Prevalence of Sleep Disturbances in Children With Neurofibromatosis Type 1.

Children with neurodevelopmental disorders are at increased risk for sleep issues, which affect quality of life, cognitive function, and behavior. To determine the prevalence of sleep problems in children with the common neurodevelopmental disorder neurofibromatosis type 1, a cross-sectional study was performed on 129 affected subjects and 89 unaffected siblings, age 2 to 17 years, using the Sleep Disturbance Scale for Children questionnaire. Children with neurofibromatosis type 1 were significantly more likely to have disturbances in initiating and maintaining sleep, arousal, sleep-wake transition, and hyperhidrosis, but not problems with abnormal sleep breathing, or excessive somnolence. Although the overall sleep scores were higher in children with neurofibromatosis type 1, this was not related to a coexisting attention deficit disorder, cognitive impairment, or stimulant medication use. Collectively, these results demonstrate that children with neurofibromatosis type 1 are more likely to have sleep disturbances, and support the use of appropriate interventions for this at-risk population.

Sirt1 extends life span and delays aging in mice through the regulation of Nk2 homeobox 1 in the DMH and LH.

The mammalian Sir2 ortholog Sirt1 plays an important role in metabolic regulation. However, the role of Sirt1 in the regulation of aging and longevity is still controversial. Here we demonstrate that brain-specific Sirt1-overexpressing (BRASTO) transgenic mice show significant life span extension in both males and females, and aged BRASTO mice exhibit phenotypes consistent with a delay in aging. These phenotypes are mediated by enhanced neural activity specifically in the dorsomedial and lateral hypothalamic nuclei (DMH and LH, respectively), through increased orexin type 2 receptor (Ox2r) expression. We identified Nk2 homeobox 1 (Nkx2-1) as a partner of Sirt1 that upregulates Ox2r transcription and colocalizes with Sirt1 in the DMH and LH. DMH/LH-specific knockdown of Sirt1, Nkx2-1, or Ox2r and DMH-specific Sirt1 overexpression further support the role of Sirt1/Nkx2-1/Ox2r-mediated signaling for longevity-associated phenotypes. Our findings indicate the importance of DMH/LH-predominant Sirt1 activity in the regulation of aging and longevity in mammals.

The ketogenic diet inhibits the mammalian target of rapamycin (mTOR) pathway.

The ketogenic diet (KD) is an effective treatment for epilepsy, but its mechanisms of action are poorly understood. We investigated the hypothesis that the KD inhibits mammalian target of rapamycin (mTOR) pathway signaling. The expression of pS6 and pAkt, markers of mTOR pathway activation, was reduced in hippocampus and liver of rats fed KD. In the kainate model of epilepsy, KD blocked the hippocampal pS6 elevation that occurs after status epilepticus. Because mTOR signaling has been implicated in epileptogenesis, these results suggest that the KD may have anticonvulsant or antiepileptogenic actions via mTOR pathway inhibition.

A ketogenic diet does not impair rat behavior or long-term potentiation.

The effect of the ketogenic diet on behavior and cognition is unclear. We addressed this issue in rats behaviorally and electrophysiologically.We fed postnatal day 21 rats a standard diet (SD), ketogenic diet (KD), or calorie-restricted diet (CR) for 2­3 weeks. CR controlled for the slower weight gain experienced by KD-fed rats. We assessed behavioral performance with a locomotor activity and a conditioned fear test. To evaluate possible parallel effects of diet on synaptic function, we examined paired-pulse modulation (PPM) and long-term potentiation (LTP) in the medial perforant path in vivo. KD-fed rats performed similarly to SD-fed rats on the behavioral tests and electrophysiologic assays. These data suggest that the KD does not alter behavioral performance or synaptic plasticity.

A physiological role for amyloid-beta protein:enhancement of learning and memory.

