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Epilepsy is a common brain disorder, characterized by spontaneous recurrent seizures, with associated neuropsychiatric and cognitive comorbidities and increased mortality. Although people at risk can often be identified, interventions to prevent the development of the disorder are not available. Moreover, in at least 30% of patients, epilepsy cannot be controlled by current antiseizure medications (ASMs). As a result of considerable progress in epilepsy genetics and the development of novel disease models, drug screening technologies and innovative therapeutic modalities over the past 10 years, more than 200 novel epilepsy therapies are currently in the preclinical or clinical pipeline, including many treatments that act by new mechanisms. Assisted by diagnostic and predictive biomarkers, the treatment of epilepsy is undergoing paradigm shifts from symptom-only ASMs to disease prevention, and from broad trial-and-error treatments for seizures in general to mechanism-based treatments for specific epilepsy syndromes. In this Review, we assess recent progress in ASM development and outline future directions for the development of new therapies for the treatment and prevention of epilepsy.

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTNgene. The co-occurrence of predicted deleterious SRPK3/TTNvariants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3−/−; ttn.1+/−) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3and ttn.1in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

IMPORTANCE: Intravenous edaravone is approved as a disease-modifying drug for patients with amyotrophic lateral sclerosis (ALS), but evidence for efficacy is limited to short-term beneficial effects shown in the MCI186-ALS19 study in a subpopulation in which efficacy was expected. OBJECTIVE: To evaluate the long-term safety and effectiveness of intravenous edaravone therapy for patients with ALS in a real-world clinical setting. DESIGN, SETTING, AND PARTICIPANTS: Multicenter, propensity score–matched cohort study conducted between June 2017 and March 2020 at 12 academic ALS referral centers associated with the German Motor Neuron Disease Network. Of 1440 patients screened, 738 were included in propensity score matching. Final analyses included 324 patients with ALS comprising 194 patients who started intravenous edaravone treatment (141 received ≥4 consecutive treatment cycles; 130 matched) and 130 propensity score–matched patients with ALS receiving standard therapy. All patients had probable or definite ALS according to the El Escorial criteria, with disease onset between December 2012 and April 2019. Subgroups were defined by applying the MCI186-ALS19 study inclusion criteria to evaluate whether patients would have been considered eligible (EFAS) or ineligible (non-EFAS). EXPOSURES: Intravenous edaravone plus riluzole vs riluzole only. MAIN OUTCOMES AND MEASURES: Patient characteristics and systematic safety assessment for patients who received at least 1 dose of intravenous edaravone. Effectiveness assessment of edaravone was conducted among patients who received at least 4 treatment cycles compared with propensity score–matched patients with ALS who received only standard therapy. Primary outcome was disease progression measured by decrease in the ALS Functional Rating Scale–Revised (ALSFRS-R) score. Secondary outcomes were survival probability, time to ventilation, and change in disease progression before vs during treatment. To account for the matched design, patients receiving edaravone and their corresponding matched controls were regarded as related samples in disease progression analyses; stratification for propensity score quintiles was used for survival probability and time to ventilation analyses. RESULTS: A total of 194 patients started intravenous edaravone treatment; 125 (64%) were male, and the median age was 57.5 years (IQR, 50.7-63.8 years). Potential adverse effects were observed in 30 cases (16%), most notably infections at infusion sites and allergic reactions. Disease progression among 116 patients treated for a median of 13.9 months (IQR, 8.9-13.9 months) with edaravone did not differ from 116 patients treated for a median of 11.2 months (IQR, 6.4-20.0 months) with standard therapy (ALSFRS-R points/month, −0.91 [95% CI, −0.69 to −1.07] vs −0.85 [95% CI, −0.66 to −0.99]; P = .37). No significant differences were observed in the secondary end points of survival probability, time to ventilation, and change in disease progression. Similarly, outcomes between patients treated with edaravone and matched patients did not differ within the EFAS and non-EFAS subgroups. CONCLUSIONS AND RELEVANCE: This cohort study using propensity score matching found that, although long-term intravenous edaravone therapy for patients with ALS was feasible and mainly well tolerated, it was not associated with any disease-modifying benefit. Intravenous edaravone may not provide a clinically relevant additional benefit compared with standard therapy alone.

ATP-binding cassette (ABC) transporters facilitate the movement of diverse molecules across cellular membranes, including those within the CNS. While most extensively studied in microvascular endothelial cells forming the blood-brain barrier (BBB), other CNS cell types also express these transporters. Importantly, disruptions in the CNS microenvironment during disease can alter transporter expression and function. Through this comprehensive review, we explore the modulation of ABC transporters in various brain pathologies and the context-dependent consequences of these changes. For instance, downregulation of ABCB1 may exacerbate amyloid beta plaque deposition in Alzheimer’s disease and facilitate neurotoxic compound entry in Parkinson’s disease. Upregulation may worsen neuroinflammation by aiding chemokine-mediated CD8 T cell influx into multiple sclerosis lesions. Overall, ABC transporters at the BBB hinder drug entry, presenting challenges for effective pharmacotherapy. Understanding the context-dependent changes in ABC transporter expression and function is crucial for elucidating the etiology and developing treatments for brain diseases.

Generally, neuropathies of peripheral nerves are a frequent condition (prevalence 2–3%) and most frequently due to alcoholism, diabetes, renal insufficiency, malignancy, toxins, or drugs. However, the vast majority of neuropathies has orphan status. This review focuses on the etiology, frequency, diagnosis, and treatment of orphan neuropathies. Literature review Rareness of diseases is not uniformly defined but in the US an orphan disease is diagnosed if the prevalence is <1:200000, in Europe if <5:10000. Most acquired and hereditary neuropathies are orphan diseases. Often the causative variant has been reported only in a single patient or family, particularly the ones that are newly detected (e.g. SEPT9, SORD). Among the complex neuropathies (hereditary multisystem disorders with concomitant neuropathies) orphan forms have been reported among mitochondrial disorders (e.g. NARP, MNGIE, SANDO), spinocerebellar ataxias (e.g. TMEM240), hereditary spastic paraplegias (e.g UBAP1), lysosomal storage disease (e.g. Schindler disease), peroxisomal disorders, porphyrias, and other types (e.g. giant axonal neuropathy, Tangier disease). Orphan acquired neuropathies include the metabolic neuropathies (e.g. vitamin-B1, folic acid), toxic neuropathies (e.g. copper, lithium, lead, arsenic, thallium, mercury), infectious neuropathies, immune-mediated (e.g. Bruns-Garland syndrome), and neoplastic/paraneoplastic neuropathies. Though orphan neuropathies are rare per definition they constitute the majority of neuropathies and should be considered as some of them are easy to identify and potentially treatable, as clarification of the underlying cause may contribute to the knowledge about etiology and pathophysiology of these conditions, and as the true prevalence may become obvious only if all ever diagnosed cases are reported.

