Glutamate induces de novo growth of functional spines in developing cortex

Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Nature (Impact Factor: 41.46). 06/2011; 474(7349):100-4. DOI: 10.1038/nature09986
Source: PubMed

ABSTRACT Mature cortical pyramidal neurons receive excitatory inputs onto small protrusions emanating from their dendrites called spines. Spines undergo activity-dependent remodelling, stabilization and pruning during development, and similar structural changes can be triggered by learning and changes in sensory experiences. However, the biochemical triggers and mechanisms of de novo spine formation in the developing brain and the functional significance of new spines to neuronal connectivity are largely unknown. Here we develop an approach to induce and monitor de novo spine formation in real time using combined two-photon laser-scanning microscopy and two-photon laser uncaging of glutamate. Our data demonstrate that, in mouse cortical layer 2/3 pyramidal neurons, glutamate is sufficient to trigger de novo spine growth from the dendrite shaft in a location-specific manner. We find that glutamate-induced spinogenesis requires opening of NMDARs (N-methyl-D-aspartate-type glutamate receptors) and activation of protein kinase A (PKA) but is independent of calcium-calmodulin-dependent kinase II (CaMKII) and tyrosine kinase receptor B (TrkB) receptors. Furthermore, newly formed spines express glutamate receptors and are rapidly functional such that they transduce presynaptic activity into postsynaptic signals. Together, our data demonstrate that early neural connectivity is shaped by activity in a spatially precise manner and that nascent dendrite spines are rapidly functionally incorporated into cortical circuits.

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    • "How does transmitter release influence the rate of synaptogenesis? It has long been proposed that transmitter release promotes synaptogenesis by encouraging the motility of pre-and postsynaptic structures, especially dendritic filopodia (Andreae and Burrone, 2014; Kwon and Sabatini, 2011; Smith and Jahr, 1992; Ziv and Smith, 1996). Filopodia are protrusive and highly motile structures and are thought to facilitate contact between axons and dendrites. "
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    ABSTRACT: Across the nervous system, neurons form highly stereotypic patterns of synaptic connections that are designed to serve specific functions. Mature wiring patterns are often attained upon the refinement of early, less precise connectivity. Much work has led to the prevailing view that many developing circuits are sculpted by activity-dependent competition among converging afferents, which results in the elimination of unwanted synapses and the maintenance and strengthening of desired connections. Studies of the vertebrate retina, however, have recently revealed that activity can play a role in shaping developing circuits without engaging competition among converging inputs that differ in their activity levels. Such neurotransmission-mediated processes can produce stereotypic wiring patterns by promoting selective synapse formation rather than elimination. We discuss how the influence of transmission may also be limited by circuit design and further highlight the importance of transmission beyond development in maintaining wiring specificity and synaptic organization of neural circuits.
    Neuron 09/2014; 83(6):1303-1318. DOI:10.1016/j.neuron.2014.08.029 · 15.05 Impact Factor
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    • "More subtle changes in astrocyte glutamate uptake may also have long term consequences on network function as astrocytes are known to regulate synaptic function (Pfrieger and Barres, 1997) and plasticity (Katagiri et al., 2001; Omrani et al., 2009). Furthermore, alterations in glutamate uptake in the neonatal cortex may affect glutamate-induced synapse formation (Kwon and Sabatini, 2011) leading to potentially permanent changes in network connectivity. Despite the importance of astrocyte glutamate uptake, surprisingly little is known about how neonatal cortical insult affects the development of this astrocytic function. "
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    ABSTRACT: Astrocytic uptake of glutamate shapes extracellular neurotransmitter dynamics, receptor activation, and synaptogenesis. During development, glutamate transport becomes more robust. How neonatal brain insult affects the functional maturation of glutamate transport remains unanswered. Neonatal brain insult can lead to developmental delays, cognitive losses, and epilepsy; the disruption of glutamate transport is known to cause changes in synaptogenesis, receptor activation, and seizure. Using the neonatal freeze-lesion (FL) model, we have investigated how insult affects the maturation of astrocytic glutamate transport. As lesioning occurs on the day of birth, a time when astrocytes are still functionally immature, this model is ideal for identifying changes in astrocyte maturation following insult. Reactive astrocytosis, astrocyte proliferation, and in vitro hyperexcitability are known to occur in this model. To probe astrocyte glutamate transport with better spatial precision we have developed a novel technique, Laser Scanning Astrocyte Mapping (LSAM), which combines glutamate transport current (TC) recording from astrocytes with laser scanning glutamate photolysis. LSAM allows us to identify the area from which a single astrocyte can transport glutamate and to quantify spatial heterogeneity in the rate of glutamate clearance kinetics within that domain. Using LSAM, we report that cortical astrocytes have an increased glutamate-responsive area following FL and that TCs have faster decay times in distal, as compared to proximal processes. Furthermore, the developmental shift from GLAST- to GLT-1-dominated clearance is disrupted following FL. These findings introduce a novel method to probe astrocyte glutamate uptake and show that neonatal cortical FL disrupts the functional maturation of cortical astrocytes.
    Frontiers in Cellular Neuroscience 09/2014; 8:277. DOI:10.3389/fncel.2014.00277 · 4.29 Impact Factor
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    • "Neuronal transfections were performed by in-utero electroporation of E15 wild-type C57BL/6J mice as described previously [17]. Briefly, an E15 pregnant mother was anesthetized using 2% isoflurane and injected with 0.1 mg/kg of buprenorphrine as anesthetic. "
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    ABSTRACT: The prokaryotic adaptive immune system CRISPR/Cas9 has recently been adapted for genome editing in eukaryotic cells. This technique allows for sequence-specific induction of double-strand breaks in genomic DNA of individual cells, effectively resulting in knock-out of targeted genes. It thus promises to be an ideal candidate for application in neuroscience where constitutive genetic modifications are frequently either lethal or ineffective due to adaptive changes of the brain. Here we use CRISPR/Cas9 to knock-out Grin1, the gene encoding the obligatory NMDA receptor subunit protein GluN1, in a sparse population of mouse pyramidal neurons. Within this genetically mosaic tissue, manipulated cells lack synaptic current mediated by NMDA-type glutamate receptors consistent with complete knock-out of the targeted gene. Our results show the first proof-of-principle demonstration of CRISPR/Cas9-mediated knock-down in neurons in vivo, where it can be a useful tool to study the function of specific proteins in neuronal circuits.
    PLoS ONE 08/2014; 9(8):e105584. DOI:10.1371/journal.pone.0105584 · 3.23 Impact Factor
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