Summary An influential striatal model postulates that neural activities in the striatal direct and indirect pathways promote and inhibit movement, respectively. Normal behavior requires coordinated activity in the direct pathway to facilitate intended locomotion and indirect pathway to inhibit unwanted locomotion. In this striatal model, neuronal population activity is assumed to encode locomotion relevant information. Here, we propose a novel encoding mechanism for the dorsal striatum. We identified spatially compact neural clusters in both the direct and indirect pathways. Detailed characterization revealed similar cluster organization between the direct and indirect pathways, and cluster activities from both pathways were correlated with mouse locomotion velocities. Using machine-learning algorithms, cluster activities could be used to decode locomotion relevant behavioral states and locomotion velocity. We propose that neural clusters in the dorsal striatum encode locomotion relevant information, and that coordinated activities of direct and indirect pathway neural clusters are required for normal striatal controlled behavior.

The insertion of alpha–amino–3–hydroxy–5–methyl–4–isoxazolepropionic acid receptors (AMPARs) into the plasma membrane is a key step in synaptic delivery of AMPARs during the expression of synaptic plasticity. However, the molecular mechanisms regulating AMPAR insertion remain elusive. By directly visualizing individual insertion events of the AMPAR subunit GluR1, we demonstrate that Protein 4.1N is required for activity dependent GluR1 insertion. PKC phosphorylation of GluR1 S816 and S818 residues enhances 4.1N binding to GluR1, and facilitates GluR1 insertion. In addition, palmitoylation of GluR1 C811 residue modulates PKC phosphorylation and GluR1 insertion. Finally, disrupting 4.1N dependent GluR1 insertion decreases surface expression of GluR1 and the expression of long–term potentiation (LTP). Our study uncovers a novel mechanism that governs activity dependent GluR1 trafficking, reveals an interesting interplay between AMPAR palmitoylation and phosphorylation, and underscores the functional significance of the 4.1N protein in AMPAR trafficking and synaptic plasticity.

PSD-95 is a major protein found in virtually all mature excitatory glutamatergic synapses in the brain. Here, we have addressed the role of PSD-95 in controlling glutamatergic synapse function by generating and characterizing a PSD-95 KO mouse. We found that the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptor (AMPAR)-mediated synaptic transmission was reduced in these mice. Two-photon (2P) uncaging of MNI-glutamate onto individual spines suggested that the decrease in AMPAR function in the PSD-95 KO mouse stems from an increase in the proportion of ''silent'' synapses i.e., synapses containing N-methyl-D-aspartate (NMDA) receptors (NMDARs) but no AMPARs. Unexpectedly, the silent synapses in the KO mouse were located onto morphologically mature spines. We also observed that a significant population of synapses appeared unaffected by PSD-95 gene deletion, suggesting that the functional role of PSD-95 displays synapse-specificity. In addition, we report that the decay of NMDAR-mediated current was slower in KO mice: The contribution of NR2B subunit containing receptors to the NMDARmediated synaptic current was greater in KO mice. The greater occurrence of silent synapses might be related to the greater magnitude of potentiation after long-term potentiation induction observed in these mice. Together, these results suggest a synapsespecific role for PSD-95 in controlling synaptic function that is independent of spine morphology.glutamate receptors ͉ hippocampus ͉ spines ͉ synaptic transmission ͉ two-photon uncaging G lutamate, the major excitatory neurotransmitter in the brain, activates ionotropic glutamate receptors of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), and kainate subtypes. There is considerable interest in elucidating the molecular mechanisms that controls synaptic targeting and trafficking of these receptors, in part because of their role in the induction and expression of various forms of synaptic plasticity (1). These receptors are imbedded in an electron-dense structure, the postsynaptic density (PSD), which is believed to contain key molecules involved in the regulation of glutamate receptor targeting and trafficking. PSD-95, a member of the membraneassociated guanylate kinase (MAGUK) superfamily of proteins, is a core component of the PSD and is thought to be important in the control of excitatory synapse function (2, 3). Because of its interaction with the cytoplasmic domains of NMDA receptor (NMDAR) subunits, it has long been suspected that PSD-95 might control the synaptic targeting of NMDARs (4, 5). However, more recent studies based on sustained or transient overexpression of PSD-95 in neurons have forced a reassessment of this view by suggesting that the primary role of PSD-95 is restricted to controlling AMPAR synaptic expression (6-10). Intriguingly, previous work on a PSD-95 KO mouse, reported no apparent changes in either AMPAR or NMDAR function (11). The interpretation of these data are, however, ...

