The relationship between severe myoclonic epilepsy of infancy (SMEI or Dravet syndrome) and the related syndrome SMEI-borderland (SMEB) with mutations in the sodium channel alpha 1 subunit gene SCN1A is well established. To explore the phenotypic variability associated with SCN1A mutations, 188 patients with a range of epileptic encephalopathies were examined for SCN1A sequence variations by denaturing high performance liquid chromatography and sequencing. All patients had seizure onset within the first 2 years of life. A higher proportion of mutations were identified in patients with SMEI (52/66; 79%) compared to patients with SMEB (25/36; 69%). By studying a broader spectrum of infantile epileptic encephalopathies, we identified mutations in other syndromes including cryptogenic generalized epilepsy (24%) and cryptogenic focal epilepsy (22%). Within the latter group, a distinctive subgroup designated as severe infantile multifocal epilepsy had SCN1A mutations in three of five cases. This phenotype is characterized by early onset multifocal seizures and later cognitive decline. Knowledge of an expanded spectrum of epileptic encephalopathies associated with SCN1A mutations allows earlier diagnostic confirmation for children with these devastating disorders.

GLT-1, GLAST, and EAAC1 are high-affinity, Na(+)-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptake Vmax, but no change in the glutamate K(m) and no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid and threo-beta-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.

We have developed a novel microfluidic device constructed from poly(dimethylsiloxane) using multilayer soft lithography technology for the analysis of single cells. The microfluidic network enables the passive and gentle separation of a single cell from the bulk cell suspension, and integrated valves and pumps enable the precise delivery of nanoliter volumes of reagents to that cell. Various applications are demonstrated, including cell viability assays, ionophore-mediated intracellular Ca2+ flux measurements, and multistep receptor-mediated Ca2+ measurements. These assays, and others, are achieved with significant improvements in reagent consumption, analysis time, and temporal resolution over macroscale alternatives.

Glucose transporter-1 deficiency syndrome is caused by mutations in the SLC2A1 gene in the majority of patients and results in impaired glucose transport into the brain. From 2004-2008, 132 requests for mutational analysis of the SLC2A1 gene were studied by automated Sanger sequencing and multiplex ligation-dependent probe amplification. Mutations in the SLC2A1 gene were detected in 54 patients (41%) and subsequently in three clinically affected family members. In these 57 patients we identified 49 different mutations, including six multiple exon deletions, six known mutations and 37 novel mutations (13 missense, five nonsense, 13 frame shift, four splice site and two translation initiation mutations). Clinical data were retrospectively collected from referring physicians by means of a questionnaire. Three different phenotypes were recognized: (i) the classical phenotype (84%), subdivided into early-onset (<2 years) (65%) and late-onset (18%); (ii) a non-classical phenotype, with mental retardation and movement disorder, without epilepsy (15%); and (iii) one adult case of glucose transporter-1 deficiency syndrome with minimal symptoms. Recognizing glucose transporter-1 deficiency syndrome is important, since a ketogenic diet was effective in most of the patients with epilepsy (86%) and also reduced movement disorders in 48% of the patients with a classical phenotype and 71% of the patients with a non-classical phenotype. The average delay in diagnosing classical glucose transporter-1 deficiency syndrome was 6.6 years (range 1 month-16 years). Cerebrospinal fluid glucose was below 2.5 mmol/l (range 0.9-2.4 mmol/l) in all patients and cerebrospinal fluid : blood glucose ratio was below 0.50 in all but one patient (range 0.19-0.52). Cerebrospinal fluid lactate was low to normal in all patients. Our relatively large series of 57 patients with glucose transporter-1 deficiency syndrome allowed us to identify correlations between genotype, phenotype and biochemical data. Type of mutation was related to the severity of mental retardation and the presence of complex movement disorders. Cerebrospinal fluid : blood glucose ratio was related to type of mutation and phenotype. In conclusion, a substantial number of the patients with glucose transporter-1 deficiency syndrome do not have epilepsy. Our study demonstrates that a lumbar puncture provides the diagnostic clue to glucose transporter-1 deficiency syndrome and can thereby dramatically reduce diagnostic delay to allow early start of the ketogenic diet.

