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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.

Growing evidence suggests that astrocytes are the active partners of neurons in many brain functions. Astrocytic mitochondria are highly motile organelles which regulate the temporal and spatial patterns of Ca 2+ dynamics, in addition to being a major source of ATP and reactive oxygen species. Previous studies have shown that mitochondria translocate to endoplasmic reticulum during Ca 2+ release from internal stores, but whether a similar spatial interaction between mitochondria and plasma membrane occurs is not known. Using total internal reflection fluorescence (TIRF) microscopy we show that a fraction of mitochondria became trapped near the plasma membrane of cultured hippocampal astrocytes during exposure to the transmitters glutamate or ATP, resulting in net translocation of the mitochondria to the plasma membrane. This translocation was dependent on the intracellular Ca 2+ rise because it was blocked by pre-incubation with BAPTA AM and mimicked by application of the Ca 2+ ionophore ionomycin. Transmembrane Ca 2+ influx induced by raising external Ca 2+ also caused mitochondrial trapping, which occurred more rapidly than that produced by glutamate or ATP. In astrocytes treated with the microtubule-disrupting agent nocodazole, intracellular Ca 2+ rises failed to induce trapping of mitochondria near plasma membrane, suggesting a role for microtubules in this phenomenon. Our data reveal the Ca 2+ -dependent trapping of mitochondria near the plasma membrane as a novel form of mitochondrial regulation, which is likely to control the perimembrane Ca 2+ dynamics and regulate signaling by mitochondria-derived reactive oxygen species.

Mouse skeletal myotubes differentiated in vitro exhibited spontaneous contractions associated with electrical activity. The ionic conductances responsible for the origin and modulation of the spontaneous activity were examined using the whole-cell patch-clamp technique and measuring [Ca(2+)](i) transients with the Ca(2+) indicator, fura 2-AM. Regular spontaneous activity was characterized by single TTX-sensitive action potentials, followed by transient increases in [Ca(2+)](i). Since the bath-application of Cd(2+) (300 microM) or Ni(2+) (50 muM) abolished the cell firing, T-type (I(Ca,T)) and L-type (I(Ca,L)) Ca(2+) currents were investigated in spontaneously contracting myotubes. The low activation threshold (around -60 mV) and the high density of I(Ca,T) observed in contracting myotubes suggested that I(Ca,T) initiated action potential firing, by bringing cells to the firing threshold. The results also suggested that the activity of I(Ca,L) could sustain the [Ca(2+)](i) transients associated with the action potential, leading to the activation of apamin-sensitive SK-type Ca(2+)-activated K(+) channels and the afterhyperpolarization (AHP) following single spikes. In conclusion, an interplay between voltage-dependent inward (Na(+) and Ca(2+)) and outward (SK) conductances is proposed to mediate the spontaneous pacemaker activity in cultured muscle myotubes during the process of myogenesis.

We have shown previously that ATP inhibits transmitter release at the neuromuscular junction through the action on metabotropic P2Y receptors coupled to specific second messenger cascades. In the present study we recorded K + or Ca 2 + currents in motor nerve endings or blocked K + or Ca 2 + channels in order to explore the nature of downstream presynaptic effectors. Endplate currents were presynaptically depressed by ATP. Blockers of Ca 2 + -activated K + -channels, such as iberiotoxin, apamin or tetraethylammonium, did not change the depressant action of ATP. By contrast, K + channel blocker 4-aminopyridine (4-AP) and raised extracellular Ca 2 + attenuated the effect of ATP. However, these effects of 4-AP and high Ca 2 + were reversed by Mg 2 + , suggesting Ca 2 + -dependence of the ATP action. Ba 2 + promoted the depressant action of ATP as did glibenclamide, a blocker of ATP-sensitive K + channels, or mild depolarization produced by 7.5 mM K + . None of the K + channel blockers affected the depressant action of adenosine. Focal recording revealed that neither ATP nor adenosine affected the fast K + currents of the motor nerve endings. However, unlike adenosine, ATP or UTP, an agonist of P2Y receptors, reversibly reduced the presynaptic Ca 2 + -current. This effect was abolished by suramin, an antagonist of P2 receptors. Depressant effect of ATP on the endplate and Ca 2 + -currents was mimicked by arachidonate, which precluded the action of ATP. ATP reduced acetylcholine release triggered by ionomycin or sucrose, suggesting inhibition of release machinery. Thus, the presynaptic depressant action of ATP is mediated by inhibition of Ca 2 + channels and by mechanism acting downstream of Ca 2 + entry.

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.

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatalNKCC1−/−mice that lack the Na–K–2Cl cotransporter isoform 1. Strikingly,NKCC1−/−slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABAAagonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K–Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in theNKCC1−/−hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

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.