The mechanistic target of rapamycin (mTOR) pathway is hyperactivated in cancer and neurological disorders. Rapalogs and mTOR kinase inhibitors (TORKi) have recently been applied to alleviate epileptic seizures in tuberous sclerosis complex (TSC). Herein, we describe a pharmacophore exploration to identify a highly potent, selective, brain penetrant TORKi. An extensive investigation of the morpholine ring engaging the mTOR solvent exposed region led to the discovery of PQR626 (8). 8displayed excellent brain penetration and was well-tolerated in mice. In mice with a conditionally inactivated Tsc1gene in glia, 8significantly reduced the loss of Tsc1-induced mortality at 50 mg/kg p.o. twice a day. 8overcomes the metabolic liabilities of PQR620 (52), the first-in-class brain penetrant TORKi showing efficacy in a TSC mouse model. The improved stability in human hepatocytes, excellent brain penetration, and efficacy in Tsc1GFAPCKO mice qualify 8as a potential therapeutic candidate for the treatment of neurological disorders.

laboratory tests for work-up of hereditary and acquired neuropathies of peripheral nerves are frequently uncritically utilized. This overview focuses on the most common laboratory tests and investigations needed for diagnosing PNPs by the general neurologist. Literature search. laboratory tests recommended for the work-up of hereditary and acquired neuropathies should be chosen according to the individual and family history, clinical presentation, and electrophysiological findings. Laboratory tests should be selected specifically according to the suspected type of neuropathy to avoid unnecessary tests and expenses. Work-up should include as few samples as necessary for uncovering the etiology and should consider the sensitivity/specificity of the tests applied.. Basic screening tests for neuropathies should include a blood cell count, thyroid, renal and liver function tests, blood glucose levels, HbA1c, vitamin-B12, and immunofixation. Other laboratory investigations should be carried out only if a specific phenotype is present or if unexpected changes of the disease course occur. In these cases referral to a neuromuscular center is recommended. Laboratory tests are helpful for the diagnosis of acquired and hereditary neuropathies but these tests should be ordered according to the history, clinical presentation and findings on electrophysiological investigations. If basic laboratory parameters fail to uncover the etiology, patients should be referred to a center specialized in neuromuscular disorders.

IMPORTANCE: A functional area associated with the piriform cortex, termed area tempestas, has been implicated in animal studies as having a crucial role in modulating seizures, but similar evidence is limited in humans. OBJECTIVE: To assess whether removal of the piriform cortex is associated with postoperative seizure freedom in patients with temporal lobe epilepsy (TLE) as a proof-of-concept for the relevance of this area in human TLE. DESIGN, SETTING, AND PARTICIPANTS: This cohort study used voxel-based morphometry and volumetry to assess differences in structural magnetic resonance imaging (MRI) scans in consecutive patients with TLE who underwent epilepsy surgery in a single center from January 1, 2005, through December 31, 2013. Participants underwent presurgical and postsurgical structural MRI and had at least 2 years of postoperative follow-up (median, 5 years; range, 2-11 years). Patients with MRI of insufficient quality were excluded. Findings were validated in 2 independent cohorts from tertiary epilepsy surgery centers. Study follow-up was completed on September 23, 2016, and data were analyzed from September 24, 2016, through April 24, 2018. EXPOSURES: Standard anterior temporal lobe resection. MAIN OUTCOMES AND MEASURES: Long-term postoperative seizure freedom. RESULTS: In total, 107 patients with unilateral TLE (left-sided in 68; 63.6% women; median age, 37 years [interquartile range {IQR}, 30-45 years]) were included in the derivation cohort. Reduced postsurgical gray matter volumes were found in the ipsilateral piriform cortex in the postoperative seizure-free group (n = 46) compared with the non–seizure-free group (n = 61). A larger proportion of the piriform cortex was resected in the seizure-free compared with the non–seizure-free groups (median, 83% [IQR, 64%-91%] vs 52% [IQR, 32%-70%]; P &lt; .001). The results were seen in left- and right-sided TLE and after adjusting for clinical variables, presurgical gray matter alterations, presurgical hippocampal volumes, and the proportion of white matter tract disconnection. Findings were externally validated in 2 independent cohorts (31 patients; left-sided TLE in 14; 54.8% women; median age, 41 years [IQR, 31-46 years]). The resected proportion of the piriform cortex was individually associated with seizure outcome after surgery (derivation cohort area under the curve, 0.80 [P &lt; .001]; external validation cohorts area under the curve, 0.89 [P &lt; .001]). Removal of at least half of the piriform cortex increased the odds of becoming seizure free by a factor of 16 (95% CI, 5-47; P &lt; .001). Other mesiotemporal structures (ie, hippocampus, amygdala, and entorhinal cortex) and the overall resection volume were not associated with outcomes. CONCLUSIONS AND RELEVANCE: These results support the importance of resecting the piriform cortex in neurosurgical treatment of TLE and suggest that this area has a key role in seizure generation.

Mechanistic target of rapamycin (mTOR) promotes cell proliferation, growth, and survival and is overactivated in many tumors and central nervous system disorders. PQR620 (3) is a novel, potent, selective, and brain penetrable inhibitor of mTORC1/2 kinase. PQR620 (3) showed excellent selectivity for mTOR over PI3K and protein kinases and efficiently prevented cancer cell growth in a 66 cancer cell line panel. In C57BL/6J and Sprague–Dawley mice, maximum concentration (Cmax) in plasma and brain was reached after 30 min, with a half-life (t1/2) > 5 h. In an ovarian carcinoma mouse xenograft model (OVCAR-3), daily dosing of PQR620 (3) inhibited tumor growth significantly. Moreover, PQR620 (3) attenuated epileptic seizures in a tuberous sclerosis complex (TSC) mouse model. In conclusion, PQR620 (3) inhibits mTOR kinase potently and selectively, shows antitumor effects in vitro and in vivo, and promises advantages in CNS indications due to its brain/plasma distribution ratio.

Because the auditory startle reaction is abnormal in disorders with substantia nigra pathology, we hypothesized that auditory startle responses (ASRs) might also be altered in restless legs syndrome (RLS).

Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB–independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1–NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP.

The adenosine triphosphate-binding cassette transporter P-glycoprotein (ABCB1/Abcb1a) restricts at the blood–brain barrier (BBB) brain distribution of many drugs. ABCB1 may be involved in drug–drug interactions (DDIs) at the BBB, which may lead to changes in brain distribution and central nervous system side effects of drugs. Positron emission tomography (PET) with the ABCB1 substrates (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide and the ABCB1 inhibitor tariquidar has allowed direct comparison of ABCB1-mediated DDIs at the rodent and human BBB. In this work we evaluated different factors which could influence the magnitude of the interaction between tariquidar and (R)-[11C]verapamil or [11C]-N-desmethyl-loperamide at the BBB and thereby contribute to previously observed species differences between rodents and humans. We performed in vitrotransport experiments with [3H]verapamil and [3H]-N-desmethyl-loperamide in ABCB1 and Abcb1a overexpressing cell lines. Moreover we conducted in vivoPET experiments and biodistribution studies with (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide in wild-type mice without and with tariquidar pretreatment and in homozygous Abcb1a/1b(−/−)and heterozygous Abcb1a/1b(+/−)mice. We found no differences for in vitrotransport of [3H]verapamil and [3H]-N-desmethyl-loperamide by ABCB1 and Abcb1a and its inhibition by tariquidar. [3H]-N-Desmethyl-loperamide was transported with a 5 to 9 times higher transport ratio than [3H]verapamil in ABCB1- and Abcb1a-transfected cells. In vivo, brain radioactivity concentrations were lower for [11C]-N-desmethyl-loperamide than for (R)-[11C]verapamil. Both radiotracers showed tariquidar dose dependent increases in brain distribution with tariquidar half-maximum inhibitory concentrations (IC50) of 1052 nM (95% confidence interval CI: 930–1189) for (R)-[11C]verapamil and 1329 nM (95% CI: 980–1801) for [11C]-N-desmethyl-loperamide. In homozygous Abcb1a/1b(−/−)mice brain radioactivity distribution was increased by 3.9- and 2.8-fold and in heterozygous Abcb1a/1b(+/−)mice by 1.5- and 1.1-fold, for (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide, respectively, as compared with wild-type mice. For both radiotracers radiolabeled metabolites were detected in plasma and brain. When brain and plasma radioactivity concentrations were corrected for radiolabeled metabolites, brain distribution of (R)-[11C]verapamil and [11C]-N-desmethyl-loperamide was increased in tariquidar (15 mg/kg) treated animals by 14.1- and 18.3-fold, respectively, as compared with vehicle group. Isoflurane anesthesia altered [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil metabolism, and this had a direct effect on the magnitude of the increase in brain distribution following ABCB1 inhibition. Our data furthermore suggest that in the absence of ABCB1 function brain distribution of [11C]-N-desmethyl-loperamide but not (R)-[11C]verapamil may depend on cerebral blood flow. In conclusion, we have identified a number of important factors, i.e., substrate affinity to ABCB1, brain uptake of radiolabeled metabolites, anesthesia, and cerebral blood flow, which can directly influence the magnitude of ABCB1-mediated DDIs at the BBB and should therefore be taken into consideration when interpreting PET results.

