NKCC1 transporter facilitates seizures in the developing brain

During development, activation of Cl(-)-permeable GABA(A) receptors (GABA(A)-R) excites neurons as a result of elevated intracellular Cl(-) levels and a depolarized Cl(-) equilibrium potential (E(Cl)). GABA becomes inhibitory as net outward neuronal transport of Cl(-) develops in a caudal-rostral progression. In line with this caudal-rostral developmental pattern, GABAergic anticonvulsant compounds inhibit motor manifestations of neonatal seizures but not cortical seizure activity. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) facilitates the accumulation of Cl(-) in neurons. The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo. Bumetanide had no effect in the presence of the GABA(A)-R antagonist bicuculline, nor in brain slices from NKCC1-knockout mice. NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat. Our results provide evidence that NKCC1 facilitates seizures in the developing brain and indicate that bumetanide should be useful in the treatment of neonatal seizures.     

Bidirectional modification of presynaptic neuronal excitability accompanying spike timing-dependent synaptic plasticity

Correlated pre- and postsynaptic activity that induces long-term potentiation is known to induce a persistent enhancement of the intrinsic excitability of the presynaptic neuron. Here we report that, associated with the induction of long-term depression in hippocampal cultures and in somatosensory cortical slices, there is also a persistent reduction in the excitability of the presynaptic neuron. This reduction requires postsynaptic Ca(2+) elevation and presynaptic PKA- and PKC-dependent modification of slow-inactivating K(+) channels. The bidirectional changes in neuronal excitability and synaptic efficacy exhibit identical requirements for the temporal order of pre- and postsynaptic activation but reflect two distinct aspects of activity-induced modification of neural circuits.     

Contrasting effects of the persistent Na+ current on neuronal excitability and spike timing

The persistent Na+ current, INaP, is known to amplify subthreshold oscillations and synaptic potentials, but its impact on action potential generation remains enigmatic. Using computational modeling, whole-cell recording, and dynamic clamp of CA1 hippocampal pyramidal cells in brain slices, we examined how INaP changes the transduction of excitatory current into action potentials. Model simulations predicted that INaP increases afterhyperpolarizations, and, although it increases excitability by reducing rheobase, INaP also reduces the gain in discharge frequency in response to depolarizing current (f/I gain). These predictions were experimentally confirmed by using dynamic clamp, thus circumventing the longstanding problem that INaP cannot be selectively blocked. Furthermore, we found that INaP increased firing regularity in response to sustained depolarization, although it decreased spike time precision in response to single evoked EPSPs. Finally, model simulations demonstrated that I(NaP) increased the relative refractory period and decreased interspike-interval variability under conditions resembling an active network in vivo.     

Febrile seizures: traffic slows in the heat

Febrile seizures, which occur in young children, have long been known to have a major inherited component. Mutations in some genes that encode sodium channel and GABA(A) receptor subunits have been found in a few families affected by febrile seizures. These mutations account only for a minority of cases, and much remains to be learnt about the molecular architecture of febrile seizures. A rare inherited cause--a mutation in the GABA(A) receptor subunit GABRG2 gene--has been recently shown to cause a temperature-dependent intracellular trafficking defect. This is an important step in unravelling the molecular pathogenesis of this common childhood disorder.     

Transplanted neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronal precursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursors in adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricle of the mouse brain in different development stages. The preliminary results indicated that the grafted cells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the third ventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amount of survival cells in different brain regions was correlated with the development stage of the recipient brain. Immunohistochemical studies showed that most of the grafted cells migrating into the specific target could express neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZ still retained immortality and ability of proliferation, which is likely to be induced by some environmental factors.     

Transplanted neuronal precursors migrate and differentiate in the developing mouse brain

The subventricular zone (SVZ), lining the lateral ventricle in forebrain, retains a population of neuronalprecursors with the ability of proliferation in adult mammals. To test the potential of neuronal precursorsin adult mice, we transplanted adult SVZ cells labeled with fluorescent dye PKH26 into the lateral ventricleof the mouse brain in different development stages. The preliminary results indicated that the graftedcells were able to survive and migrate into multiple regions of the recipient brain, including SVZ, the thirdventricle, thalamus, superior colliculus, inferior colliculus, cerebellum and olfactory bulb etc; and the amountof survival cells in different brain regions was correlated with the development stage of the recipient brain.Immunohistochemical studies showed that most of the grafted cells migrating into the specific target couldexpress neuronal or astrocytic marker. Our results revealed that the neuronal precursors in adult SVZstill retained immortality and ability of proliferation, which is likely to be induced by some environmentalfactors.     