Amyloid-beta protein (Abeta) is well recognized as having a significant role in the pathogenesis of Alzheimer's disease (AD). The reason for the presence of Abeta and its physiological role in non-disease states is not clear. In these studies, low doses of Abeta enhanced memory retention in two memory tasks and enhanced acetylecholine production in the hippocampus in vivo. We then tested whether endogenous Abeta has a role in learning and memory in young, cognitively intact mice by blocking endogenous Abeta in healthy 2-month-old CD-1 mice. Blocking Abeta with antibody to Abeta or DFFVG (which blocks Abeta binding) or decreasing Abeta expression with antisense directed at the Abeta precursor, AbetaPP, all resulted in impaired learning in T-maze foot-shock avoidance. Finally, Abeta 1-42 facilitated induction and maintenance of long term potentiation in hippocampal slices, whereas antibodies to Abeta inhibited hippocampal LTP. In conclusion, these results indicate that in normal healthy young animals the presence of Abeta is important for learning and memory.

FGF14 regulates the intrinsic excitability of cerebellar Purkinje neurons.

A missense mutation in the fibroblast growth factor 14 (FGF14) gene underlies SCA27, an autosomal dominant spinocerebellar ataxia in humans. Mice with a targeted disruption of the Fgf14 locus (Fgf14(-/-)) develop ataxia resembling human SCA27. We tested the hypothesis that loss of FGF14 affects the firing properties of Purkinje neurons, which play an important role in motor control and coordination. Current clamp recordings from Purkinje neurons in cerebellar slices revealed attenuated spontaneous firing in Fgf14(-/-) neurons. Unlike in the wild type animals, more than 80% of Fgf14(-/-) Purkinje neurons were quiescent and failed to fire repetitively in response to depolarizing current injections. Immunohistochemical examination revealed reduced expression of Nav1.6 protein in Fgf14(-/-) Purkinje neurons. Together, these observations suggest that FGF14 is required for normal Nav1.6 expression in Purkinje neurons, and that the loss of FGF14 impairs spontaneous and repetitive firing in Purkinje neurons by altering the expression of Nav1.6 channels.

Calorie restriction and glucose regulation.

Ketogenic diets (KDs) are effective treatments for epilepsy. The mechanisms of action are poorly understood. In some experimental seizure models, calorie restriction and hypoglycemia may augment the antiseizure effects of KDs. In addition, inhibiting glycolysis or diverting glucose from the glycolytic pathway inhibits seizures and possibly epileptogenesis, suggesting an interaction between energy regulation and the anticonvulsant actions of these interventions. Children on KDs frequently exhibit poor weight gain and have lower blood glucose levels compared to children on standard, balanced diets. Young rodents on a KD also exhibit slow weight gain, lower blood glucose and insulin levels, and elevated leptin levels. This review considers the possibility that calorie restriction, low serum glucose, and KDs share common cell signaling pathways to alter brain excitability. AMP-activated protein kinase (AMPK) is an attractive candidate signaling protein that could link energy balance to gene expression in such a way so as to reduce brain excitability.

Increased severity of pentylenetetrazol induced seizures in leptin deficient ob/ob mice.

Leptin modulates multiple ion channels making its net effect on brain excitability difficult to predict. One method of determining leptin's net effect on brain excitability is to examine brain excitability during chronic leptin deficiency. We compared the susceptibility of leptin deficient ob/ob and wild type mice to pentylenetetrazol (PTZ) induced seizures using continuous video electroencephalogram (EEG) recordings. We found that ob/ob mice were more likely to die and were more susceptible to generalized clonic and clonic-tonic seizures than wild type mice at submaximal PTZ doses. These findings suggest that chronic leptin deficiency in vivo increases seizure susceptibility.

Obesity and hypertriglyceridemia produce cognitive impairment.

Obesity is associated with cognitive impairments. Long-term mechanisms for this association include consequences of hyperglycemia, dyslipidemia, or other factors comprising metabolic syndrome X. We found that hypertriglyceridemia, the main dyslipidemia of metabolic syndrome X, is in part responsible for the leptin resistance seen in obesity. Here we determined whether triglycerides have an immediate and direct effect on cognition. Obese mice showed impaired acquisition in three different cognitive paradigms: the active avoidance T-maze, the Morris water maze, and a food reward lever press. These impairments were not attributable to differences in foot shock sensitivity, swim speed, swimming distance, or voluntary milk consumption. Impaired cognition in obese mice was improved by selectively lowering triglycerides with gemfibrozil. Injection into the brain of the triglyceride triolein, but not of the free fatty acid palmitate, impaired acquisition in normal body weight mice. Triolein or milk (97% of fats are triglycerides), but not skim milk (no triglycerides), impaired maintenance of the N-methyl-d-aspartate component of the hippocampal long-term synaptic potential. Measures of oxidative stress in whole brain were reduced by gemfibrozil. We conclude that triglycerides mediate cognitive impairment as seen in obesity, possibly by impairing maintenance of the N-methyl-d-aspartate component of hippocampal long-term potentiation, and that lowering triglycerides can reverse the cognitive impairment and improve oxidative stress in the brain.

