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Neuronal ceroid lipofuscinoses (NCLs) are a group of genetic childhood-onset progressive brain diseases characterized by a decline in mental and motor capacities, epilepsy, visual loss and premature death. Using patch clamp, fluorescence imaging and caged Ca 2+ photolysis, we evaluated the mechanisms of neuronal Ca 2+ clearance in Cln8 mnd mice, a model of the human NCL caused by mutations in the CLN8 gene. In Cln8 mnd hippocampal slices, Ca 2+ clearance efficiency in interneurons and, to some extent, principal neurons declined with age. In cultured Cln8 mnd hippocampal neurons, clearance of large Ca 2+ loads was inefficient due to impaired mitochondrial Ca 2+ uptake. In contrast, neither Ca 2+ uptake by sarco/endoplasmic reticulum Ca 2+ ATPase, nor Ca 2+ extrusion through plasma membrane was affected by the Cln8 mutation. Excitotoxic glutamate challenge caused Ca 2+ deregulation more readily in Cln8 mnd than in wt neurons. We propose that neurodegeneration in human CLN8 disorders is primarily caused by reduced mitochondrial Ca 2+ buffering capacity.

SummaryThe neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl− transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells. The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

Functional expression of the K-Cl cotransporter KCC2 in developing central neurons is crucial for the maturation of Cl−-dependent, GABAAreceptor-mediated inhibitory responses. In pyramidal neurons of the rodent hippocampus, GABAergic postsynaptic responses are typically depolarizing and often excitatory during the first postnatal week. Here, we show that a single neonatal seizure episode induced by kainate injection during postnatal days 5–7 results in a fast increase in the Cl−extrusion capacity of rat hippocampal CA1 neurons, with a consequent hyperpolarizing shift of the reversal potential of GABAA-mediated currents (EGABA). A significant increase in the surface expression of KCC2 as well as the α2 subunit of the Na-K-ATPase parallels the seizure-induced increase in the Cl−extrusion capacity. Exposing hippocampal slices to kainate resulted in a similar increase in the neuronal Cl−extrusion and in the surface expression of KCC2. Both effects were blocked by the kinase inhibitor K252a. Hence, in the neonatal hippocampus the overall KCC2 expression level is high enough to promote a rapid functional activation of K-Cl cotransport and a consequent negative shift in EGABAclose to the adult level. The activity-dependent regulation of KCC2 function and its effect on GABAergic transmission may represent an intrinsic antiepileptogenic mechanism.

GABAergic terminals of axo-axonic cells (AACs) are exclusively located on the axon initial segment (AIS) of cortical principal neurons, and they are generally thought to exert a powerful inhibitory action. However, recent work (Szabadics et al., 2006) indicates that this input from AACs can be depolarizing and even excitatory. Here, we used local photolysis of caged GABA to measure reversal potentials (EGABA) of GABAAreceptor-mediated currents and to estimate the local chloride concentration in the AIS compared with other cellular compartments in dentate granule cells and neocortical pyramidal neurons. We found a robust axo-somato-dendritic gradient in which theEGABAvalues from the AIS to the soma and dendrites become progressively more negative. Data fromNKCC1−/−and bumetanide-exposed neurons indicated that the depolarizingEGABAat the AIS is set by chloride uptake mediated by the Na–K–2Cl cotransporter NKCC1. Our findings demonstrate that spatially distinct interneuronal inputs can induce postsynaptic voltage responses with different amplitudes and polarities as governed by the subcellular distributions of plasmalemmal chloride transporters.

Reactive oxygen species (ROS) constitute important signaling molecules in the central nervous system. They regulate a number of different functions both under physiological conditions and under pathological conditions. Here we tested the hypothesis that in the immature hippocampus ATP, the most diffuse neurotransmitter in the brain, modulates synaptic transmission via ROS. We show that ATP, acting on metabotropic P2Y1 receptors, increased the frequency of GABA(A)-mediated spontaneous postsynaptic currents (SPSCs) in CA3 principal cells, an effect that was prevented by the antioxidant N-acetyl-cysteine or by catalase, an enzyme that breaks down H2O2. The effect of ATP on SPSCs was mimicked by H2O2 or by the pro-oxidant, Fe2+, which, through the Fentol reaction, catalyzes the conversion of H2O2 into highly reactive hydroxyl radicals. MRS-2179, a P2Y1 receptor antagonist, removed the facilitatory action of Fe2+ on SPSCs, suggesting that endogenous ATP acting on P2Y1 receptors is involved in Fe2+-induced modulation of synaptic transmission. Imaging ROS with the H2O2-sensitive dye DCF revealed that ATP induces generation of peroxide in astrocytes via activation of P2Y1 receptors coupled to intracellular calcium rise. Neither N-acetyl-cysteine nor catalase prevented Ca2+ transients induced by ATP in astrocytes. Since a single hippocampal astrocyte can contact many neurons, ATP-induced ROS signaling may control thousands of synapses. This may be crucial for information processing in the immature brain when GABAergic activity is essential for the proper wiring of the hippocampal network.