Ethanol-induced neuronal apoptosis in vivo requires BAX in the developing mouse brain

A single episode of ethanol intoxication triggers widespread apoptotic neurodegeneration in the infant rat or mouse brain. The cell death process occurs over a 6-16 h period following ethanol administration, is accompanied by a robust display of caspase-3 enzyme activation, and meets ultrastructural criteria for apoptosis. Two apoptotic pathways (intrinsic and extrinsic) have been described, either of which may culminate in the activation of caspase-3. The intrinsic pathway is regulated by Bax and Bcl-XL and involves Bax-induced mitochondrial dysfunction and release of cytochrome c as antecedent events leading to caspase-3 activation. Activation of caspase-8 is a key event preceding caspase-3 activation in the extrinsic pathway. In the present study, following ethanol administration to infant mice, we found no change in activated caspase-8, which suggests that the extrinsic pathway is not involved in ethanol-induced apoptosis. We also found that ethanol triggers robust caspase-3 activation and apoptotic neurodegeneration in C57BL/6 wildtype mice, but induces neither phenomenon in homozygous Bax-deficient mice. Therefore, it appears that ethanol-induced neuroapoptosis is an intrinsic pathway-mediated phenomenon involving Bax-induced disruption of mitochondrial membranes and cytochrome c release as early events leading to caspase-3 activation.     

The infraslow potential oscillations in developing chick embryo brain. Correlations with neuronal activity.

The correlations between infraslow potential oscillations [ISPO] and EEG activity were studied in the brain hemispheres and optic lobes of chick embryos from the 15th to 21st day of incubation. 1. The ISPO of the optic lobes remained unchanged during and after peripheral optic stimulation -- not only in 17-day-old embryos (the prefunctional stage), but also in 19- and 21-day-old embryos, in which optic evoked activity in the optic tectum is already well developed. 2. The intracerebral administration of strychnine, GABA and sodium glutamate had no effect on the ISPO of the brain hemispheres in 15-day-old embryos. 3. The effect of these neurotropic drugs in 20-day-old embryos varied. Strychnine evoked concomitant activation of ISPO and the EEG, sodium glutamate simultaneously depressed both activities, while GABA inhibited EEG activity without affecting ISPO patterns. 4. These results supported our conclusion that neuronal activity plays a secondary role in the ISPO generation process in developing brain tissue.     

O-GlcNAc modification of radial glial vimentin filaments in the developing chick brain

We examined the post-translational modification of intracellular proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) with regard to neurofilament phosphorylation in the developing chick optic tectum. A regulated developmental pattern of O-GlcNAcylation was discovered in the developing brain. Most notably, discernible staining occurs along radial glial filaments but not along neuronal filaments in vivo. Immunohistochemical analyses in sections of progressive stages of development suggest upregulation of O-GlcNAc in the ependyma, tectofugal neuron bodies, and radial glial processes, but not in axons. In contrast, double-label immunostaining of monolayer cultures made from dissociated embryonic day (E) 7 optic tecta revealed O-GlcNAcylation of most axons. Labeling of brain sections together with Western blot analyses showed O-GlcNAc modification of a few discrete proteins throughout development, and suggested vimentin as the protein in radial glia. Immunoprecipitation of vimentin from E9 whole brain lysates confirmed O-GlcNAcylation of vimentin in development. These results indicate a regulated pattern of O-GlcNAc modification of vimentin filaments, which in turn suggests a role for O-GlcNAc-modified intermediate filaments in radial glia, but not in neurons during brain development. The control mechanisms that regulate this pattern in vivo, however, are disrupted when cells are placed in vitro.     

Spatiotemporal specificity of neuronal activity directs the modification of receptive fields in the developing retinotectal system

The precise temporal relation between pre- and postsynaptic activity modulates the strength of synaptic connections. Despite its extensive characterization in vivo and in vitro, the degree to which spike timing-dependent plasticity can shape receptive field properties is unclear. We use in vivo patch-clamp recordings of tectal neurons in developing Xenopus tadpoles to control the precise timing of action potentials with respect to the arrival of a subset of visual inputs evoked by local light stimulation on the retina. The pattern of visual inputs onto a tectal neuron was tracked over time by rapid reverse correlation mapping of receptive fields. Spike timing-dependent potentiation or depression of a subset of synapses reliably shifts the spatial receptive fields toward or away from the trained subregion of visual space, respectively. These results demonstrate that natural patterns of activity evoked by sensory stimuli play an instructive role in the developing nervous system.     

Gonadal steroid-dependent neuronal circuitries in avian limbic and preoptic regions.