Leptin inhibits 4-aminopyridine- and pentylenetetrazole-induced seizures and AMPAR-mediated synaptic transmission in rodents.

Leptin is a hormone that reduces excitability in some hypothalamic neurons via leptin receptor activation of the JAK2 and PI3K intracellular signaling pathways. We hypothesized that leptin receptor activation in other neuronal subtypes would have anticonvulsant activity and that intranasal leptin delivery would be an effective route of administration. We tested leptin's anticonvulsant action in 2 rodent seizure models by directly injecting it into the cortex or by administering it intranasally. Focal seizures in rats were induced by neocortical injections of 4-aminopyridine, an inhibitor of voltage-gated K+ channels. These seizures were briefer and less frequent upon coinjection of 4-aminopyridine and leptin. In mice, intranasal administration of leptin produced elevated brain and serum leptin levels and delayed the onset of chemical convulsant pentylenetetrazole-induced generalized convulsive seizures. Leptin also reduced neuronal spiking in an in vitro seizure model. Leptin inhibited alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor-mediated synaptic transmission in mouse hippocampal slices but failed to inhibit synaptic responses in slices from leptin receptor-deficient db/db mice. JAK2 and PI3K antagonists prevented leptin inhibition of AMPAergic synaptic transmission. We conclude that leptin receptor activation and JAK2/PI3K signaling may be novel targets for anticonvulsant treatments. Intranasal leptin administration may have potential as an acute abortive treatment for convulsive seizures in emergency situations.

Abnormal glutamate homeostasis and impaired synaptic plasticity and learning in a mouse model of tuberous sclerosis complex.

Mice with inactivation of the Tuberous sclerosis complex-1 (Tsc1) gene in glia (Tsc1 GFAP CKO mice) have deficient astrocyte glutamate transporters and develop seizures, suggesting that abnormal glutamate homeostasis contributes to neurological abnormalities in these mice. We examined the hypothesis that Tsc1 GFAP CKO mice have elevated extracellular brain glutamate levels that may cause neuronal death, abnormal glutamatergic synaptic function, and associated impairments in behavioral learning. In vivo microdialysis documented elevated glutamate levels in hippocampi of Tsc1 GFAP CKO mice and several cell death assays demonstrated neuronal death in hippocampus and neocortex. Impairment of long-term potentiation (LTP) with tetanic stimulation was observed in hippocampal slices from Tsc1 GFAP CKO mice and was reversed by low concentrations of NMDA antagonist, indicating that excessive synaptic glutamate directly inhibited LTP. Finally, Tsc1 GFAP CKO mice exhibited deficits in two hippocampal-dependent learning paradigms. These results suggest that abnormal glutamate homeostasis predisposes to excitotoxic cell death, impaired synaptic plasticity and learning deficits in Tsc1 GFAP CKO mice.

Impaired spatial learning and defective theta burst induced LTP in mice lacking fibroblast growth factor 14.

Spinocerebellar ataxia 27 (SCA27) is a recently described syndrome characterized by impaired cognitive abilities and a slowly progressive ataxia. SCA27 is caused by an autosomal dominant missense mutation in Fibroblast Growth Factor 14 (FGF14). Mice lacking FGF14 (Fgf14(-/-) mice) have impaired sensorimotor functions, ataxia and paroxysmal dyskinesia, a phenotype that led to the discovery of the human mutation. Here we extend the similarities between Fgf14(-/-) mice and FGF14(F145S) humans by showing that Fgf14(-/-) mice exhibit reliable acquisition (place learning) deficits in the Morris water maze. This cognitive deficit appears to be independent of sensorimotor disturbances and relatively selective since Fgf14(-/-) mice performed similarly to wild type littermates during cued water maze trials and on conditioned fear and passive avoidance tests. Impaired theta burst initiated long-term synaptic potentiation was also found in hippocampal slices from Fgf14(-/-) mice. These results suggest a role for FGF14 in certain spatial learning functions and synaptic plasticity.

