Ligia A. Papale

Altered Function of the SCN1A Voltage-gated Sodium Channel Leads to γ-Aminobutyric Acid-ergic (GABAergic) Interneuron Abnormalities

Voltage-gated sodium channels are required for the initiation and propagation of action potentials. Mutations in the neuronal voltage-gated sodium channel SCN1A are associated with a growing number of disorders including generalized epilepsy with febrile seizures plus (GEFS+),7 severe myoclonic epilepsy of infancy, and familial hemiplegic migraine. To gain insight into the effect of SCN1A mutations on neuronal excitability, we introduced the human GEFS+ mutation SCN1A-R1648H into the orthologous mouse gene. Scn1aRH/RH mice homozygous for the R1648H mutation exhibit spontaneous generalized seizures and premature death between P16 and P26, whereas Scn1aRH/+ heterozygous mice exhibit infrequent spontaneous generalized seizures, reduced threshold and accelerated propagation of febrile seizures, and decreased threshold to flurothyl-induced seizures. Inhibitory cortical interneurons from P5-P15 Scn1aRH/+ and Scn1aRH/RH mice demonstrated slower recovery from inactivation, greater use-dependent inactivation, and reduced action potential firing compared with wild-type cells. Excitatory cortical pyramidal neurons were mostly unaffected. These results suggest that this SCN1A mutation predominantly impairs sodium channel activity in interneurons, leading to decreased inhibition. Decreased inhibition may be a common mechanism underlying clinically distinct SCN1A-derived disorders.

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability.

Heterozygous mutations of the voltage-gated sodium channel SCN8A are associated with spike-wave discharges and absence epilepsy in mice

In a chemical mutagenesis screen, we identified the novel Scn8a8J allele of the gene encoding the neuronal voltage-gated sodium channel Nav1.6. The missense mutation V929F in this allele alters an evolutionarily conserved residue in the pore loop of domain 2 of Nav1.6. Electroencephalography (EEG) revealed well-defined spike-wave discharges (SWD), the hallmark of absence epilepsy, in Scn8a8J heterozygotes and in heterozygotes for two classical Scn8a alleles, Scn8amed (null) and Scn8amed-jo (missense). Mouse strain background had a significant effect on SWD, with mutants on the C3HeB/FeJ strain showing a higher incidence than on C57BL/6J. The abnormal EEG patterns in heterozygous mutant mice and the influence of genetic background on SWD make SCN8A an attractive candidate gene for common human absence epilepsy, a genetically complex disorder.

Preferential inactivation of Scn1a in parvalbumin interneurons increases seizure susceptibility.

Voltage-gated sodium channels (VGSCs) are essential for the generation and propagation of action potentials in electrically excitable cells. Dominant mutations in SCN1A, which encodes the Nav1.1 VGSC α-subunit, underlie several forms of epilepsy, including Dravet syndrome (DS) and genetic epilepsy with febrile seizures plus (GEFS+). Electrophysiological analyses of DS and GEFS+ mouse models have led to the hypothesis that SCN1A mutations reduce the excitability of inhibitory cortical and hippocampal interneurons. To more directly examine the relative contribution of inhibitory interneurons and excitatory pyramidal cells to SCN1A-derived epilepsy, we first compared the expression of Nav1.1 in inhibitory parvalbumin (PV) interneurons and excitatory neurons from P22 mice using fluorescent immunohistochemistry. In the hippocampus and neocortex, 69% of Nav1.1 immunoreactive neurons were also positive for PV. In contrast, 13% and 5% of Nav1.1 positive cells in the hippocampus and neocortex, respectively, were found to co-localize with excitatory cells identified by CaMK2α immunoreactivity. Next, we reduced the expression of Scn1a in either a subset of interneurons (mainly PV interneurons) or excitatory cells by crossing mice heterozygous for a floxed Scn1a allele to either the Ppp1r2-Cre or EMX1-Cre transgenic lines, respectively. The inactivation of one Scn1a allele in interneurons of the neocortex and hippocampus was sufficient to reduce thresholds to flurothyl- and hyperthermia-induced seizures, whereas thresholds were unaltered following inactivation in excitatory cells. Reduced interneuron Scn1a expression also resulted in the generation of spontaneous seizures. These findings provide direct evidence for an important role of PV interneurons in the pathogenesis of Scn1a-derived epilepsies.

Sleep pattern in rats under different stress modalities.

The present study was designed to evaluate the sleep pattern of rats submitted to chronic stressors (restraint, electrical footshock, swimming and cold) applied to male rats. After 48 h-baseline recording, rats were submitted to 4 days of chronic stress, and electrocorticogram recordings were carried out continuously. The stressors (footshock, swimming and cold) were applied twice a day for periods of 1 h at 9:00 and 16:00 h. Restrained animals were maintained in plastic cylinders for 22 h/day. The findings indicated that sleep efficiency, slow wave sleep (SWS) and paradoxical sleep (PS) were decreased on the third and fourth days of unpredictable shocks compared to baseline while immobilization and swimming presented reduced sleep efficiency in all 4-day recordings. Swimming led to decreased SWS, whereas augmented PS was observed on the first day compared to baseline. Immobilization produced drastic alterations in sleep patterns since it reduced SWS during the 4 days and PS at days 1 to 4 in relation to baseline. Of all stressors, cold was the only one that did not result in any statistical differences in sleep pattern during the light periods. Regarding the effect of stress compared to baseline on the dark recordings, PS was higher during cold stress periods, whereas footshock increased PS on days 2 to 4 and swimming only on day 2. Immobilization decreased PS throughout the 4 days of the stress sessions. Thus, the data suggest that different stress modalities result in distinct sleep responses, with immobilization producing the most dramatic alterations.

