In vivo cocaine experience generates silent synapses
Studies over the past decade have enunciated silent synapses as prominent cellular substrates for synaptic plasticity in the developing brain. However, little is known about whether silent synapses can be generated postdevelopmentally. Here, we demonstrate that highly salient in vivo experience, such as exposure to cocaine, generates silent synapses in the nucleus accumbens (NAc) shell, a key brain region mediating addiction-related learning and memory. Furthermore, this cocaine-induced generation of silent synapses is mediated by membrane insertions of new, NR2B-containing N-methyl-D-aspartic acid receptors (NMDARs). These results provide evidence that silent synapses can be generated de novo by in vivo experience and thus may act as highly efficient neural substrates for the subsequent experience-dependent synaptic plasticity underlying extremely long-lasting memory.
CREB modulates the functional output of nucleus accumbens neurons: a critical role of N-methyl-D-aspartate glutamate receptor (NMDAR) receptors
Nucleus accumbens (NAc) medium spiny neurons cycle between two states, a functionally inactive downstate and a functionally active upstate. Here, we show that activation of the transcription factor cAMP-response element-binding protein (CREB), a common molecular response to several drugs of abuse, increases both duration of the upstate and action potential firing during the upstate. This effect of CREB is mediated by enhanced N-methyl-d-aspartate glutamate receptor (NMDAR) function: increased CREB activity increases both NMDAR-mediated synaptic currents and surface level of NMDARs, while inhibition of NMDARs abolishes the effect of CREB on upstate duration. Furthermore, mimicking the effect of CREB by pharmacological enhancement of NMDAR function in the NAc in vivo suppressed novelty- and cocaine-elicited locomotor activity. These findings suggest that by enhancing NMDAR-mediated synaptic transmission, CREB activation promotes the proportion of time NAc neurons spend in the upstate. This effect, along with the CREB enhancement of NAc membrane excitability (Dong, Y., Green, T., Saal, D., Marie, H., Neve, R., Nestler, E. J., and Malenka, R. C. (2006) Nat. Neurosci. 9, 475-477), may counteract drug-induced maladaptations in the NAc and thus ameliorate the addictive state.
PSD-95 and PKC converge in regulating NMDA receptor trafficking and gating
Neuronal NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a putative NMDAR anchoring protein and core component of the PSD, at excitatory synapses. PKC activation and PSD-95 expression each enhance NMDAR channel opening rate and number of functional channels at the cell surface. Here we show in Xenopus oocytes that PSD-95 and PKC potentiate NMDA gating and trafficking in a nonadditive manner. PSD-95 and PKC each enhance NMDA channel activity, with no change in single-channel conductance, reversal potential or mean open time. PSD-95 and PKC each potentiate NMDA channel opening rate (k(beta)) and number of functional channels at the cell surface (N), as indicated by more rapid current decay and enhanced charge transfer in the presence of the open channel blocker MK-801. PSD-95 and PKC each increase NMDAR surface expression, as indicated by immunofluorescence. PKC potentiates NMDA channel function and NMDAR surface expression to the same final absolute values in the absence or presence of PSD-95. Thus, PSD-95 partially occludes PKC potentiation. We further show that Ser-1462, a putative phosphorylation target within the PDZ-binding motif of the NR2A subunit, is required for PSD-95-induced potentiation and partial occlusion of PKC potentiation. Coimmunoprecipitation experiments with cortical neurons in culture indicate that PKC activation promotes assembly of NR2 with NR1, and that the newly assembled NMDARs are not associated with PSD-95. These findings predict that synaptic scaffolding proteins and protein kinases convergently modulate NMDAR gating and trafficking at synaptic sites.