Neural transplantation of GABA-producing cells into key structures within seizure-suppressing circuits holds promise for medication-resistant epilepsy patients not eligible for resection of the epileptic focus. The substantia nigra pars reticulata (SNr), a basal ganglia output structure, is well known to modulate different seizure types. A recent microinjection study by our group indicated that the subthalamic nucleus (STN), which critically regulates nigral activity, might be a more promising target for focal therapy in epilepsies than the SNr. As a proof of principle, we therefore assessed the anticonvulsant efficacy of bilateral and unilateral allografting of GABA-producing cell lines into the STN using the timed intravenous pentylenetetrazole seizure threshold test, which allows repeated seizure threshold determinations in individual rats. We observed (a) that grafted cells survived up to the end of the experiments, (b) that anticonvulsant effects can be induced by bilateral transplantation into the STN using immortalized GABAergic cells derived from the rat embryonic striatum and cells additionally transfected to obtain higher GABA synthesis than the parent cell line, and (c) that anticonvulsant effects were observed even after unilateral transplantation into the STN. Neither grafting of control cells nor transplantation outside the STN induced anticonvulsant effects, emphasizing the site and cell specificity of the observed anticonvulsant effects. To our knowledge, the present study is the first showing anticonvulsant effects by grafting of GABA-producing cells into the STN. The STN can be considered a highly promising target region for modulation of seizure circuits and, moreover, has the advantage of being clinically established for functional neurosurgery.

Five patients received a bilateral hand (n = 3), a bilateral forearm (n = 1) and a unilateral hand transplant (n = 1) between 03/2000 and 03/2014. We herein describe the long-term outcome with emphasis on function, immunosuppression (IS), histomorphology and graft vascular changes.Induction therapy with antithymocyte globulin or alemtuzumab was followed by tacrolimus, prednisolone ± mycophenolate mofetil (MMF) or tacrolimus and MMF maintenance IS. Later, an mTOR-Inhibitor was added under simultaneous withdrawal or dose reduction of tacrolimus or MMF. Steroids were avoided in one and withdrawn in 2 patients.Range of motion reached up to 70% of normal with a grip strength up to 10kg. Hand function correlated with time after transplantation and amputation level and remained stable after year 5 in all cases. Intrinsic hand muscle function recovery, discriminative sensation and temperature sensation were observed after hand transplantation. Three, 7, 6, 3 and one rejection episodes were successfully treated with steroids, anti-CD25, anti-CD52 and anti-CD20 antibodies and/or intensified maintenance IS. Repetitive events of skin rejection/inflammation late after transplantation were observed in one case. Skin histology at current shows no or mild perivascular lymphocytic infiltrates without signs of progression. Vessels are patent without signs for luminal narrowing or intimal proliferation. The overall functional outcome and patient satisfaction are highly encouraging. All patients are now free of rejection with moderate levels of IS.

Elacridar (ELC) and tariquidar (TQD) are generally thought to be nontransported inhibitors of P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP), but recent data indicate that they may also be substrates of these multidrug transporters (MDTs). The present study was designed to investigate potential transport of ELC and TQD by MDTs at the blood-brain barrier at tracer doses as used in positron emission tomography (PET) studies. We performed PET scans with carbon-11-labeled ELC and TQD before and after MDT inhibition in wild-type and transporter-knockout mice as well as in in vitro transport assays in MDT-overexpressing cells. Brain entrance of [11C]ELC and [11C]TQD administered in nanomolar tracer doses was found to be limited by Pgp- and Bcrp1-mediated efflux at the mouse blood-brain barrier. At higher, MDT-inhibitory doses, i.e., 15 mg/kg for TQD and 5 mg/kg for ELC, brain activity uptake of [11C]ELC at 25 minutes after tracer injection was 5.8 ± 0.3, 2.1 ± 0.2, and 7.5 ± 1.0-fold higher in wild-type, Mdr1a/b(−/−,)and Bcrp1(−/−)mice, respectively, but remained unchanged in Mdr1a/b(−/−)Bcrp1(−/−)mice. Activity uptake of [11C]TQD was 2.8 ± 0.2 and 6.8 ± 0.4-fold higher in wild-type and Bcrp1(−/−)mice, but remained unchanged in Mdr1a/b(−/−)and Mdr1a/b(−/−)Bcrp1(−/−)mice. Consistent with the in vivo findings, in vitro uptake assays in Pgp- and Bcrp1-overexpressing cell lines confirmed low intracellular accumulation of ELC and TQD at nanomolar concentrations and increased uptake at micromolar concentrations. As this study shows that microdoses can behave pharmacokinetically differently from MDT-inhibitory doses if a compound interacts with MDTs, conclusions from microdose studies should be drawn carefully.

Diverse brain insults, including traumatic brain injury, stroke, infections, tumors, neurodegenerative diseases, and prolonged acute symptomatic seizures, such as complex febrile seizures or status epilepticus (SE), can induce "epileptogenesis," a process by which normal brain tissue is transformed into tissue capable of generating spontaneous recurrent seizures. Furthermore, epileptogenesis operates in cryptogenic causes of epilepsy. In view of the accumulating information about cellular and molecular mechanisms of epileptogenesis, it should be possible to intervene in this process before the onset of seizures and thereby either prevent the development of epilepsy in patients at risk or increase the potential for better long-term outcome, which constitutes a major clinical need. For identifying pharmacological interventions that prevent, interrupt or reverse the epileptogenic process in people at risk, two groups of animal models, kindling and SE-induced recurrent seizures, have been recommended as potentially useful tools. Furthermore, genetic rodent models of epileptogenesis are increasingly used in assessing antiepileptogenic treatments. Two approaches have been used in these different model categories: screening of clinically established antiepileptic drugs (AEDs) for antiepileptogenic or disease-modifying potential, and targeting the key causal mechanisms that underlie epileptogenesis. The first approach indicated that among various AEDs, topiramate, levetiracetam, carisbamate, and valproate may be the most promising. On the basis of these experimental findings, two ongoing clinical trials will address the antiepileptogenic potential of topiramate and levetiracetam in patients with traumatic brain injury, hopefully translating laboratory discoveries into successful therapies. The second approach has highlighted neurodegeneration, inflammation and up-regulation of immune responses, and neuronal hyperexcitability as potential targets for antiepileptogenesis or disease modification. This article reviews these areas of progress and discusses the challenges associated with discovery of antiepileptogenic therapies.

