Scientific Understanding of Consciousness
Neural Development of Spines
Nature, 474, 100–104 (02 June 2011)
Glutamate induces de novo growth of functional spines in developing cortex
Hyung-Bae Kwon1 & Bernardo L. Sabatini1
Howard Hughes Medical Institute, Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA
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.
During postnatal development, the formation and elimination of glutamatergic synapses are thought to be reflected in the growth and retraction of dendritic spines. In cortical pyramidal neurons, waves of new spine growth (spinogenesis) and synapse formation (synaptogenesis) occur at specific developmental stages, followed by pruning as the brain matures. Many signals have been proposed to trigger and regulate de novo spine growth in a developing circuit including neurotrophins, neurotransmitters and cell-adhesion molecules. To uncover the triggers for and mechanisms of spinogenesis, we imaged dendrites of enhanced green fluorescent protein (EGFP)-expressing cortical layer 2/3 pyramidal neurons while releasing glutamate at a specific dendritic location by two-photon laser-induced photolysis of (4-methoxy-7-nitroindolinyl)-glutamate (MNI-glutamate). Analysis was performed in acute cortical brain slices from young mice (postnatal day (P) 8–12), a period in which spinogenesis occurs in vivo.
In this study, we established a protocol for the reliable and spatiotemporally precise induction of spinogenesis. These experiments demonstrate that glutamate is sufficient to trigger rapid spine formation and suggest that neurons use glutamate release to establish circuit wiring. Thus these data support the hypothesis that axonal growth and glutamate release may be the triggering event in synapse formation such that axonal bouton localization is an important early step for precise neuronal circuit formation. Given the involvement of NMDARs, Ca2+ stores, cAMP, PKA and MAPK in activity-dependent spinogenesis, it is likely that many neuromodulators that regulate these molecules may influence the capacity or threshold for new spine formation. For instance, activation of dopaminergic, serotonergic or adrenergic receptors that signal by Gαs may facilitate spinogenesis, whereas receptors that activate Gαi-coupled signalling may function as inhibitory signals.
Lastly, we provide experimental evidence that spines that grow de novo in developing cortical tissue become rapidly functionally integrated into the circuit such that they sense synaptically released glutamate through AMPARs and NMDARs. Whether these nascent spines are rapidly physically associated with a presynaptic bouton and display the ultrastructural correlates of a synapse is unknown. Our results indicate that spines can grow de novo without the need for a filopodial intermediate and probably without a dendritic-shaft synapse stage. In total, this study demonstrates that synaptic activity can rapidly modify neuronal connectivity with high accuracy by generating new circuit elements.
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