Changes in surface trafficking of AMPA receptors play an important role in synaptic plasticity. Phosphorylation of the C terminus of the AMPA receptor (AMPAR) subunit glutamate receptor 2 (GluR2) and the binding of GluR2 to the PDZ [postsynaptic density-95/Discs large/zona occludens-1]-domain containing protein, protein interacting with protein kinase C (PICK1), have been proposed to play an important role in NMDA receptor dependent internalization of GluR2. However, the fate of internalized GluR2 after NMDA receptor (NMDAR) activation is still unclear. Both recycling and degradation of GluR2 after the activation of NMDAR have been reported. Here, we used a pH-sensitive green fluorescent protein variant, pHluorin, tagged to the N terminus of GluR2 (pH-GluR2) to study the dynamic internalization and recycling of GluR2 after NMDAR activation. Using fluorescence recovery after photobleach (FRAP), we directly demonstrate that internalized pH-GluR2 subunits recycle back to the cell surface after NMDAR activation. We further demonstrate that changing the phosphorylation state of the S880 residue at the C terminus of GluR2 does not affect NMDAR-dependent GluR2 internalization, but alters the recycling of GluR2 after NMDAR activation. In addition, mutation of the N-ethylmaleimide-sensitive fusion protein (NSF) binding site in the pH-GluR2 slows receptor recycling. Finally, neurons lacking PICK1 display normal NMDAR dependent GluR2 internalization compared with wild-type neurons, but demonstrate accelerated GluR2 recycling after NMDAR activation. These results indicate that phosphorylation of GluR2 S880 and NSF and PICK1 binding to GluR2 dynamically regulate GluR2 recycling.

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.

Regulation of AMPA receptor (AMPAR) membrane trafficking plays a critical role in synaptic plasticity and learning and memory. However, how AMPAR trafficking occurs in vivo remains elusive. Using in vivo two-photon microscopy in the mouse somatosensory barrel cortex, we found that acute whisker stimulation leads to a significant increase in the surface expression of the AMPAR GluA1 subunit (sGluA1) in both spines and dendritic shafts and small increases in spine size. Interestingly, initial spine properties bias spine changes following whisker stimulation. Changes in spine sGluA1 are positively correlated with changes in spine size and dendritic shaft sGluA1 following whisker stimulation. The increase in spine sGluA1 evoked by whisker stimulation is NMDA receptor dependent and long lasting, similar to major forms of synaptic plasticity in the brain. These results reveal experience dependent AMPAR trafficking in real time and characterize, in vivo, a major form of synaptic plasticity in the brain.

Cyclophilin D (CyPD) is thought to sensitize opening of the mitochondrial permeability transition pore (mPTP) based on the findings that cyclosporin A (CsA), a pseudo-CyPD substrate, hyperpolarizes the mitochondrial membrane potential (⌬⌿) and inhibits apoptosis. We provide evidence that contrasts with this model. Using live cell imaging and two photon microscopy, we report that overexpression of CyPD desensitizes HEK293 and rat glioma C6 cells to apoptotic stimuli. By site-directed mutagenesis of CyPD that compromises peptidyl-prolyl cis-trans isomerase (PPIase) activity, we demonstrate that the mechanism involved in this protective effect requires PPIase activity. Furthermore, we show that, under resting conditions, ⌬⌿ is hyperpolarized in CyPD wild type-overexpressing cells but not in cells overexpressing mutant forms of CyPD that lack PPIase activity. Finally, in glutathione S-transferase (GST) pull-down assays, we demonstrate that CyPD binding to the adenine nucleotide translocator (ANT), which is considered to be the core component of the mPTP, is not affected by the loss of PPIase activity. Collectively, our data suggest that CyPD should be viewed as a cell survival-signaling molecule and indicate a protective role of CyPD against apoptosis that is mediated by one or more targets other than the ANT.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping neurodegenerative disorders whose pathogenesis remains largely unknown. Here using TDP-43A315T mice, an ALS and FTD model with profound cortical pathology, we demonstrated that hyperactive somatostatin interneurons disinhibited layer 5 pyramidal neurons (L5-PN) and contributed to their excitotoxicity. Focal ablation of somatostatin interneurons efficiently restored normal excitability of L5-PN and alleviated neurodegeneration, suggesting a novel therapeutic target for ALS and FTD.