Leptin contributes to slower weight gain in juvenile rodents on a ketogenic diet.

The ketogenic diet (KD) is an efficacious therapy for medically refractory childhood epilepsy that also slows weight gain. We tested the hypothesis that the KD slows weight gain via neurohormones involved in energy homeostasis. We found that juvenile rodents fed a KD had slower weight gain than those fed a standard diet (SD). Rats fed a KD had higher serum leptin levels and lower insulin levels compared with those fed an SD. We investigated the increase in leptin further because this change was the only one consistent with slower weight gain. Although rats fed the SD experienced slower weight gain when calorie restricted, they had serum leptin levels similar to those fed the SD ad libitum. Furthermore, leptin deficient (ob/ob) and leptin receptor deficient (db/db) mice did not show slower weight gain on the KD. All animals on the KD had elevated serum beta-hydroxybutyrate (betaHB) levels. Thus, ketosis is insufficient and a functioning leptin signaling system appears necessary for the KD to slow weight gain. The increase in leptin may contribute to the anticonvulsant effects of the KD.

Ghrelin controls hippocampal spine synapse density and memory performance.

The gut hormone and neuropeptide ghrelin affects energy balance and growth hormone release through hypothalamic action that involves synaptic plasticity in the melanocortin system. Ghrelin binding is also present in other brain areas, including the telencephalon, where its function remains elusive. Here we report that circulating ghrelin enters the hippocampus and binds to neurons of the hippocampal formation, where it promotes dendritic spine synapse formation and generation of long-term potentiation. These ghrelin-induced synaptic changes are paralleled by enhanced spatial learning and memory. Targeted disruption of the gene that encodes ghrelin resulted in decreased numbers of spine synapses in the CA1 region and impaired performance of mice in behavioral memory testing, both of which were rapidly reversed by ghrelin administration. Our observations reveal an endogenous function of ghrelin that links metabolic control with higher brain functions and suggest novel therapeutic strategies to enhance learning and memory processes.

Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo.

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is central to Alzheimer's disease pathogenesis. Abeta aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Abeta levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Abeta levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Abeta metabolism and ultimately region-specific Abeta deposition. The findings also have important implications for treatment development.

In vivo imaging of dendritic spines during electrographic seizures.

Epilepsy is associated with significant neurological morbidity, including learning disabilities, motor deficits, and behavioral problems. Although the causes of neurological dysfunction in epilepsy are multifactorial, accumulating evidence indicates that seizures in themselves may directly cause brain injury. Although it is clear that seizures can result in neuronal death, it is likely that under some circumstances seizures can induce more subtle functional or structural alterations in neurons. We induced focal neocortical seizures with 4-aminopyridine in transgenic mice expressing green fluorescent protein in cortical neurons and sequentially imaged individual dendrites in living animals with two-photon laser-scanning microscopy to determine whether these seizures caused acute alterations in dendritic spine morphology. No dendritic alterations were observed in anesthetized animals during electrographic seizures over a 3-hour period. Similarly, in unanesthetized mice, low-stage, clinical electrographic seizures had minimal effect on dendritic spines. More severe, high-stage seizures in unanesthetized mice were associated with a moderate loss of spines and dendritic swelling, but this effect may have been contingent on a synergistic action of phototoxicity from the imaging method itself. Overall, our results suggest that most neocortical seizures have minimal acute effects on dendrites over several hours, but may predispose to dendritic injury under extreme conditions.

Ketogenic diet reduces hypoglycemia-induced neuronal death in young rats.

Hypoglycemia is an important complication of insulin treatment in diabetic children and may contribute to lasting cognitive impairment. Previous studies demonstrated that 21-day-old rats (P21) subjected to brief, repetitive episodes of hypoglycemia sustain cortical neuronal death. The developing brain is capable of utilizing alternative energy substrates acetoacetate and beta-hydroxybutyrate. In these studies we tested the hypothesis that the developing brain adapted to ketone utilization and provided with ketones during hypoglycemia by eating a ketogenic diet would sustain less brain injury compared to littermates fed a standard diet. Supporting this hypothesis, P21 rats weaned to a ketogenic diet and subjected to insulin-induced hypoglycemia at P25 had significantly less neuronal death than rats on a standard diet. This animal model may provide insight into the determinants influencing the brain's susceptibility to hypoglycemic injury.