BACKGROUND To date, 13 different neurofilament light-chain polypeptide gene (NEFL) mutations have been identified in 55 patients with Charcot-Marie-Tooth disease (CMT) from 16 families. NEFL mutations were found to be associated with axonal and demyelinating variants of CMT. OBJECTIVES To describe the clinical features of 11 patients with CMT and NEFL mutations and to explore possible genotype-phenotype correlations. DESIGN Standardized neuromuscular and nerve conduction studies were performed, and the coding regions of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), gap junction β-1 protein (GJB1), and NEFL genes were analyzed by direct DNA sequencing. SETTING Two university hospitals in Austria (referral centers for neuromuscular disorders). PATIENTS Eleven patients with CMT and NEFL mutations. MAIN OUTCOME MEASURE We genotyped NEFL in all of the patients and healthy relatives and correlated the genotype with the phenotype. RESULTS A novel NEFL mutation (p.L93P) was detected in 1 family with 4 affected individuals exhibiting a severe CMT phenotype. Nerve conduction velocities were intermediately slowed to a range of 35 to 39 m/s. In a second family and in a sporadic patient, a p.P8R mutation was identified with intermediate and severe nerve conduction slowing. CONCLUSION The results argue against an obvious genotype-phenotype correlation regarding disease onset, degree of muscle weakness, and nerve conduction slowing caused by NEFL mutations.Arch Neurol. 2007;64(7):938-944 --

The antiepileptic drug valproic acid (VPA) is widely used in the treatment of epilepsy, bipolar disorders, and migraine. However, rather high doses are required for the clinical effects of VPA, which is due to its relatively inefficient delivery to the brain. The poor brain distribution of VPA is thought to reflect an asymmetric transport system at the blood-brain barrier (BBB). Based on recent data from in vitro experiments, multidrug resistance proteins (MRPs) have been proposed to be involved in the efflux transport of VPA at the BBB. In the present study, we used different experimental in vitro and in vivo strategies to evaluate whether VPA is a substrate for MRPs or the efflux transporter P-glycoprotein (Pgp). In contrast to known Pgp or MRP substrates, such as cyclosporin A or vinblastine, no directional transport of VPA was observed in cell monolayer efflux assays using the kidney cell lines Madin Darby canine kidney II and LLC-PK1, which had been transfected with either human or mouse cDNAs for the genes encoding Pgp, MRP1, or MRP2. Likewise, no indication for efflux transport of VPA was obtained in a rat microdialysis model, using inhibitors of either Pgp or MRPs. Furthermore, a significant role of MRP2 in brain efflux of VPA was excluded by using MRP2-deficient rats. Our data do not support the hypothesis that MRP1 or MRP2 is involved in the efflux of VPA from the brain. Thus, the molecular identity of the putative transporter(s) mediating the active efflux of VPA from the brain remains to be elucidated.

Multipotent neural precursors have been suggested to exist in many parts of the adult mammalian brain. In the present study, we characterized the neurogenic potential in the piriform cortex of adult rats. Proliferation rates as detected by 5′-bromodeoxyuridine-labeling proved to be low when compared with the major neurogenic brain regions (i.e. the hippocampus and the subventricular zone). 5′-BromodeoxyuridineNeuN-labeling in accordance with doublecortin, polysialylated neural cell adhesion molecule, and TUC-4-labeling indicated that neuronal differentiation of newborn cells occurs predominantly in layer II of the piriform cortex. Many of the cells exhibited a pyramidal cell morphology. The lack of 5′-bromodeoxyuridineNeuN-labeled cells 12 weeks after 5′-bromodeoxyuridine administration argued against long-term survival of newborn neurons in the piriform cortex.

Despite the use of new antiepileptic drugs, approximately one third of patients with epilepsy have seizures that cannot be controlled satisfactorily by medical treatment. Drug resistance may exist at the time of the first seizure or may develop later as result of the disease process. The mechanisms of these different scenarios are likely to be multifactorial, and may include alterations in brain uptake or brain targets of antiepileptic drugs. Such alterations may be constitutive (intrinsic), thus underlying de novodrug resistance in epilepsy, or induced, e.g., as a consequence of recurrent seizures or disease progression. Alterations in drug efflux (“multidrug”) transporters and drug targets, such as voltage‐gated sodium channels, have been found in epileptogenic brain tissue from both patients with epilepsy, and rodent models of epilepsy. However, although the multidrug transporter and target hypotheses are biologically plausible, proof‐of‐principle is lacking for these hypotheses. An advantage of the multidrug transporter hypothesis is that it can be validated both experimentally and clinically by combining antiepileptic drugs with inhibitors of such transporters. Selective inhibitors of the major efflux transporter P‐glycoprotein are currently in clinical trials for reversing chemotherapy resistance in oncology and may soon be used to determine whether such inhibitors can prevent or reverse drug resistance in epilepsy.

Medical intractability, i.e. the absence of any response to anti-epileptic drug (AED) therapy, is an unresolved problem in many patients with epilepsy. Mechanisms of intractability are not well understood, but may include alterations of pharmacological targets and poor penetration of AEDs into the brain because of increased expression of multiple drug-resistance proteins, such as P-glycoprotein (Pgp; ABCB1), capable of active brain extrusion of various drugs, including AEDs. Increased expression of Pgp has been reported in brain tissue of patients with refractory epilepsy, but there is a lack of adequate controls, i.e. brain tissue from patients with drug-responsive epilepsy. In the present study, we used a rat model of temporal lobe epilepsy to examine whether AED responders differ from non-responders in their expression of Pgp in the brain. In this model, spontaneous recurrent seizures develop after status epilepticus induced by prolonged electrical stimulation of the basolateral amygdala. The frequency of these seizures was recorded by continuous video-EEG monitoring before, during and after daily treatment with phenobarbital, which was given at maximum tolerated doses for 2 weeks. Based on their individual response to phenobarbital, rats were grouped into responders (n = 7) and non-responders (n = 4). Pgp expression was studied by immunohistochemistry and showed striking overexpression in non-responders compared with responders in limbic brain regions, including the hippocampus. The Pgp overexpression was confined to brain capillary endothelial cells which form the blood–brain barrier. The present data are the first to demonstrate that rats with drug-resistant spontaneous seizures differ from rats with drug-responsive seizures in their Pgp expression in the brain, thereby substantiating the multidrug transporter hypothesis of intractable epilepsy.

The MDR1gene product, P-glycoprotein (P-gp), was shown to confer multi-drug resistance to cancer cells, but its overexpression is also suggested to be involved in pharmacoresistance of epilepsy by acting as an energy-dependent drug-efflux pump in the blood-brain barrier (BBB). In normal brain tissue, P-gp is almost exclusively expressed by capillary endothelial cells (EC) of the BBB, whereas little or no expression is detected in other cell types. Increased P-gp expression was observed after seizures, but localization of this increase, i.e., within brain capillary EC or within parenchymal or perivascular astrocytes, which contribute to the BBB function, is controversial. To test whether these antithetic data arise from unusual properties of the antigen itself, we compared different immunohistochemical techniques and monoclonal or polyclonal antibodies to P-gp in normal rat brain and rat brain after kainate-induced seizures. Using acetone-fixed cryostat sections of snap-frozen tissue, strong P-gp labeling was detected in EC and, after seizures, in hippocampal neurons, but not in astrocytes. In contrast, EC and neuronal P-gp immunolabeling were not seen in paraformaldehyde-fixed sections, whereas both perivascular and parenchymal astrocytes exhibited strong P-gp labeling after seizures. The lack of P-gp labeling in EC by paraformaldehyde fixation, was reversed by treatment of the sections with acetate/ethanol. These experiments demonstrate that various fixation conditions have a striking effect on the immunohistochemical localization of P-gp in rat brain and detection of its increased expression by seizures. When data obtained from different immunohistochemical techniques are taken together, seizures seem to induce overexpression of P-gp in four different cell types, i.e., EC, perivascular astrocytes, parenchymal astrocytes, and neurons.