Protein kinase A regulates calcium permeability of NMDA receptors
Calcium (Ca2+) influx through NMDA receptors (NMDARs) is essential for synaptogenesis, experience-dependent synaptic remodeling and plasticity. The NMDAR-mediated rise in postsynaptic Ca2+ activates a network of kinases and phosphatases that promote persistent changes in synaptic strength, such as long-term potentiation (LTP). Here we show that the Ca2+ permeability of neuronal NMDARs is under the control of the cyclic AMP-protein kinase A (cAMP-PKA) signaling cascade. PKA blockers reduced the relative fractional Ca2+ influx through NMDARs as determined by reversal potential shift analysis and by a combination of electrical recording and Ca2+ influx measurements in rat hippocampal neurons in culture and hippocampal slices from mice. In slices, PKA blockers markedly inhibited NMDAR-mediated Ca2+ rises in activated dendritic spines, with no significant effect on synaptic current. Consistent with this, PKA blockers depressed the early phase of NMDAR-dependent LTP at hippocampal Schaffer collateral-CA1 (Sch-CA1) synapses. Our data link PKA-dependent synaptic plasticity to Ca2+ signaling in spines and thus provide a new mechanism whereby PKA regulates the induction of LTP.
Postsynaptic density protein-95 regulates NMDA channel gating and surface expression
NMDA receptors (NMDARs) colocalize with postsynaptic density protein-95 (PSD-95), a multivalent synaptic scaffolding protein and core component of the postsynaptic density, at excitatory synapses. Although much is known about the identity and properties of scaffolding proteins, little is known about their actions on NMDAR function. Here we show that association of PSD-95 with NMDARs modulates channel gating and surface expression. PSD-95 increases the number of functional channels at the cell surface and channel opening rate of NMDARs, with little or no change in conductance, reversal potential, or mean open time. We show further that PSD-95 increases NMDAR surface expression by increasing the rate of channel insertion and decreasing the rate of channel internalization. The PDZ (PSD-95, discs large, zona occludens-1) binding motif at the distal end of the NR2 C-terminal tail is critical to the actions of PSD-95 on NMDAR function and surface expression. Given that activity bi-directionally modifies synaptic levels of PSD-95, our findings suggest a novel mechanism for activity-dependent regulation of NMDARs at central synapses.
Ischemic insults derepress the gene silencer REST in neurons destined to die
A subset of genes implicated in genetic and acquired neurological disorders encode proteins essential to neural patterning and neurogenesis. The gene silencing transcription factor neuronal repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF) plays a critical role in elaboration of the neuronal phenotype. In neural progenitor and non-neural cells, REST acts by repression of a subset of neural genes important to synaptic plasticity and synaptic remodeling, including the AMPA receptor (AMPAR) subunit GluR2. Here we show that global ischemia triggers REST mRNA and protein expression. REST suppresses GluR2 promoter activity and gene expression in neurons destined to die. Because the GluR2 subunit governs AMPAR Ca2+ permeability, these changes are expected to have profound effects on neuronal survival. In keeping with this concept, acute knockdown of the REST gene by antisense administration prevents GluR2 suppression and rescues post-ischemic neurons from ischemia-induced cell death in an in vitro model. To our knowledge, our study represents the first example of ischemia-induced induction of a master transcriptional regulator gene and its protein expression critical to neural differentiation and patterning in adult neurons. Derepression of REST is likely to be an important mechanism of insult-induced neuronal death.
