[Show abstract][Hide abstract] ABSTRACT: Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
[Show abstract][Hide abstract] ABSTRACT: How the billions of synapses in the adult mammalian brain are precisely specified remains one of the fundamental questions of neuroscience. Although a genetic program is likely to encode the basic neural blueprint, much evidence suggests that experience-driven activity through NMDA receptors wires up neuronal circuits by inducing a process similar to long-term potentiation. To test this notion directly, we eliminated NMDA receptors before and during synaptogenesis in single cells in vitro and in vivo. Although the prevailing model would predict that NMDA receptor deletion should strongly inhibit the maturation of excitatory circuits, we find that genetic ablation of NMDA receptor function profoundly increases the number of functional synapses between neurons. Conversely, reintroduction of NMDA receptors into NR1-deficient neurons reduces the number of functional inputs, a process requiring network activity and NMDA receptor function. Although NMDA receptor deletion increases the strength of unitary connections, it does not alter neuronal morphology, suggesting that basal NMDA receptor activation blocks the recruitment of AMPA receptors to silent synapses. Based on these results we suggest a new model for the maturation of excitatory synapses in which ongoing activation of NMDA receptors prevents premature synaptic maturation by ensuring that only punctuated bursts of activity lead to the induction of a functional synapse for the activity-dependent wiring of neural circuitry.
Preview · Article · May 2008 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Interneurons are born in subcortical germinative zones and tangentially migrate in multiple streams above and below the developing cortex, and then, at the appropriate developmental stage, migrate radially into the cortex. The factors that control the formation of and the timing of exit from the streams remain obscure; moreover, the rationale for this complicated developmental plan is unclear. We show that a chemokine, Cxcl12, is an attractant for interneurons during the stage of stream formation and tangential migration. Furthermore, the timing of exit from the migratory streams accompanies loss of responsiveness to Cxcl12 as an attractant. Mice with mutations in Cxcr4 have disorganized migratory streams and deletion of Cxcr4 after the streams have formed precipitates premature entry into the cortical plate. In addition, constitutive deletion of Cxcr4 specifically in interneurons alters the regional distribution of interneurons within the cortex and leads to interneuron laminar positioning defects in the postnatal cortex. To examine the role of interneuron distribution on the development of cortical circuitry, we generated mice with focal defects in interneuron distribution and studied the density of postnatal inhibitory innervation in areas with too many and too few interneurons. Interestingly, alterations in IPSC frequency and amplitude in areas with excess interneurons tend toward normalization of inhibitory tone, but in areas with reduced interneuron density this system fails. Thus, the processes controlling interneuron sorting, migration, regional distribution, and laminar positioning can have significant consequences for the development of cortical circuitry and may have important implications for a range of neurodevelopmental disorders.
Preview · Article · Feb 2008 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Long-term potentiation (LTP) at hippocampal synapses is thought to involve the insertion of AMPA receptors into the postsynaptic membrane. Conflicting evidence exists as to whether calcium-permeable receptors are inserted during LTP and whether synaptic activity mediated by the newly inserted AMPA receptors is required to maintain the increase in synaptic strength. Here, we rigorously test these hypotheses and conclude that calcium-permeable AMPA receptors are not inserted during LTP nor does potentiation require ongoing activity to be maintained.
Preview · Article · May 2007 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
[Show abstract][Hide abstract] ABSTRACT: Abnormally synchronized synaptic transmission in the brain causes epilepsy. Most inherited forms of epilepsy result from mutations in ion channels. However, one form of epilepsy, autosomal dominant partial epilepsy with auditory features (ADPEAF), is characterized by mutations in a secreted neuronal protein, LGI1. We show that ADAM22, a transmembrane protein that when mutated itself causes seizure, serves as a receptor for LGI1. LGI1 enhances AMPA receptor-mediated synaptic transmission in hippocampal slices. The mutated form of LGI1 fails to bind to ADAM22. ADAM22 is anchored to the postsynaptic density by cytoskeletal scaffolds containing stargazin. These studies in rat brain indicate possible avenues for understanding human epilepsy.