Summary: The blood-brain barrier (BBB) contributes to brain homeostasis by protecting the brain from potentially harmful endogenous and exogenous substances. BBB active drug efflux transporters of the ATP-binding cassette (ABC) gene family are increasingly recognized as important determinants of drug distribution to, and elimination from, the CNS. The ABC efflux transporter P-glycoprotein (Pgp) has been demonstrated as a key element of the BBB that can actively transport a huge variety of lipophilic drugs out of the brain capillary endothelial cells that form the BBB. In addition to Pgp, other ABC efflux transporters such as members of the multidrug resistance protein (MRP) family and breast cancer resistance protein (BCRP) seem to contribute to BBB function. Consequences of ABC efflux transporters in the BBB include minimizing or avoiding neurotoxic adverse effects of drugs that otherwise would penetrate into the brain. However, ABC efflux transporters may also limit the central distribution of drugs that are beneficial to treat CNS diseases. Furthermore, neurological disorders such as epilepsy may be associated with overexpression of ABC efflux transporters at the BBB, resulting in pharmacoresistance to therapeutic medication. Therefore, modulation of ABC efflux transporters at the BBB forms a novel strategy to enhance the penetration of drugs into the brain and may yield new therapeutic options for drug-resistant CNS diseases.

Multidrug transporter over-expression is considered to limit access of antiepileptic drugs to the epileptic focus region and to be one cause of intractable epilepsy. To reach further proof for this multidrug transporter hypothesis, we compared P-glycoprotein expression rates in subgroups of Wistar rats which are sensitive or resistant to the anticonvulsant effect of the antiepileptic drug phenytoin in the amygdala-kindling model of temporal lobe epilepsy. In the electrode-implanted amygdala of phenytoin-resistant rats, the area labelled for P-glycoprotein was more than twice as large than that in phenytoin-sensitive rats. The data indicate that P-glycoprotein expression levels in the kindled focus have a critical impact on the anticonvulsant response to antiepileptic drugs.

Previous lesion studies have indicated a role of the amygdala in the central regulation of food intake. In the present experiments, twice-daily electrical stimulation of the basolateral nucleus of the amygdala in female Wistar rats was found to be associated with a significant body weight gain compared to unstimulated controls. On average, significant increases in body weight were observed after 25 amygdala stimulations, using a kindling paradigm for stimulation. Compared to kindled rats, in which amygdala stimulations were terminated after about 20 stimulations, extended kindling of the amygdala with up to 280 stimulations led to progressive weight increases and compulsive hyperphagia. No gross neuronal damage was seen in thionin-stained sections of the amygdala after extended kindling, but degeneration of a specific type of neurons can not be excluded. The results substantiate that amygdaloid nuclei are an important extrahypothalamic site for the regulation of food intake and body weight. The extensive weight gain over extended amygdala kindling provides an interesting new model for experimentally induced obesity.

The blood-brain barrier (BBB) is a physical and metabolic barrier between the brain and the systemic circulation, which functions to protect the brain from circulating drugs, toxins, and xenobiotics. ATP-dependent multidrug transporters such as P-glycoprotein (Pgp; ABCB1), which are found in the apical (luminal) membranes of brain capillary endothelial cells, are thought to play an important role in BBB function by limiting drug penetration into the brain. More recently, the multidrug resistance protein MRP2 (ABCC2) has been found in the luminal surface of brain capillary endothelium of different species, including humans. In endothelial cells from patients with drug-resistant epilepsy, MRP2 was shown to be overexpressed, indicating that it may be critically involved in multidrug resistance of such patients. However, the role of MRP2 in drug disposition into the brain is defined poorly. Herein, we used different strategies to study the contribution of MRP2 to BBB function. First, the MRP inhibitor probenecid was shown to increase extracellular brain levels of the major antiepileptic drug phenytoin in rats, indicating that phenytoin is a substrate of MRP2 in the BBB. This was substantiated by using MRP2-deficient TR– rats, in which extracellular brain levels of phenytoin were significantly higher compared with the normal background strain. In the kindling model of epilepsy, coadministration of probenecid significantly increased the anticonvulsant activity of phenytoin. In kindled MRP2-deficient rats, phenytoin exerted a markedly higher anticonvulsant activity than in normal rats. These data indicate that MRP2 substantially contributes to BBB function.

We describe a patient with intermittent, at times rhythmic trunk flexion movements. Neurophysiological assessment excluded a psychogenic movement disorder. The segmental spinal myoclonus occurred 6 years after radiation therapy of the brain and entire spinal cord, and we suggest this patient to be the first case of a late‐delayed sequela of spinal cord irradiation presenting as segmental spinal myoclonus. © 2002 Movement Disorder Society

A decline in voluntary muscle activation and adaptations in motor cortical excitability contribute to the progressive decline in voluntary force during sustained isometric contractions. However, the neuronal control of muscle activation differs between isometric and dynamic contractions. This study was designed to investigate voluntary activation, motor cortex excitability, and intracortical inhibition during fatiguing concentric and eccentric actions. Eight subjects performed 143 torque motor–controlled, repeated shortening and lengthening actions of the elbow flexor muscles. Transcranial magnetic stimulation (TMS) was applied three times every 20 cycles. Magnetic evoked motor potentials (MEP), duration of the silent period (SP), and the torque increase due to TMS were analyzed. TMS resulted in a small torque increase in unfatigued actions. With repeated actions, voluntary torque dropped rapidly and the amplitude of the TMS‐induced twitches increased, especially during repeated lengthening actions. MEP area of biceps brachii and brachioradialis muscles increased during repeated actions to a similar extent during lengthening and shortening fatigue. The duration of biceps and brachioradialis SP did not change with fatigue. Thus, voluntary activation became suboptimal during fatiguing dynamic actions and motor cortex excitability increased without any changes in intracortical inhibition. The apparent dissociation of voluntary activation and motor cortex excitability suggests that the central fatigue observed, especially during lengthening actions, did not result from changes in motor cortex excitability. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 000–000, 2002

Epilepsy, one of the most common neurologic disorders, is a major public health issue. Despite more than 20 approved antiepileptic drugs (AEDs), about 30% of patients are refractory to treatment. An important characteristic of pharmacoresistant epilepsy is that most patients with refractory epilepsy are resistant to several, if not all, AEDs, even though these drugs act by different mechanisms. This argues against epilepsy-induced alterations in specific drug targets as a major cause of pharmacoresistant epilepsy, but rather points to nonspecific and possibly adaptive mechanisms, such as decreased drug uptake into the brain by intrinsic or acquired over-expression of multidrug transporters in the blood-brain barrier (BBB). There is accumulating evidence demonstrating that multidrug transporters such as P-glycoprotein (PGP) and members of the multidrug resistance-associated protein (MRP) family are over-expressed in capillary endothelial cells and astrocytes in epileptogenic brain tissue surgically resected from patients with medically intractable epilepsy. PGP and MRPs in the BBB are thought to act as an active defense mechanism, restricting the penetration of lipophilic substances into the brain. A large variety of compounds, including many lipophilic drugs, are substrates for either PGP or MRPs or both. It is thus not astonishing that several AEDs, which have been made lipophilic to penetrate into the brain, seem to be substrates for multidrug transporters in the BBB. Over-expression of such transporters in epileptogenic tissue is thus likely to reduce the amount of drug that reaches the epileptic neurons, which would be a likely explanation for pharmacoresistance. PGP and MRPs can be blocked by specific inhibitors, which raises the option to use such inhibitors as adjunctive treatment for medically refractory epilepsy. However, although over-expression of multidrug transporters is a novel and reasonable hypothesis to explain multidrug resistance in epilepsy, further studies are needed to establish this concept. Furthermore, there are certainly other mechanisms of pharmacoresistance that need to be identified.