In the avian limbic and preoptic region, the sexually dimorphic medial preoptic nucleus and nucleus of the stria terminalis are characterized by the presence of a testosterone-dependent aromatase-immunoreactive neuronal population. In situ hybridization studies confirmed that testosterone is modulating the expression of aromatase gene. Both nuclei are also characterized by a sexually dimorphic, testosterone-dependent vasotocin system. Immunocytochemical and in situ hybridization data, demonstrated that dimorphism and testosterone-sensitivity of this system are both dependent by an embryonic organizational effect of the estradiol. Intracerebroventricularly injected vasotocin, has a profound inhibitory effect on the male sexual behaviour, and immunocytochemical investigations revealed close associations among vasotocin fibres and aromatase cell bodies. These data suggest that this neuropeptidergic system could have a key role in the circuitries controlling different aspects of male reproductive behaviour in the Japanese quail.     

The influence of sodium and potassium dynamics on excitability,seizures, and the stability of persistent states: I. Single neuron dynamics

In these companion papers, we study how the interrelated dynamics of sodium and potassium affect the excitability of neurons, the occurrence of seizures, and the stability of persistent states of activity. In this first paper, we construct a mathematical model consisting of a single conductance-based neuron together with intra- and extracellular ion concentration dynamics. We formulate a reduction of this model that permits a detailed bifurcation analysis, and show that the reduced model is a reasonable approximation of the full model. We find that competition between intrinsic neuronal currents, sodium-potassium pumps, glia, and diffusion can produce very slow and large-amplitude oscillations in ion concentrations similar to what is seen physiologically in seizures. Using the reduced model, we identify the dynamical mechanisms that give rise to these phenomena. These models reveal several experimentally testable predictions. Our work emphasizes the critical role of ion concentration homeostasis in the proper functioning of neurons, and points to important fundamental processes that may underlie pathological states such as epilepsy.

Effects of bicuculline-induced seizures on benzodiazepine and adenosine receptors in developing rat brain

The effects of seizures induced by an acute administration of bicuculline have been investigated on the central benzodiazepine and adenosine receptors in developing rats and in adults. Generalized seizures rapidly increased the total number of both benzodiazepine binding sites and adenosine A1 receptors, without changes in receptor affinity (KD). It was concluded that such a phenomenon may facilitate the anticonvulsant action of benzodiazepine and adenosine via receptor binding and that it could be an adaptative process to protect subjects against recurrent seizures, especially in newborns.     

Age-dependent inhibition of neuronal protein synthesis by hypothyroidism in the developing rat brain cortex

Neuronal perikarya isolated from developing rat brain cortex were employed for studying the effect of hypothyroidism on RNA and protein synthesis in vitro. Neuronal protein synthesis was inhibited by hypothyroidism during the second week of brain development. Thyroxine treatment in vivo stimulated neuronal protein synthesis in hypothyroid rats. The synthesis of neuronal RNA was depressed by hypothyroidism in 7-day old rats. The inhibition of neuronal protein synthesis due to the lack of thyroid hormaones was restricted to membrane-bound ribosomes. The results suggest that the maturation of the neurone is very sensitive to hormonal imbalance during the critical period of brain development.     

Ethanol impairs insulin-stimulated neuronal survival in the developing brain: role of PTEN phosphatase

Gestational exposure to ethanol causes fetal alcohol syndrome, which is associated with cerebellar hypoplasia. Previous in vitro studies demonstrated ethanol-impaired neuronal survival with reduced signaling through the insulin receptor (IRbeta). We examined insulin signaling in an experimental rat model of chronic gestational exposure to ethanol in which the pups exhibited striking cerebellar hypoplasia with increased apoptosis. Immunoprecipitation and Western blot analyses detected reduced levels of tyrosyl-phosphorylated IRbeta, tyrosyl-phosphorylated insulin receptor substrate-1 (IRS-1), and p85-associated IRS-1 but no alterations in IRbeta, IRS-1, or p85 protein expression in cerebellar tissue from ethanol-exposed pups. In addition, ethanol exposure significantly reduced the levels of total phosphoinositol 3-kinase, Akt kinase, phospho-BAD (inactive), and glyceraldehyde-3-phosphate dehydrogenase and increased the levels of glycogen synthase kinase-3 activity, activated BAD, phosphatase and tensin homolog deleted in chromosome 10 (PTEN) protein, and PTEN phosphatase activity in cerebellar tissue. Cerebellar neurons isolated from ethanol-exposed pups had reduced levels of insulin-stimulated phosphoinositol 3-kinase and Akt kinase activities and reduced insulin inhibition of PTEN and glycogen synthase kinase-3 activity. The results demonstrate that cerebellar hypoplasia produced by chronic gestational exposure to ethanol is associated with impaired survival signaling through insulin-regulated pathways, including failure to suppress PTEN function.