[Show abstract][Hide abstract] ABSTRACT: AMPA receptors mediate the majority of the fast excitatory transmission in the central nervous system. Much evidence suggests that the fast trafficking of AMPA receptors into and out of the postsynaptic membrane underlies changes in synaptic strength thought to be necessary for higher cognitive functions such as learning and memory. Despite the abundance of research conducted in this area, a direct, real-time functional assay that measures the trafficking of native AMPA receptors has been lacking. Toward this aim, we use a photoreactive, irreversible antagonist of AMPA receptors, ANQX, to rapidly silence surface AMPA receptors and investigate directly the trafficking of native AMPA receptors in real time. We find that the most dynamic movement of AMPA receptors occurs by lateral movement across the surface of neurons. Fast cycling of surface AMPA receptors with receptors from internal stores does occur but exclusively at extrasynaptic somatic sites. The cycling of synaptic AMPA receptors only occurs on a much longer timescale with complete exchange requiring at least 16 hr. This cycling is not dependent on protein synthesis or action potential driven network activity. These data suggest a revised model of AMPA receptor trafficking wherein a large internal store of AMPA receptors exchanges rapidly with extrasynaptic somatic AMPA receptors, and these newly inserted AMPA receptors then travel laterally along dendrites to reside stably at synapses.
[Show abstract][Hide abstract] ABSTRACT: Synaptic plasticity involves activity-dependent trafficking of AMPA-type glutamate receptors. Numerous cytoplasmic scaffolding proteins are postulated to control AMPA receptor trafficking, but the detailed mechanisms remain unclear. Here, we show that the transmembrane AMPA receptor regulatory protein (TARP) gamma-8, which is preferentially expressed in the mouse hippocampus, is important for AMPA receptor protein levels and extrasynaptic surface expression. By controlling the number of AMPA receptors, gamma-8 is also important in long-term potentiation, but not long-term depression. This study establishes gamma-8 as a critical protein for basal AMPA receptor expression and localization at extrasynaptic sites in the hippocampus and raises the possibility that TARP-dependent control of AMPA receptors during synapse development and plasticity may be widespread.
Full-text · Article · Dec 2005 · Nature Neuroscience
[Show abstract][Hide abstract] ABSTRACT: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors mediate fast excitatory synaptic transmission in the brain. These ion channels rapidly deactivate and desensitize, which determine the time course of synaptic transmission. Here, we find that the AMPA receptor interacting protein, stargazin, not only mediates AMPA receptor trafficking but also shapes synaptic responses by slowing channel deactivation and desensitization. The cytoplasmic tail of stargazin determines receptor trafficking, whereas the ectodomain controls channel properties. Stargazin alters AMPA receptor kinetics by increasing the rate of channel opening. Disrupting the interaction of stargazin ectodomain with hippocampal AMPA receptors alters the amplitude and shape of synaptic responses, establishing a crucial function for stargazin in controlling the efficacy of synaptic transmission in the brain.
[Show abstract][Hide abstract] ABSTRACT: Palmitoylation is a lipid modification that plays a critical role in protein trafficking and function throughout the nervous system. Palmitoylation of PSD-95 is essential for its regulation of AMPA receptors and synaptic plasticity. The enzymes that mediate palmitoyl acyl transfer to PSD-95 have not yet been identified; however, proteins containing a DHHC cysteine-rich domain mediate palmitoyl acyl transferase activity in yeast. Here, we isolated 23 mammalian DHHC proteins and found that a subset specifically palmitoylated PSD-95 in vitro and in vivo. These PSD-95 palmitoyl transferases (P-PATs) showed substrate specificity, as they did not all enhance palmitoylation of Lck, SNAP-25b, Galpha(s), or H-Ras in cultured cells. Inhibition of P-PAT activity in neurons reduced palmitoylation and synaptic clustering of PSD-95 and diminished AMPA receptor-mediated neurotransmission. This study suggests that P-PATs regulate synaptic function through PSD-95 palmitoylation.