Temporal lobe epilepsy is often refractory to antiepileptic drugs (AEDs). Accordingly, amygdala-kindled rats, a widely used model of temporal lobe epilepsy, have previously been found to be less responsive to AED treatment than non-kindled rats. In view of recent findings of over-expression of multidrug transporters in the blood–brain barrier of patients with pharmacoresistant epilepsy, one explanation for the finding of difficult-to-treat seizures in kindled rats would be a reduced penetration of AEDs into epileptogenic brain tissue. For evaluation of this possibility, we used brain microdialysis in order to compare extracellular levels of the AED phenytoin in amygdala and hippocampus of conscious, unrestrained amygdala-kindled and non-kindled rats. Consistent with the lower anticonvulsant efficacy of phenytoin in kindled rats, average phenytoin levels in dialysates of kindled rats were lower (up to 30) than in non-kindled controls, but the differences were not statistically significant. This indicates that either the relatively large interindividual variation in dialysate levels of phenytoin masks functionally significant differences in individual kindled rats or that alterations in brain drug penetration are not involved in the lowered response of kindled rats to AEDs.

Despite considerable advances in the pharmacotherapy of epilepsy, about 30 of epileptic patients are refractory to antiepileptic drugs (AEDs). In most cases, a patient who is resistant to one major AED is also refractory to other AEDs, although these drugs act by different mechanisms. The mechanisms that lead to drug resistance in epilepsy are not known. Recently, over-expression of multidrug transporters, such as P-glycoprotein (PGP) and multidrug resistance-associated protein (MRP), has been reported in surgically resected epileptogenic human brain tissue and suggested to contribute to the drug resistance of epilepsy. However, it is not known to what extent multidrug transporters such as PGP or MRP are involved in transport of AEDs. In the present study, we used in vivomicrodialysis in rats to study whether the concentration of carbamazepine in the extracellular fluid of the cerebral cortex can be enhanced by inhibition of PGP or MRP, using the PGP inhibitor verapamil and the MRP inhibitor probenecid. Local perfusion with verapamil or probenecid via the microdialysis probe increased the extracellular concentration of carbamazepine. The data indicate that both PGP and MRP participate in the regulation of extracellular brain concentrations of the major AED carbamazepine.

In a hamster model (genetic symbol dtsz) of primary paroxysmal non‐kinesiogenic dystonic choreoathetosis, recent studies have shown beneficial effects of glutamate and dopamine receptor antagonists. Nitric oxide (NO), synthesized from L‐arginine by NO synthase in response to glutamate receptor activation, elicits cyclic GMP and modulates glutamate‐mediated processes and striatal dopamine release.Therefore, the effects of NO synthase inhibitors and of L‐arginine on severity of dystonia were investigated in dtszhamsters in which dystonic attacks, characterized by twisting movements and postures, can be induced by stress.The NO synthase inhibitors NG‐nitro‐L‐arginine (L‐NNA), NG‐nitro‐L‐arginine methyl ester (L‐NAME) and 7‐nitroindazole significantly reduced the severity of dystonia. At antidystonic effective doses neither L‐NNA nor L‐NAME caused observable side effects, whereas 7‐nitroindazole exerted moderate reduction of locomotor activity.The antidystonic effect of L‐NAME was reversed by co‐administration of the NO precursor L‐arginine. However, L‐arginine administered alone did not exert any effect on severity of dystonia.Cerebellar cyclic GMP levels in brains of mutant hamsters in comparison to non‐dystonic control hamsters did not significantly differ, but the cerebellar cyclic GMP levels tended to be increased in dtszhamsters during a dystonic attack. L‐NAME significantly decreased the cerebellar cyclic GMP levels in both dtszand control hamsters.Although an overproduction of NO is probably not critically involved in the pathogenesis of paroxysmal dystonia, it may contribute to the manifestation of dystonic attacks, as indicated by the antidystonic effects of NO synthase inhibitors.Peripheral side effects may limit the clinical use of NO synthase inhibitors, but more selective inhibitors of the neuronal NO synthase should be considered as interesting candidates for the treatment of paroxysmal dystonia.

1 The centrally acting analgesic tramadol has recently been reported to cause seizures at re-commended dosages in patients, whereas animal experiments had indicated that seizures only occur in high, toxic doses. Tramadol has a dual mechanism of action that includes weak agonistic effects at the mu-opioid receptor as well as inhibition of monoamine (serotonin, norepinephrine) re-uptake. Its major (M1) metabolite mono-O-desmethyltramadol, which is rapidly formed in vivo, has a markedly higher affinity for mu receptors and may thus contribute to the effects of the parent compound. Furthermore, the pharmacological effects of tramadol appear to be related to the different, but complementary and interactive pharmacologies of its enantiomers. In the present study, we evaluated (±)-tramadol, its enantiomers, and its M1 metabolite ((+)-enantiomer) in the amygdala kindling model of epilepsy in rats. Adverse effects determined in kindled rats were compared to those in nonkindled rats. 2 At doses within the analgesic range, (±)-tramadol and its enantiomers induced anticonvulsant effects in kindled rats. However, at only slightly higher doses seizures occurred. With (±)-tramadol, generalized seizures were observed at 30 mg kg−1 in most kindled but not in nonkindled rats. The (−)-enantiomer induced myoclonic seizures at 30 mg kg−1 in most kindled but not in nonkindled rats, although myoclonic seizure activity was observed in some nonkindled rats at 10 or 20 mg kg−1. Seizures were also observed after the (+)-enantiomer and the (+)-enantiomer of the M1 metabolite, but experiments with higher doses of these compounds were limited by marked respiratory depression. 3 The data demonstrate that kindling enhances the susceptibility of rats to convulsant adverse effects of tramadol and its enantiomers, indicating that a preexisting lowered seizure threshold increases the risk of tramadol-induced seizures. British Journal of Pharmacology (2000) 131, 203 – 212

We report an Austrian family with proximal muscle weakness and wasting predominantly of the shoulder girdle musculature, normal or slightly reduced distal muscle power, mild foot deformity, absent or reduced tendon reflexes in the lower limbs, and normal or slightly diminished sensation. Electrophysiologically, motor nerve conduction velocities were slowed to less than 33 m/s, distal latencies were prolonged, and compound motor action potentials were low. Sensory nerve conduction velocities were extremely reduced or no sensory potentials were recordable. Genetic testing in three affected individuals revealed a duplication of the chromosomal region 17p11.2. In addition, genetic testing for facioscapulohumeral muscular dystrophy (FSHD) revealed a 33 kb EcoRI fragment on chromosome 4q35 in one affected individual and in the clinically normal parent, whereas in a second affected person normal DNA‐sizes were observed. These clinical findings define a new phenotypic variant associated with the Charcot‐Marie‐Tooth 1A duplication. This may be due to a mutation in another gene contained in the 1.5 Mb duplication although mutations in the peripheral myelin protein 22 gene have been excluded. Alternatively, the genetic background of other genes in the family may modify the phenotypic expression, as found in other inherited diseases.The unusual phenotype cannot be explained by the concomitant presence of FSHD despite some evidence for coexistance in one individual. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1243–1249, 2000.