Differential presynaptic modulation of excitatory and inhibitory autaptic currents in cultured hippocampal neurons.

Short-term synaptic plasticity has an important role in higher cortical function. Hyperpolarization may effect a form of short-term plasticity by promoting recovery from sodium channel inactivation or by activating axonal A-type potassium channels. To determine whether one or both processes occur, we examined the effect of hyperpolarizing prepulses on autaptic currents in cultured postnatal rat hippocampal neurons. As expected of enhanced recovery from sodium channel inactivation, hyperpolarizing prepulses reversibly increased fast excitatory autaptic currents (eacs) mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), slow eacs mediated by N-methyl-D-aspartate receptors (NMDARs), and inhibitory autaptic currents (iacs) mediated by gamma-aminobutyric acidA receptors (GABAARs). Hyperpolarizing prepulses augmented nearly all fast and slow eacs but only half of the iacs. This change occurred without a change in autaptic current kinetics. Of note, hyperpolarizing prepulses did not significantly reduce autaptic currents in any neuron studied. The rapidly dissociating competitive antagonists kynurenate and L-2-amino-5-phosphonovaleric acid (LAPV) inhibited fast and slow eacs, respectively, to the same extent with and without a hyperpolarizing prepulse. In addition, hyperpolarizing prepulses revealed a slow eac even after the slow eac evoked without a prepulse was completely blocked by the open channel blocker, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801). Finally, hyperpolarizing prepulses did not alter currents evoked by exogenous applications of glutamate and GABA. These findings suggest that hyperpolarizing prepulses preferentially enhance eacs over iacs, and that they do so, in part, by overcoming conduction block or by activating silent synapses.

Effects of seven clinically important antiepileptic drugs on inhibitory glycine receptor currents in hippocampal neurons.

Although potentiation of the inhibitory glycine receptor (GlyR) may contribute to the mechanism of action of antiepileptic drugs (AEDs), the effects of AEDs on GlyRs have not been investigated in detail in forebrain neurons. We examined the effects of seven clinically important AEDs on GlyR-mediated currents using whole-cell patch clamp recordings from cultured embryonic mouse hippocampal neurons. At high therapeutic concentrations, topiramate (in 24% of neurons) and pentobarbital reversibly decreased glycine currents to 89+/-6 % and 81+/-7 % of control, respectively. At or below therapeutic concentrations, carbamazepine, felbamate, gabapentin, phenytoin, and valproate had no effect on glycine currents, while at supratherapeutic concentrations these agents produced modest reversible inhibition. We conclude that GlyR potentiation does not contribute to the antiepileptic action of the seven AEDs examined.

Repetitive hypoglycemia in young rats impairs hippocampal long-term potentiation.

Mechanisms underlying cognitive dysfunction in young diabetic children are poorly understood, and may include synaptic dysfunction from insulin-induced hypoglycemia. We developed a model of repetitive insulin-induced hypoglycemia in young rats and examined hippocampal long-term potentiation, an electrophysiologic assay of synaptic plasticity, 3-5 d after the last hypoglycemic event. Three hypoglycemic events between postnatal d 21-25 produced modest cortical (17 +/- 2.9 dead neurons per section in parasagittal cortex), but not hippocampal, neuron death quantified by Fluoro-Jade B staining. There was no change in neurogenesis in the hippocampal dentate granule cell region by quantification of bromodeoxyuridine incorporation. Although normal baseline hippocampal synaptic responses were elicited from hippocampal slices from hypoglycemic animals, long-term synaptic potentiation could not be induced in hippocampal slices from rats subjected to hypoglycemia. These results suggest that repetitive hypoglycemia in the developing brain can cause selective impairment of synaptic plasticity in the absence of cell death, and without complete disruption of basal synaptic transmission. We speculate that impaired synaptic plasticity in the hippocampus caused by repetitive hypoglycemia could underlie memory and cognitive deficits observed in young diabetic children, and that cortical neuron death caused by repetitive hypoglycemia in the developing brain may contribute to other neurologic, cognitive, and psychological problems sometimes encountered in diabetic children.