We tested the hypothesis that astrocytic glycogen sustains axon function during and enhances axon survival after 60 min of glucose deprivation. Axon function in the rat optic nerve (RON), a CNS white matter tract, was monitored by measuring the area of the stimulus-evoked compound action potential (CAP). Switching to glucose-free artificial CSF (aCSF) had no effect on the CAP area for ϳ30 min, after which the CAP rapidly failed. Exposure to glucose-free aCSF for 60 min caused irreversible injury, which was measured as incomplete recovery of the CAP. Glycogen content of the RON fell to a low stable level 30 min after glucose withdrawal, compatible with rapid use in the absence of glucose. An increase of glycogen content induced by high-glucose pretreatment increased the latency to CAP failure and improved CAP recovery. Conversely, a decrease of glycogen content induced by norepinephrine pretreatment decreased the latency to CAP failure and reduced CAP recovery. To determine whether lactate represented the fuel derived from glycogen and shuttled to axons, we used the lactate transport blockers quercetin, ␣-cyano-4-hydroxycinnamic acid (4-CIN), and p-chloromercuribenzene sulfonic acid ( pCMBS). All transport blockers, when applied during glucose withdrawal, decreased latency to CAP failure and decreased CAP recovery. The inhibitors 4-CIN and pCMBS, but not quercetin, blocked lactate uptake by axons. These results indicated that, in the absence of glucose, astrocytic glycogen was broken down to lactate, which was transferred to axons for fuel. Key words: astrocytes; ␣-cyano-4-hydroxycinnamate; glucose; hypoglycemia; lactate; p-chloromercuribenzene sulfonic acid; quercetin; rat optic nerveThe function of brain glycogen is not well understood. Glycogen turns over rapidly in the brain, however, and turnover is enhanced when adjacent neural activity is increased (Orkand et al., 1973;Pentreath and Kai-Kai, 1982;Swanson et al., 1992). It is appealing to imagine that glycogen might serve to provide fuel to the brain when glucose is in short supply. Indeed, astrocytic glycogen in vitro is degraded rapidly when glucose is withdrawn (Dringen et al., 1993), and glycogen falls rapidly in vivo during ischemia, with a time course that is closely related to the depletion of ATP and the accumulation of lactate (Swanson et al., 1989a). These observations are consistent with the action of glycogen as a fuel source during glucose shortage, but they do not prove this hypothesis. Glycogen content varies by a factor of two or more between brain regions [it is highest in the brainstem and cerebellum and lowest in the striatum and white matter (Swanson et al., 1989a)]. Energy metabolism also varies significantly between different brain regions (Sokoloff et al., 1977). Therefore, glycogen could be more protective against glucose depletion in some areas than in others.Given all of the above, it is natural to wonder whether glycogen can enhance the survival and function of brain tissue in the absence of glucose. Surprisingly, only a single st...

The antiproliferative action of vinblastine at low concentrations appears to result from modulation of the polymerization dynamics of spindle microtubules rather than from depolarization of the microtubules [Jordan, M. A., Thrower, D., & Wilson, L. (1991) Cancer Res. 51, 2212-2222; (1992) J. Cell. Sci. 102, 401-416]. In the present study, we used differential interference contrast video microscopy to analyze the effects of vinblastine on the growing and shortening dynamics (dynamic instability) of individual bovine brain microtubules in vitro. With microtubules which were either depleted of microtubule-associated proteins (MAPs) or rich in MAPs, low concentrations of vinblastine (0.2 microM-1 microM) suppressed the growing and shortening rates and increased the percentage of time that the microtubules spent a state of attenuated activity, neither growing nor shortening detectably. Vinblastine also suppressed the duration of microtubule growing and shortening, and increased the duration of the attenuated state, during which the microtubules neither grew nor shortened detectably. Consistent with previous data obtained using radiolabeled nucleotide exchange in microtubule suspensions [Jordan, M. A., & Wilson, L. (1990) Biochemistry 29, 2730-2739], vinblastine suppressed growing and shortening dynamics at the kinetically more rapid plus ends. The results suggest that vinblastine kinetically stabilizes microtubule ends by modulating the gain and loss of the stabilizing GTP or GDP-Pi "cap", which is believed to be responsible for the transitions between the growing and shortening phases. The data support the hypothesis that (1) low concentrations of vinblastine inhibit mitosis by kinetically stabilizing the polymerization dynamics of spindle microtubules and that (2) the dynamics of spindle microtubules are critical for the proper progression of mitosis.

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