We report on a large four-generation Austrian family with autosomal dominant distal hereditary motor neuronopathy type V (distal HMN V). Forty-seven at-risk family members, of whom 21 were definitely affected, underwent detailed clinical, electrophysiological and genetic studies. The age at onset was in the second decade of life in most affected individuals, but clinical presentation was rather variable. While the majority of patients were primarily disabled by progressive asymmetrical wasting of the thenar and the first dorsal interosseus muscles, others had marked foot deformity and gait disturbance with the occasional absence of hand involvement. Sensation sense was normal except for the reduced response to vibration. Many individuals showed brisk tendon reflexes and some elevated muscle tone in the lower limbs, but extensor plantar responses were rarely observed. Electrophysiological evaluation revealed normal or reduced motor nerve conduction velocities, normal or prolonged distal motor latencies, and low compound motor action potentials, depending on the degree of muscle wasting. Sensory nerve studies were usually within the normal range or slightly to moderately abnormal in older or severely affected persons. Electromyography showed high-amplitude motor unit potentials and reduced recruitment compatible with anterior horn cell degeneration. Central motor conduction times were prolonged in two-thirds of the patients. Molecular genetic studies excluded Charcot–Marie–Tooth 1A syndrome and proximal spinal muscular atrophy linked to chromosome 5q as well as the known gene loci for distal HMN II on chromosome 12q, HMN V on chromosome 7p and juvenile amyotrophic lateral sclerosis on chromosome 9q. The findings in this family thus provide detailed clinical and electrophysiological information on HMN V and demonstrate broad phenotypic variability in this disorder. Hallmark features are discussed that appear to be most reliable to differentiate this type of HMN V from other variants of hereditary neuropathies, and a set of diagnostic criteria is proposed. Furthermore, this is the first report of prolonged central motor conduction times in HMN V, which indicates additional involvement of the central motor pathways in this disease. Finally, molecular genetic studies demonstrate genetic heterogeneity, suggesting the existence of at least a second genetic subtype in HMN V.

Seizures in human temporal lobe epilepsy are characterized by paroxysmal activity in the limbic system. The primary olfactory or piriform cortex is a central part of the limbic system. Since a relationship between olfactory sensation and limbic seizures has been described, we were interested in the effect of strong olfactory stimulation on the seizure susceptibility of amygdala-kindled rats, a model of human temporal lobe epilepsy. Olfactory stimulation with toluene was able to suppress seizures in most kindled rats after stimulation at 20% above the threshold for eliciting epileptic afterdischarges. Olfactory stimulation with toluene or ammonia increased the threshold by 27 and 25% compared to control conditions. Our data substantiate that olfactory brain regions, such as the piriform cortex, are involved in amygdala-kindled seizures and suggest that strong physiological stimulation of this nucleus interferes with on-going seizure activity in the limbic system. Thus, olfactory stimulation could contribute to anticonvulsant therapy.

Objectives: The aim of the study was to investigate the effects of different loads on tremor around 10 Hz during fatiguing contractions.

The anticonvulsant activity of inhibitors of monoamine oxidase (MAO) was reported early after the development of irreversible MAO inhibitors such as tranylcypromine, but was never clinically used because of the adverse effects of these compounds. The more recently developed reversible MAO inhibitors with selectivity for either the MAO-A or MAO-B isoenzyme forms have not been studied extensively in animal models of epilepsy, so it is not known which type of MAO inhibitor is particularly effective in this respect. We compared the following drugs in the kindling model of epilepsy: 1) l-deprenyl (selegiline), i.e., an irreversible inhibitor of MAO-B, which, however, also inhibits MAO-A at higher doses, 2) the novel reversible MAO-B inhibitor LU 53439 (3,4-dimethyl-7-(2-isopropyl-1,3,4-thiadiazol-5-yl)-methoxy-coumarin), which is much more selective for MAO-B than l-deprenyl, 3) the novel reversible and highly selective MAO-A inhibitor LU 43839 (esuprone; 7-hydroxy-3,4-dimethylcoumarin ethanesulfonate), and 4) the irreversible nonselective MAO inhibitor tranylcypromine. Esuprone proved to be an effective anticonvulsant in the kindling model with a similar potency as l-deprenyl. In contrast to esuprone and l-deprenyl, the selective MAO-B inhibitor LU 53439 was not effective in the kindling model; this substantiates the previous notion that the anticonvulsant activity ofl-deprenyl is not related to MAO-B inhibition, but to other effects of this drug, such as inhibition of MAO-A. Drugs inhibiting both MAO-A and MAO-B to a similar extent (tranylcypromine) or combinations of selective MAO-A and MAO-B inhibitors (esuprone plus LU 53439) had no advantage over MAO-A inhibition alone, but were less well tolerated. The data thus suggest that selective MAO-A inhibitors such as esuprone may be an interesting new approach for the treatment of epilepsy.

Dystonic attacks, including twisting movements, can be initiated by mild stress in mutant (gene symbol dt^sz) Syrian golden hamsters, an animal model of idiopathic paroxysmal dystonia. Previous studies suggested that dysfunctions in basal ganglia, which are not restricted to periods of attacks, are involved in the dystonic syndrome in mutant hamsters. Therefore, in the present study in anesthetized animals, we examined whether the spontaneous firing rate of extracellularly recorded neurons of the substantia nigra pars reticulata (SNr) differs between dt^sz and age-matched nondystonic control hamsters. Furthermore, we investigated the responsiveness of these nondopaminergic, presumably GABAergic neurons to a subconvulsive dose (25mg/kg i.p.) of systemically applied pentylenetetrazole (PTZ), which exerts prodystonic effects in mutant hamsters. The mean basal (spontaneous) firing rate of SNr neurons was not altered in mutant hamsters. However, within 5 min after i.p. injection of PTZ, the mean firing rate of SNr neurons significantly increased to about 160% of predrug control values in dt^sz but not in control hamsters. Although the present study failed to reveal changes in the basal firing rate of SNr neurons in mutant hamsters, the abnormal response to PTZ is in line with previous pharmacological and biochemical data indicating disturbed function of the GABAergic system. Synapse 33:259–267, 1999. © 1999 Wiley-Liss, Inc.

We have previously shown that the novel anticonvulsant levetiracetam exerts potent anticonvulsant activity against both focal and secondarily generalized seizures in fully amygdala-kindled rats, i.e., a model of temporal lobe epilepsy. We examined whether levetiracetam also exhibits antiepileptogenic activity, i.e., prevents or retards acquisition or development of amygdala-kindling in rats. Before the experiments with chronic administration of levetiracetam at different doses, we determined the pharmacokinetics of the drug after i.p. injection. Levetiracetam had a relatively short half-life (about 2-3 hr) in rats, so that any lasting effects of the drug after chronic administration were certainly not due to drug accumulation. When rats were treated with levetiracetam during kindling acquisition at daily i.p. doses of 13, 27 or 54 mg/kg, the drug dose-dependently suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation. After termination of daily treatment with 54 mg/kg, duration of behavioral seizures and of afterdischarges recorded from the amygdala remained to be significantly shorter compared to vehicle controls, although amygdala stimulations were continued in the absence of drug. These data thus indicate that levetiracetam not simply masked the expression of kindled seizures through an anticonvulsant action, but exerted a true antiepileptogenic effect. Adverse effects were not observed at any dose of levetiracetam tested in kindled rats. The powerful antiepileptogenic activity of levetiracetam in the kindling model indicates that levetiracetam is not only an interesting novel drug for symptomatic treatment of epilepsy but might be suited for pharmacological prevention of this disease in patients with a high prospective risk of the development of epilepsy.

A modified cortical ramp stimulation (CRS) model has been developed allowing repeated determinations of seizure threshold at short time intervals in individual rats without inducing postictal threshold increases. Anticonvulsant potency of the standard antiepileptic drugs carbamazepine, phenytoin, phenobarbital, valproate, diazepam and ethosuximide in the CRS model was compared with respective drug potencies in two more traditional seizure models with transcorneal stimulus application, i.e., the minimal electroshock seizure threshold (minEST) and the maximal electroshock seizure threshold (maxEST). In the CRS model, two different types of threshold were determined, the threshold for localized seizures (TLS) and the threshold for generalized seizures (TGS). When screw electrodes were implanted over the primary motor cortex, TLS was characterized by unilateral forelimb clonus, tonic abduction of contralateral forelimb, and head adversion. When ramp-shaped stimulation was continued above the TLS current, bilateral clonic forelimb seizures with loss of posture developed, which was defined as TGS. In contrast to TLS, TGS could not be repeatedly determined at short time intervals because of postictal threshold increase. TLS was dose-dependently increased by carbamazepine, phenobarbital, valproate and diazepam, although phenytoin showed a truncated dose-response, and ethosuximide was ineffective. In comparison to TLS, drug-induced increases in TGS were more marked. All drugs dose-dependently increased minEST and, except ethosuximide, maxEST. For comparison of drug potencies, doses increasing seizure thresholds by 20 or 50% were calculated from dose-response curves. Respective comparisons showed marked differences in drug potencies between models, indicating that the CRS method presents a model of another, more pharmacoresistant seizure type than seizure types induced in traditional models, such as transcorneal electroshock. Based on the location of electrodes in the frontal neocortex, the characteristic seizure pattern, and the low pharmacological sensitivity of the seizures to standard antiepileptics, the modified CRS model most likely represents a new model of localization-related seizures occurring in frontal lobe epilepsy and may thus be used in the search for novel drugs with higher efficacy against this difficult-to-treat type of epilepsy.