Global ischemia-induced increases in the gap junctional proteins connexin 32 (Cx32) and Cx36 in hippocampus and enhanced vulnerability of Cx32 knock-out mice
Gap junctions are conductive channels that connect the interiors of coupled cells. In the hippocampus, GABA-containing hippocampal interneurons are interconnected by gap junctions, which mediate electrical coupling and synchronous firing and thereby promote inhibitory transmission. The present study was undertaken to examine the hypothesis that the gap junctional proteins connexin 32 (Cx32; expressed by oligodendrocytes, interneurons, or both), Cx36 (expressed by interneurons), and Cx43 (expressed by astrocytes) play a role in defining cell-specific patterns of neuronal death in hippocampus after global ischemia in mice. Global ischemia did not significantly alter Cx32 and Cx36 mRNA expression and slightly increased Cx43 mRNA expression in the vulnerable CA1, as assessed by Northern blot analysis and in situ hybridization. Global ischemia induced a selective increase in Cx32 and Cx36 but not Cx43 protein abundance in CA1 before onset of neuronal death, as assessed by Western blot analysis. The increase in Cx32 and Cx36 expression was intense and specific to parvalbumin-positive inhibitory interneurons of CA1, as assessed by double immunofluorescence. Protein abundance was unchanged in CA3 and dentate gyrus. The finding of increase in connexin protein without increase in mRNA suggests regulation of Cx32 and Cx36 expression at the translational or post-translational level. Cx32(Y/-) null mice exhibited enhanced vulnerability to brief ischemic insults, consistent with a role for Cx32 gap junctions in neuronal survival. These findings suggest that Cx32 and Cx36 gap junctions may contribute to the survival and resistance of GABAergic interneurons, thereby defining cell-specific patterns of global ischemia-induced neuronal death.
Protein kinase C modulates NMDA receptor trafficking and gating
Regulation of neuronal N-methyl-D-aspartate receptors (NMDARs) by protein kinases is critical in synaptic transmission. However, the molecular mechanisms underlying protein kinase C (PKC) potentiation of NMDARs are uncertain. Here we demonstrate that PKC increases NMDA channel opening rate and delivers new NMDA channels to the plasma membrane through regulated exocytosis. PKC induced a rapid delivery of functional NMDARs to the cell surface and increased surface NR1 immunofluorescence in Xenopus oocytes expressing NMDARs. PKC potentiation was inhibited by botulinum neurotoxin A and a dominant negative mutant of soluble NSF-associated protein (SNAP-25), suggesting that receptor trafficking occurs via SNARE-dependent exocytosis. In neurons, PKC induced a rapid delivery of functional NMDARs, assessed by electrophysiology, and an increase in NMDAR clusters on the surface of dendrites and dendritic spines, as indicated by immunofluorescence. Thus, PKC regulates NMDAR channel gating and trafficking in recombinant systems and in neurons, mechanisms that may be relevant to synaptic plasticity.
Mutation of structural determinants lining the N-methyl-D-aspartate receptor channel differentially affects phencyclidine block and spermine potentiation and block
Spermine and other endogenous polyamines potentiate, block and permeate the N-methyl-D-aspartate receptor channel. To identify structural determinants of the N-methyl-D-aspartate channel that mediate spermine's actions, we generated mutant receptors with asparagine (N) to glutamine (Q) or arginine (R) substitutions in the selectivity filter of the channel. We demonstrate that mutation of the three critical asparagines in this domain differentially affects block by phencyclidine and both potentiation and block by spermine. N-to-Q and N-to-R mutations in the N site of the NR1 subunit (N598 in NR1(011), N619 in NR1(100)) and N-to-Q mutations in the N and N + 1 sites (N595 and N596 in NR2A, respectively) of the NR2 subunit (Q/NN, R/NN, N/QN, N/NQ, Q/QN and Q/NQ receptors) reduced affinity for phencyclidine. The Q/NN receptor showed markedly reduced potentiation by spermine, with little or no change in spermine block. The R/NN receptor showed markedly reduced spermine potentiation and affinity for spermine at its block site. The N/QN, N/NQ and Q/QN mutant receptors showed somewhat enhanced spermine block, while the Q/ NQ double mutant exhibited significantly more enhanced spermine block. Thus, the asparagine residues critical to Ca2+ permeability and Mg2+ block of N-methyl-D-aspartate channels are also critical to block by spermine and phencyclidine. To examine the interaction of spermine and phencyclidine within the channel, we performed competition studies. Spermine appeared to compete with phencyclidine for binding to the receptor; however, blocks by phencyclidine and by spermine were not additive. The findings suggest that spermine can bind to a site in the external vestibule of the channel to impede phencyclidine binding, but allow Na+ influx.