneous (s.c.) administration of KA in normal rats. AVP mRNA levels in the PVN and the SON did not change significantly at 3h, 24h and 1 week after s.c. administration of KA in normal rats. In KA-administered transgenic rats, AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON were significantly stronger compared to vehicle-administered transgenic rats. By pretreatment with MK-801 (nonselective N-methyl-D-aspartate, NMDA, receptor antagonist), AVP-eGFP transgenic rats after administration of KA did not show preconvulsive symptoms or convulsions and AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON of these rats was significantly reduced. These results suggested that KA-induced increases in AVP transcripts and AVP were prevented by MK801 because seizure activity was prevented or reduced.

An in vitro model for testing drugs to treat tinnitus.

Tinnitus affects approximately 50 million people in the USA alone, with 10 million being highly debilitated. Pharmacotherapy for tinnitus is still in emerging stages due to time consuming clinical trials and/or animal experiments. We tested a new cellular model where induced rapid neuronal firing or spiking was used as a mimic for the type of aberrant activity that may occur in tinnitus. Spontaneously active auditory cortical networks growing on microelectrode arrays were exposed to pentylenetetrazol (PTZ), a proconvulsant and an antagonist of GABA(A) receptor, which is implicated in tinnitus. Auditory cortical networks were then exposed to experimental tinnitus drugs linopirdine (Dup966, a potassium channel blocker), L-carnitine (an antioxidant), or selective Ca(2+) channel antagonists pregabalin (Lyrica), or gabapentin (Neurontin) at various concentrations. PTZ increased spike rate by 139.6±27% and burst rate by 129.7±28% in auditory cortical networks with a phenotypic high firing of excitable neurons. Reductions of increased activity were observed to varying degrees using the experimental tinnitus drugs. The potency of the drugs was linopirdine (EC(50): 176±7.0 µM)>L-carnitine (EC(50): 1569±41 µM)>pregabalin (EC(50): 8360±340 µM), >gabapentin, with 34.2±7.5% efficacy (EC(50): 2092±980 µM). These studies provide proof of principle for the use of auditory cortical networks on microelectrode array as a feasible platform for semi-high throughput application for screening of drugs that might be used for the treatment of tinnitus.