Effects of the trans-isomer of 2-en-valproate (trans-2-en-NaVP; E-Δ2-en-valproate or 2-en-valproate), an unsaturated metabolite of valproic acid (VPA), on intracellularly recorded sodium-dependent action potentials of cultured mouse spinal cord and cortical neurons were compared with those of the anticonvulsant sodium valproate (NaVP). The maximal rate of rise of action potentials triggered by trains of 1-msec or 400-msec pulses declined progressively until failure to fire in both cell types during exposure to trans-2-en-NaVP or NaVP was observed. The limitation of firing by both drugs was concentration, voltage, rate and time dependent. The IC50of trans-2-en-NaVP was 1.2 × 10−3at ≤1 hr and 4.8 × 10−5M at 24 to 48 hr. Trans-2-en-NaVP did not limit sustained repetitive firing in all cortical neurons. This may reflect slower rates of firing during 400-msec depolarizations in neurons of this type. In paired-pulse experiments, the absolute refractory period was 7 msec in control solution and 15 msec (P < .01 vs.control;n= 9) in solution containing 6 × 10−4M trans-2-en-NaVP. Firing was limited in all spinal cord neurons after exposure to 0.5 mM NaVP for 24 to 48 hr; 80% were limited by 1 mM NaVP at ≤1 hr. Coincubation of the spinal cord neurons with trans-2-en-NaVP and NaVP for 24 hr showed no hyperadditive effect of these two drugs in vitro.Limitation of sustained repetitive firing was reversed by hyperpolarization in the continuing presence of either drug and incubation in drug-free medium. Limitation of sodium-dependent action potential firing rates could contribute, at least in part, to the anticonvulsant effect of trans-2-en-NaVP.

There is good evidence in some species, including rats, that circannual rhythms are innate and can occur even under constant environmental conditions. Such circannual rhythms, e.g. in hormone levels and immune system function, may influence tumourigenesis. This prompted us to study 7,12‐dimelhylbenz[a]anthracene(DMBA)‐induced mammary carcinogenesis at different seasons of the year in female Sprague‐Dawley rats under constant environmental conditions (photoperiod, temperature, air humidity, food). DMBA was administered orally at a dose of 5 mg per rat at the first day of the experiment and then at weekly intervals up to a total dose of 20 mg per rat. Rats were palpated once weekly for the presence of mammary tumours. After 13 weeks, they were necropsied for examination of the number and size of mammary tumours. Age‐matched groups of 36‐99 rats were used per experiment. When the experiment was performed twice within 2 years during the same season (spring/summer), tumour incidence (56 and 61%) and tumour burden were almost equal, indicating that data obtained in this way were reproducible. However, the same experiment performed in autumn yielded a significantly lower tumour incidence (34%) and tumour burden. When the experiment was started during winter, tumour incidence was similar to the spring/summer groups, but tumour burden was lower. The data indicate a seasonal variation in the development and growth of DMBA‐induced breast cancer in Sprague‐Dawley rats. One possible explanation for this phenomenon may be the seasonal variation in pineal melatonin production and immune function previously reported in rodents under constant environmental conditions.

Valproate is currently one of the major antiepileptic drugs in clinical use. Because of its wide spectrum of anticonvulsant activity against different seizure types, it has repeatedly been suggested that valproate acts through a combination of several mechanisms. As shown in this review, there is substantial evidence that valproate increases GABA turnover and thereby potentiates GABAergic functions in some specific brain regions, such as substantia nigra, thought to be involved in the control of seizure generation and propagation. Furthermore, valproate seems to reduce the release of the epileptogenic amino acid gamma-hydroxybutyric acid and to block cell firing induced by NMDA-type glutamate receptors. In addition to effects on amino acidergic neurotransmission, valproate presumably exerts a direct action on ion channels, thereby limiting sustained repetitive neuronal firing. Recent microdialysis data suggest that valproate also alters dopaminergic and serotonergic functions. These diverse effects of valproate might explain why the drug not only exerts anticonvulsant activity but also other pharmacodynamic and pharmacotherapeutic actions, such as antipsychotic and antidystonic efficacy.

Concentrations of 11 amino acids, including the neurotransmitters GABA, glutamate, aspartate, glycine and taurine, were determined in 12 brain regions of female rats during different stages of the estrous cycle. In addition, amino acids and sex hormone levels were determined in plasma. All sample collections were done in the forenoon between 9 and 11 a.m. Most regional amino acid levels measured did not change signficantly during estrous cycle, but significant alterations were found for GABA and glutamate in hypothalamus. Both amino acids were slightly decreased in hypothalamus during proestrus, which might reflect an alteration of GABA turnover in response to the high estrogen levels during this stage. A decreased glutamate level during proestrus was also found in thalamus, while both glutamate and GABA did not vary throughout estrous cycle in any of the other examined regions, including substantia nigra, amygdala, striatum, cortex and hippocampus. When diestrus was subdivided according to progesterone levels, high levels of this hormone seemed to be associated with effects on metabolism of certain amino acids, including glycine in substantia nigra, alanine in thalamus and threonine in pons/medulla. However, the few changes in regional amino acid concentrations found during the estrous cycle were so small that the functional significance of these changes cannot be ascertained without further determination of the cellular or subcellular compartments of brain tissue involved.

Kindling is a chronic model of epilepsy characterized by a progressive increase in response to the same regularly applied stimulus. The biological basis of the kindling phenomenon requires to be determined, but several studies indicate that alterations in amino acidergic neurotransmission may be involved. In the present experiments, levels of glutamate, aspartate, GABA, glycine, and taurine were determined in 12 brain regions by HPLC in 3 groups of animals: (a) a group which was kindled via electrical stimulation of intraamygdala electrodes and was sacrificed 36 days after the last fully kindled seizure for neurochemical determinations; (b) a group of implanted but nonstimulated rats (surgical control group) in which neurochemical measurements were done at the same time after electrode implantation as the kindled group, and (c) a group of non-implanted, naive control rats. Compared to surgical controls, kindling induced a significant reduction of glutamate, GABA, and taurine in the brain stem (pons/medulla), whereas no differences between both groups were found in any of the other regions. However, both electrode-implanted groups differed significantly from non-implanted naive rats in several regions, indicating that electrode-implantation per se induced long-lasting alterations in transmitter amino acids. The most striking difference to naive controls was an increase of glycine levels in several regions in which this amino acid is known to potentiate glutamatergic transmission. In order to examine the functional consequences of prolonged electrode implantation, seizure thresholds were determined in groups of rats with short and prolonged electrode implantation. Data from these experiments indicated that prolonged electrode implantation per se induces pro-kindling effects, i.e. a dramatic decrease of seizure threshold. The data of this study thus demonstrate that the choice of adequate controls is critical in neurochemical and functional studies on the kindling phenomenon.