Dysfunction of the Scn8a Voltage-gated Sodium Channel Alters Sleep Architecture, Reduces Diurnal Corticosterone Levels, and Enhances Spatial Memory

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of transient depolarizing currents and play a critical role in the electrical signaling between neurons. A null mutation in the VGSC gene SCN8A, which encodes the transmembrane protein Nav1.6, was identified previously in a human family. Heterozygous mutation carriers displayed a range of phenotypes, including ataxia, cognitive deficits, and emotional instability. A possible role for SCN8A was also proposed in studies examining the genetic basis of attempted suicide and bipolar disorder. In addition, mice with a Scn8a loss-of-function mutation (Scn8amed-Tg/+) show altered anxiety and depression-like phenotypes. Because psychiatric abnormalities are often associated with altered sleep and hormonal patterns, we evaluated heterozygous Scn8amed-jo/+ mutants for alterations in sleep-wake architecture, diurnal corticosterone levels, and behavior. Compared with their wild-type littermates, Scn8amed-jo/+ mutants experience more non-rapid eye movement (non-REM) sleep, a chronic impairment of REM sleep generation and quantity, and a lowered and flattened diurnal rhythm of corticosterone levels. No robust differences were observed between mutants and wild-type littermates in locomotor activity or in behavioral paradigms that evaluate anxiety or depression-like phenotypes; however, Scn8amed-jo/+ mutants did show enhanced spatial memory. This study extends the spectrum of phenotypes associated with mutations in Scn8a and suggests a novel role for altered sodium channel function in human sleep disorders.

Altered Sleep Regulation in a Mouse Model of SCN1A-Derived Genetic Epilepsy with Febrile Seizures Plus (GEFS+)

Mutations in the voltage‐gated sodium channel (VGSC) gene SCN1A are responsible for a number of epilepsy disorders, including genetic epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. In addition to seizures, patients with SCN1A mutations often experience sleep abnormalities, suggesting that SCN1A may also play a role in the neuronal pathways involved in the regulation of sleep. However, to date, a role for SCN1A in the regulation of sleep architecture has not been directly examined. To fill this gap, we tested the hypothesis that SCN1A contributes to the regulation of sleep architecture, and by extension, that SCN1A dysfunction contributes to the sleep abnormalities observed in patients with SCN1A mutations.

Influence of long-term food restriction on sleep pattern in male rats

The present purpose was to determine the effects of different schedules of long-term food restriction (FR) applied to rats from weaning to the 8th week. Rats were distributed into FR and ad libitum groups at weaning and fed at 7 am, at 7 pm, and finally, restricted rats fed ad libitum. The restricted rats started with 6 g/day and the food was increased by 1 g per week until reaching 15 g/day by adulthood. The rats were implanted with electrodes to record electrocorticogram/eletromyogram signals. Their wake-sleep cycles were monitored over 3 consecutive days (72 h of recording). The FR group fed at 7 am showed an increase in awake time, and decrease in slow wave sleep (SWS) and paradoxical sleep (PS) during the three light periods compared with the control recordings whereas in the dark periods, these sleep parameters were the opposite. The restricted group fed in the evening showed no statistical significances at diurnal periods; however, a significant decrease was observed in the dark recordings for awake time, but the SWS and PS were increased in relation to controls. The analysis of the 24-h period demonstrated that both FR groups presented increase in SWS time. After being FR, the rats were fed ad libitum and their sleep was monitored for 3 additional days. During the first dark recording, the decrease in awake time and increase in SWS were still present; however, as ad libitum food continued, these sleep parameters returned to control values, reestablishing the normal sleep pattern. These results suggest that dietary restriction, regardless to the feeding schedule, caused increase in total sleep time, during the active period.

Glyoxalase 1 and its substrate methylglyoxal are novel regulators of seizure susceptibility.

Epilepsy is a complex disease characterized by a predisposition toward seizures. There are numerous barriers to the successful treatment of epilepsy. For instance, current antiepileptic drugs have adverse side effects and variable efficacies. Furthermore, the pathophysiologic basis of epilepsy remains largely elusive. Therefore, investigating novel genes and biologic processes underlying epilepsy may provide valuable insight and enable the development of new therapeutic agents. We previously identified methylglyoxal (MG) as an endogenous γ‐aminobutyric acid (GABAA) receptor agonist. Here, we investigated the role of MG and its catabolic enzyme, glyoxalase 1 (GLO1), in seizures.

Age-related effects on genital reflexes induced by paradoxical sleep deprivation and cocaine in rats

Although sexual function often decreases with age, a recent study demonstrated that paradoxical sleep deprivation (PSD) was effective in inducing penile erection (PE) in 60% of 22-month-old PSD rats after acute cocaine injection, whereas this behavior was absent in non-PSD control rats. The present study sought to compare alterations in genital reflexes (PE and ejaculation-EJ) induced by PSD followed by acute cocaine or saline at different points in the life span of male rats. Rats of different ages (3, 6, 10, 12, 18, 22 and 28 months), were given a single injection of either saline or cocaine (7 mg/kg, i.p.) after a 4-day period of PSD, and then evaluated for genital reflexes. Results indicated that genital reflexes in PSD rats given saline become less frequent with age and disappear after the age of 18 months. However, cocaine potentiated and prolonged these behaviors until the age of 22 months. Although a number of factors are involved in such a complex phenomenon as PE, we suggest that the previously documented dopamine receptor supersensitivity induced by PSD may be an important contributor to the potentiation by cocaine of genital reflexes after sleep deprivation.